gp37 3’ UTR reduces protein production at the polyhedrin locus of Autographa californica multicapsid nucleopolyhedrovirus

gp37 3’ UTR reduces protein production at the polyhedrin locus of Autographa californica multicapsid nucleopolyhedrovirus | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article gp37 3’ UTR reduces protein production at the polyhedrin locus of Autographa californica multicapsid nucleopolyhedrovirus Xiao-Wen Cheng, Jianli Xue, Hui Shang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7992808/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Baculovirus is a double-stranded DNA virus widely used in agriculture for the biological control of insect pests and in the pharmaceutical industry for vaccine production, due to its safety in humans. The type species, Autographa californica multiple nucleopolyhedrovirus (AcMNPV), has a broad insect host range and is the most genetically characterized baculovirus. During infection of insect cells, early genes are transcribed by the host RNA polymerase II, while late and very late genes are transcribed by a virus-encoded RNA polymerase. One such late gene, gp37 , is considered non-essential for viral replication, though its biological function remains unclear. In this study, we constructed three pairs of recombinant viruses, each containing complementary reporter genes, to investigate the regulatory role of the gp37 3' untranslated region (3' UTR). Our results show that the gp37 3' UTR modulates the expression of polyhedrin, green fluorescent protein and luciferase genes even when driven by the same promoter. This suggests a post-transcriptional regulatory role for the gp37 3' UTR in gene expression. Gene transcription gene regulation protein expression Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Baculoviruses are large, double-stranded DNA viruses widely utilized both as biological control agents against insect pests and vectors for protein expression in biotechnology applications [ 1 , 2 ]. The type species of the family Baculoviridae , Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV), has served as a key model system for baculovirus genetic research due to the high permissiveness of serval insect cell lines to AcMNPV infection [ 1 ]. Notably, AcMNPV was the first baculovirus to have its genome fully sequenced, revealing a compact 134-kilobase (kb) genome [ 3 ]. During a productive infection cycle in permissive insect cells, AcMNPV enters the cells likely through receptor-mediated endocytosis. Once internalized, virion-containing endosomes are transported to the nucleus along actin filaments [ 4 ]. Fusion of the viral envelope with the endosomal membrane releases the nucleocapsids into the cytoplasm near the nucleus, after which they are imported into the nucleus through the nuclear pore complex. Inside the nucleus, the nucleocapsids uncoat, releasing the viral DNA genome to initiate transcription. Viral gene expression occurs in distinct temporal phases: early transcribed by host RNA polymerase II, late transcribed by virus-encoded RNA polymerase, and an undefined phase characterized by transcripts lacking a canonical 3′ end processing signal [ 5 ]. During the late phase of infection, the polh gene is highly expressed, leading to the production of large polyhedrin protein complexes known as occlusion bodies. These structures encapsulate or occlude enveloped virions [ 6 ]. The formation of occlusion bodies is believed to be an evolutionary adaptation that protects virions from environmental degradation, thereby enhancing baculovirus transmission within insect populations under natural condition [ 7 ]. The gp37 gene of AcMNPV , a homologue of the spindlin gene of Choristoneura fumiferana defective nucleopolyhedrovirus ( CfDEFNPV ), is classified as a late gene, but is not highly expressed during cell infection. In contrast, CfDEFNPV expresses the spindlin gene at high levels in permissive cells, resulting in the formation of distinctive spindle-shaped inclusions in the cytoplasm. Similar spindle-shaped inclusions have also been reported in cells infected with other nucleopolyhedroviruses (NPVs), including Choristoneura murinana NPV, Orgyia pseudotsugata MNPV, Cadra cautella NPV, and Archips cerasivoranus NPV [ 8 , 9 ]. However, AcMNPV -infected cells do not exhibit spindle-shaped inclusions, suggesting that either gp37 is not expressed at sufficient levels, or it is expressed abundantly but fails to crystallize into visible structures. The exact function of AcMNPV gp37 remains unclear, though it has been shown to be non-essential for viral replication [ 10 ]. Interestingly, the gp37 gene from Cydia pomonella granulovirus ( CpGV ) was recently reported to encode a toxin that enhances baculovirus efficacy in insect control [ 11 ]. Transcriptional analysis of AcMNPV gp37 during infection of permissive Sf21 cells has revealed multiple transcript variants, differing in the length of their 3′ untranslated regions (3′ UTRs) [ 12 ], suggesting complex post-transcriptional regulation. The 3′ untranslated regions (3′ UTRs) of eukaryotic mRNAs play multiple regulatory roles, including directing the formation of the poly(A) tail, influencing mRNA stability, and providing binding sites for microRNAs [ 13 , 14 ]. Proper 3′ end formation is crucial for mRNA maturation and gene expression. This process typically requires cleavage and polyadenylation signals, located near the end of the 3′ UTR, which facilitate the addition of the poly(A) tail to most transcripts [ 15 , 16 ]. Alternative polyadenylation (APA) , the use of multiple polyadenylation signals within a single transcript, can significantly impact gene regulation. APA may result in the removal of large portions of the 3′ UTR, thereby reducing the transcript's exposure to regulatory elements such as microRNA binding sites or AU-rich elements, which are often associated with decreased stability [ 17 ]. Depending on the polyadenylation site used, APA can produce transcripts with subtle changes, such as variable 3′ UTR lengths, or more dramatic consequences, including altered RNA stability, localization, or even changes in the coding sequence that yield protein isoforms with distinct domains. Most alternative polyadenylation occurred in cellular organisms. Given the compact and highly efficient nature of viral genomes, APA has been observed in numerous viruses, including human papillomavirus type 16 (HPV-16) and avian retroviruses, where it contributes to the diversity and regulation of viral gene expression [ 18 – 20 ]. Alternative polyadenylation has also been reported in viruses, such as adeno-associated virus type 5 RNA [ 21 ]. More recently, alternative polyadenylation has been reported in other viruses. For example, alternative polyadenylation has been reported in vesicular stomatitis virus, macrophages, influenza A and parvovirus [ 22 – 25 ]. Therefore, alternative polyadenylation might be widely used by viruses to increase their coding capacity, considering in general, the virus genome is much smaller and more compact than cellular genomes. Three transcript isoforms of the AcMNPV gp37 gene, differing in their 3′ untranslated regions (3′ UTRs), have been detected during infection [ 12 ]. These transcripts terminate at three distinct positions, each associated with a canonical polyadenylation signal (AAUAAA) located downstream of the gp37 coding sequence. All three transcript variants are detectable from 6 hours post-infection (h p.i.), but their relative abundances change over time. The two shorter transcripts dominate in the early stages of infection, whereas the longest transcript becomes increasingly abundant as the infection progresses [ 12 ]. This shift from shorter to longer transcripts reflects a switch in polyadenylation site usage, resulting in alternative 3′ UTRs for gp37 . A similar pattern where shorter transcript forms are more prevalent in the early stages of infection has been observed for other baculovirus mRNAs that undergo alternative polyadenylation [ 6 , 33 ]. Interestingly, the spindlin gene of CfDEFNPV , which shares 59.09% similarity with AcMNPV gp37 , contains only a single polyadenylation signal downstream of its stop codon [ 9 ]. Despite this homology, the expression level of CfDEFNPV spindlin is markedly higher than that of AcMNPV gp37 in infected cells. Given the regulatory significance of alternative polyadenylation in modulating transcript stability, localization, and translation, we hypothesize that the different 3′ UTR architecture of gp37 contributes to its lower protein expression levels of AcMNPV gp37 compared to CfDEFNPV spindlin . Specifically, the use of multiple polyadenylation sites and resulting 3′ UTR heterogeneity may negatively impact gp37 transcript stability or translation efficiency, thereby reducing overall protein expression. Materials and Methods Cell lines and viruses. The insect cell lines Sf21 and Hi5 were maintained at 27°C in Grace’s medium supplemented with 0.33% yeastolate, 0.33% lactalbumin hydrolysate and 10% fetal bovine serum. AcMNPV Genomic DNA was used as the template in PCR amplification to clone the open reading frames (ORFs) of 3’ UTR of the polh and gp37 genes. Comparison of 3’ downstream sequences of AcMNPV gp37 , polyhedrin gene and CfDEFNPV spindlin gene. AcMNPV gp37 , AcMNPV polh , and CfDEFNPV spindlin were retrieved from GenBank. To identify putative polyadenylation signals, the DNASTAR software was used to search for the canonical hexamer motif AATAAA within the 3′ downstream sequences. For AcMNPV gp37 and polh , where transcript ends have been previously mapped, the search was limited to the known 3′ untranslated regions (3′ UTRs) [ 6 , 12 ]. For CfDEFNPV spindlin , whose transcript boundaries are unknown, the search was conducted within 1,000 nucleotides downstream of the stop codon (TAA or TGA). Additionally, GT-rich downstream elements, which are typically located 10–30 nucleotides downstream of polyadenylation signals and are known to facilitate cleavage and polyadenylation, were visually inspected and compared the three genes. Construction of viruses. Specific primers were designed to amplify the polh ORF and its 3′ downstream sequence (DS) from AcMNPV DNA. PCR products were verified by agarose gel electrophoresis, excised, and purified using the glassmilk method [ 26 ]. These fragments were first cloned into the pGEM-T Easy vector (Promega) and confirmed via DNA sequencing, then subcloned into the pFastBac1 vector (Invitrogen) to generate pFastBacpolh-polhUTR. To construct a second recombinant, primers containing restriction enzyme (REN) sites were designed to separately amplify the polyhedrin ORF and the gp37 3′ DS from AcMNPV DNA. After PCR amplification and agarose gel verification, the fragments were purified (glassmilk method) and cloned into pGEM-T Easy, resulting in pGEMT-polh and pGEMT-gp37UTR. Sequencing confirmed the integrity of both inserts. Using restriction enzymes EcoRI/XbaI (for polyhedrin ORF) and XbaI/XhoI (for gp37 DS), the two fragments were excised and ligated into the pFastBac1 vector in a three-piece ligation, yielding pFastBacpolh-gp37UTR. To produce a construct for green fluorescent protein ( gfp ) expression, specific primers including REN sites were designed to amplify the GFP ORF using pBlueGFP (Table 1 ) [ 10 ] as the template and to amplify the polyhedrin 3’ DS using AcMNPV DNA as the template and verified by agarose gel electrophoresis. The amplified PCR products were gel-extracted by the glassmilk method (Vogelstein and Gillespie, 1979), then cloned to the pGEM-T Easy vector to construct pGEMT-gfp, which has the GFP ORF and pGEMT -polhUTR, which has the polyhedrin 3’ UTR. The GFP ORF and polyhedrin 3’UTR were retrieved from the plasmids pGEMT-gfp and pGEMT-polhUTR; then these two fragments were ligated to pFastBac1 in a three-piece DNA ligation reaction to construct pFastBacGFP-polhUTR. The GFP ORF and gp37 3’UTR fragments were retrieved from the plasmids pGEMT-gfp and pGEMT-gp37UTR; then the two fragments were ligated to pFastBac1 in a three-piece DNA ligation reaction to produce plasmid pFastBacGFP-gp37UTR. Table 1 A primer list in this project GFP-F-EcoRI 5’-GAATTCATGGTGAGCAAGGGCGAG-3’ GFP-R-BamHI 5’-GGATCCGGAACCACCACCACCCTTGTACAGCTCGTCCATG-3 Ac-gp37UTR-XbaI 5’-TCT AGA TAA AAC AAA CAA AAT TTT AAT TAC ATA TTA TAT TTA GCA AGA AG-3 Ac-gp37UTR-XhoI 5’-CTC GAG GAC GCA ATG GAG GCG TTG-3 Ac-Polh-F-NotI 5’-GCG GCC GCG TCT ATC AAT ATA TAG TTG CTG-3 Ac-Polh-R-BclI 5’-TGA TCA TAA CAC GCC CGA TGT TAA A-3 In addition to using GFP as a reporter, we also used luciferase ( luc ) as a reporter gene to further support our discovery on UTR regulating gene expression. To construct a plasmid with the luciferase gene, we retrieved the luciferase gene from pGL-basic vector (Promega) by digestion with restriction enzyme BglII/XbaI. This REN fragment was purified by agarose gel electrophoresis using the glassmilk method. This fragment was cloned together with gp37UTR or polhUTR fragment to pFastBac1 similar to the GFP construct to produce pFastBacluc-polhUTR and pFastBacluc-gp37UTR. The plasmids pFastBac-polhUTR and pFastBac-gp37UTR, pFastBacgfp-polhUTR and pFastBacgfp-gp37UTR as well as pFastBacluc-polhUTR and pFastBacluc-gp37UTR were used to transform competent DH10Bac E. coli cells (Invitrogen) following the protocol from the kit manufacturer. White colonies from each transformation were picked and confirmed by PCR with specific primers. DNAs were extracted from the bacteria that contain the right insertion in the bacmid and then used to transfect Sf21 cells to produce the viruses AcBacpolh-polhUTR, AcBacpolh-gp37UTR, AcBacgfp-polyhUTR, AcBacgfp-gp37UTR, AcBacluc-polhUTR and AcBacluc-gp37UTR. The newly constructed viruses were confirmed by polyhedra formation or by showing green fluorescence post-infection, or luciferase activity before propagation and tittered (O'Reilly et al., 1992). Infection and polyhedra production assay . Sf21 or Hi5 insect cells (3×10⁶ cells/flask) were infected with either Acpolh-polhUTR or Acpolh-gp37UTR recombinant baculoviruses at a multiplicity of infection ( MOI ) of 5 plaque-forming units (p.f.u.) per cell in 25 cm² tissue culture flasks. Infections were conducted at 27°C for 96 hours. Cell images were acquired using a SPOT Insight digital camera mounted on a Nikon Eclipse TE2000-U inverted microscope. After imaging, media were removed, and 1 ml of 0.1% SDS (w/v) was added to lyse the cells and release polyhedra. Flasks were gently rocked at 27°C for 1 hour. Cell lysates were transferred to 1.5 ml centrifuge tubes, and 10 µl aliquots were taken for polyhedra yield estimation using a hemocytometer under a microscope. Polyhedra size measurement and comparison. To assess differences in polyhedron size, 50 polyhedra were randomly selected and measured from Sf21 cells infected with either Acpolh-polhUTR or Acpolh-gp37UTR. Measurements were performed using a Nikon Eclipse TE2000-U inverted microscope equipped with an ocular micrometer in one of the ocular lens. The same procedure was carried out on Hi5 cells infected with the same recombinant viruses. The purpose of these tests was to evaluate the effect of 3′ downstream sequences on polyhedron size in different insect cell lines. Protein yield assay . Polyhedrin protein yields were estimated using the Bio-Rad protein assay kit based on the Bradford method. A standard curve was generated using bovine serum albumin (BSA) standards ranging from 0.2 mg/ml to 1.4 mg/ml in 0.1 M Na₂CO₃ (pH 10.5). The 5× dye reagent was diluted to 1× , and then added to each BSA dilution. The absorbance at 595 nm (OD₅₉₅) was measured using a spectrophotometer, with triplicate readings taken for each sample to ensure accuracy. For sample analysis, 50 µl of purified polyhedra from Sf21 cells infected with either Acpolh-polhUTR or Acpolh-gp37UTR were mixed with 50 µl of 0.1 M Na₂CO₃, followed by the addition of the diluted dye reagent. The absorbance at OD₅₉₅ was recorded, and polyhedrin concentrations were determined using the previously constructed standard curve. Estimated polyhedrin yields from Acpolh - polhUTR- and Acgpolh - p37UTR-derived polyhedra were statistically analyzed to assess differences in protein production between the two constructs by Microsoft Excel. Estimation of reporter protein expression levels . Since polyhedra may not be completely dissolved and this may affect protein yield measurement accuracy, we then measured GFP expression levels using a spectrofluorometer. Sf21 cells infected with either Acgfp-polhUTR or Acgfp-gp37UTR followed the same infection protocol used for polyhedrin yield experiments, except that cells were processed for fluorescence analysis instead of protein quantification. After 96 hours of incubation, infected cells were dislodged from the tissue culture (TC) flasks by jet-flushing with media using a Pasteur pipette. The cell suspensions were centrifuged at 500 × g for 5 minutes, and the supernatants were discarded. Residual media were carefully blotted from the cell pellets. Cells were lysed in 500 µl of 0.1% SDS, and the resulting lysates were used to measure GFP fluorescence using a Shimadzu RF-5301PC spectrofluorometer (excitation at 488 nm, emission at 507 nm). Fluorescence readings were taken in triplicate f or each sample. Emission values were used to quantify and compare GFP expression levels between the two viral constructs (Acgfp-polhUTR vs Acgfpg-gp37UTR ) using a two-tailed Student's t-test performed in Microsoft Excel. To complement GFP measurement, we also used luciferase as a reporter gene for protein yield comparison between the two viral constructs (AcBacluc-polhUTR and AcBacluc-gp37UTR.). Results 3’ UTR sequence analysis. The 3′ downstream sequences (DS) of the AcMNPV gp37 and polh genes, as well as the spindlin gene from CfDEFNPV, were analyzed for polyadenylation signal s . The 3′ DS of AcMNPV gp37 contains three potential polyadenylation sites , including a canonical AATAAA signal and associated GT-rich downstream sequences (Fig. 1 A,). In contrast, the spindlin gene, a gp37 homologue from CfDEFNPV, has only a single polyadenylation site ( Fig. 1 B). The 3′ DS of the AcMNPV polyhedrin gene contains two AATAAA motifs; however, only the second polyadenylation signal is followed by GT-rich downstream sequences, indicating that it may serve as the functional polyadenylation site (Fig. 1 C). A summary comparison of the UTR regions of the three baculovirus genes were presented in Table D, E, and F. Construction of three sets of viruses. In order to understand the regulatory effect of the alternative polyadenylation signals in the 3’ DS of AcMNPV gp37 , the 3’ DS of gp37 was fused to the downstream of three reporter genes, the polh gene, the gfp gene and luc gene. The 3’ DS of the polh gene was used as a strong polyadenylation signal with high protein expression to compare with the gp37 3’ DS. The reporter genes and the 3’ DSs were under the control of a strong polh gene promoter. The Acpolh-polhUTR had the polh ORF followed by 3’ DS of polh whereas the Acpolh-gp37UTR had polh ORF followed by the 3’ DS of gp37 at the polh locus. Since these two viruses were constructed using the AcMNPV bacmid, the endogenous polh gene is not present. The Acgfp-polhUTR and Acgfp-gp37UTR viruses had the gfp gene followed by the 3’ DS of polh or the 3’ DS of gp37 at the polh locus, respectively. At the same time we also successfully constructed two viruses (Acluc-polhUTR and Acluc-gp37UTR) that express luciferase to further support our discovery (Fig. 2 ). Polyhedron production and size comparisons between Acpolh-polhUTR and Acpolh-gp37UTR. The 3’ DS of gp37 increased polyhedron sizes but reduced polyhedron yields. In the polh virus set, the polh gene was used as the reporter gene. Polyhedra are formed in the nuclei of infected cells by baculovirus at the late stage of infection, which give a good visible tracking of the dynamics of polyhedrin expression under the microscope. The Sf21cells infected with Acpolh - gp37UTR showed much larger but fewer polyhedra in the nuclei of infected cells compared to Acpolh-polhUTR (Fig. 3 a and b). When the polyhedral numbers produced in the Sf21 cells infected with Acpolh - polhUTR and Acpolh-gp37UTR were compared, production of polyhedra by Ac polh- polhUTR showed twice as much as that produced by Acpolh-UTR (Fig. 3 c). When the sizes of polyhedra formed by Acpolh-polhUTR and Acpolh-gp37UTR in Sf21 cells were measured and compared, polyhedral size formed by Acpolh-gp37UTR is 1.63 times larger in diameter than that of Acpolh-polhUTR (Fig. 3 d). Therefore we see in inverse relationship between polyhedral number and size. Then we further compared polyhedrin protein yield form Sf21 cells infected by the two viruses. It is unknown if the size and number difference is cell type dependence or not, we further confirmed your discovery in Sf21 in Hi5 cells. To further confirm the reduction of polyhedra number and increase of polyhedron size from Acpolh-gp37UTR infected cells, Hi5 cells were infected by Acpolh-polhUTR or Acpolh-gp37UTR for polyhedron production comparison in terms of polyhedron number and size. Similar phenomena were observed in Hi5 cells infection as in Sf21 cells infection (Fig. 4 a and b). To quantify our observation, the infection of Hi5 cells by Acpolh-gp37UTR produced 1.44-fold larger polyhedra in diameter than Acpolh-polhUTR (Fig. 4 c) and the reduction of total polyhedron production is about 6-fold (Fig. 4 d). Therefore, infections of both Sf21 cells and Hi5 cells by the two viruses confirmed that the gp37 3’ DS reduces polyhedron yield but increases polyhedron size compared to the polh 3’ DS, an inverse relationship. The next question we asked is if the total protein production is affected by the UTR regions. Comparison of protein production . Although polyhedron yields were reduced when the gp37 DS replaced the polh 3’ DS, the total protein yield was not affected. This conclusion is drawn from two independent assays. The Bio-Rad protein assay was used to compare the total protein expression levels of polh from Sf21 cells infected with Acpolh-gp37UTR and Acpolh-polhUTR. It was found that the total polh amounts from Sf21 cells infected with Acpolh-gp37UTR and Sf21 cells infected with Acpolh-polhUTR were at the same level (Fig. 5 a). This result is supported by comparing GFP expression levels in Sf21 cells infected by Acgfp-polhUTR and Acgfp-gp37UTR. GFP expression by the two viral constructs in Sf21 cells showed no statistic difference, although it appeared different when the images of Sf21 cells infected with the two viruses were compared (Fig. 5 d). Furthermore, luciferase assay also did not show difference between AcBacluc-gp37UTR and AcBacluc-gp37UTR. This result further confirms that the total protein production levels either by polh or GFP or luciferase were not affected by the 3’ DS of gp37. Discussion mRNA processing plays a major regulatory role in eukaryotic cells. Alternative polyadenylation has been recently identified as an important contributor to gene regulation [ 28 ]. Alternative polyadenylation is known to regulate gene expression in various ways [ 12 , 28 ]. Other than producing different proteins, alternative polyadenylation can be used to produce the same coding region of an mRNA but with different 3’ UTRs [ 17 ]. It has only been recently appreciated that nearly all genes have additional polyadenylation signals in their 3’ UTRs, and more than half of the human genes are alternatively polyadenylated [ 29 ]. Many alternative polyadenylation signals are evolutionarily conserved [ 29 ]. The use of alternative polyadenylation signals can protect the transcript from the stronger regulatory potential of longer 3’ UTRs. For example, the shorter 3’ UTR can lose microRNA (miRNA) complementary sites [ 17 ]. It was reported that alternative polyadenylation can activate oncogenes in cancer cells by shortening the 3’ UTRs of oncogenes; these shorter isoforms of oncogenes have higher stability and produce more proteins in part through the loss of miRNA-mediated repression [ 17 ]. The alternative polyadenylated isoforms of the same gene can also have different translation efficiency [ 30 ]. In this study, we demonstrated that 3’ DS of the spindlin gene from CfDEFNPV has one putative polyadenylation site whereas the 3’ DS of AcMNPV gp37 has multiple polyadenylation sites (Fig. 1 ). It was also reported that the AcMNPV gp37 transcripts have different lengths and the different 3’ end of the transcripts match well with the three polyadenylation signals in the 3’ UTR of gp37 . The percentage of the largest transcript decreases during later times post-infection while the percentage of the smaller transcripts increases [ 12 ]. Whether baculovirus late gene late gene mRNA 3’end processing follows cellular gene using AAUAAA as the polyadenylation positioning signal and G/U rich region for stimulation of polyadenylation is not certain. Early studies suggest baculovirus does not use AAUAAA and G/U signal for polyadenylation for cleavage before polyadenylation, and it was reported that a stretch of seven Us (UUUUUUU) is the signal for termination [ 31 , 32 ]. However, these were performed using a combination of 4 AcMNPV RNA polymerase subunits in vitro [ 31 ]. Previously, we showed a stretch of seven Us was found in SV40 polyA [ 33 ]. A more recent transcriptome analysis of AcMNPV in vivo showed that 82.5% gene is associated with the cellular canonical polyadenylation signals; only about 13% carry the T-rich (U-rich in RNA) sequence as suggested by [ 34 ]. Another search for seven U in AcMNPV polh , gp37 , and CfDEFNPV spindlin did not yield any stretch of seven U (in RNA) or T in DNA. AcMNPV RNA polymerase (LEF 4, LEf 8, LEf 9 and P47) for late gene transcription may not represent what is really happening in the AcMNPV transcription in vivo . There might be other proteins joining the AcMNPV polymerase for AcMNPV late gene transcript 3’ processing, considering cellular transcription and AcMNPV gene transcription are all in the nucleus. These proteins might be either viral or cellar origin. A more recent AcMNPV transcriptome analysis in fact supports the idea of cellular factors involved in the 3’ end processing of AcMNPV later transcripts by cleavage similar to the cellular mRNA 3’ processing [ 34 ]. In addition, a search in the AcMNPV genome did find a polyA polymerase, the enzyme to add a stretch of adenine. Of course this is not conclusive since there are many genes in the AcMNPV genome without gene function assignment. In order to test the regulatory function of the gp37 gene 3’ DS, the 3’ DS of polh from AcMNPV, which is similar to 3’ DS of the spindlin gene and has a high expression level, was used for comparison. When the polh gene from AcMNPV was used as the marker gene, the virus Acpolh-gp37UTR that has gp37 3’ DS makes fewer, but larger polyhedra in the nuclei of both infected Sf21 and Hi5 cells than does Acpolh-polhUTR, which has the polh 3’ DS. In Sf21 cells the reduction is 2-fold, and in Hi5 cells the reduction is 6-fold, both of which are significant. Surprisingly the total polh protein levels from Acpolh-polhUTR- and Acpolh-gp37UTR-infected Sf21 cells are the same. This may reflect the size difference between polyhedra in Acpolh-polhUTR and Acpolh-gp37UTR infected cells. When GFP was used as the reporter gene instead of polh in order to confirm that there is no difference at the final protein amount, the fluorescence of the cells infected with virus were very similar, which confirmed the observation that there is no difference in the final protein amount. The different size and number of polyhedra from the two virus infections indicates that there is a regulatory effect of the gp37 3’ UTR since the coding regions of the two viruses are identical, the only difference being in the 3’ DS of the reporter gene, polh . But the regulation is not at the level of total protein production. Polyhedra are crystals and the formation of the crystals has two components, nucleation and growth. High protein concentration will lead to the supersaturating of the solution and drive the nucleation. The growth is favored by then reducing the protein concentration since the reduced protein concentration can prevent further nucleation from competing with the growth of established nuclei [ 35 ]. A possible explanation for the fewer, larger polyhedra from Acpolh-gp37UTR-infected cells is that the 3’ UTR of gp37 changes the protein synthesis kinetics. During the early stage of the infection, Acpolh-polhUTR may have higher polh expression than Acpolh-gp37UTR, making it possible for Acpolh-polhUTR to produce more polyhedra. At later stages of the infection, Acpolh-gp37UTR catches up in polh expression but can only build on the established polyhedra. It will be interesting to test whether the different lengths of gp37 transcripts have different RNA stability and translation efficiency. How the alternative polyadenylation occurs in gp37 transcripts and regulates gene expression is unknown. It was reported that the efficiency of using different polyadenylation sites is controlled by the interaction of the regulatory cis-acting RNA elements and the trans-acting protein factors. Among the factors, the cleavage and stimulation factor CstF-64 binds to the GU-rich region downstream of the AAUAAA signal and then recruits other factors to assemble the polyadenylation complex. One possible explanation for the regulatory effect of gp37 3’ UTR is that the downstream GU-rich regions of each AAUAAA bind to CstF-64 weakly, allowing the alternative polyadenylation to take place. It was reported the sequences in the GU-rich region controls the binding [ 36 ], so by controlling the sequences in the GU-rich region downstream of AAUAAA, AcMNPV can regulate the gp37 expression without requirement of additional regulatory proteins. It was reported that multiple genes have different 3’ end sites and the proportion of the various forms of these mRNAs may vary temporally, although often the shortest forms are more common in the earlier stages of transcription [ 6 ]. It has been well known that viruses have very compact genomes and use their genetic information very efficiently, so alternative polyadenylation should be commonly employed in many viral genomes. The results in this study show that alternative polyadenylation regulates the polyhedra formation without changing the total amount of the protein expressed, suggesting a possible impact on changing protein expression kinetics. This study can serve as an example of the regulatory effect of alternative polyadenylation in baculovirus and other viruses. Declarations Conflicts of interest The authors declare no financial or commercial conflict of interest. Author contributions X-W C, HS and JL Xue planned experiments; X-W C and JL Xue wrote the manuscript ACKNOWLEDGEMENTS We would like to thank Miami University CBFG for help in using the equipment in luciferase assay. Dr. Gary R. Janssen is credited for proofreading this manuscript. References Doerfler W (1986) The Molecular Biology of Baculoviruses. Springer-Verlag Wien & New York Miller LK (1988) Baculoviruses as Gene Expression Vectors. in Annu Rev Microbiol p. pp. 177–199 Ayres MD et al (1994) The complete DNA sequence of Autographa californica nuclear polyhedrosis virus. Virology 202(2):586–605 Charlton CA, Volkman LE (1993) Penetration of Autographa californica nuclear polyhedrosis virus nucleocapsids into IPLB Sf 21 cells induces actin cable formation. Virology 197(1):245–254 Peros IG et al (2020) Advances in the Bioinformatics Knowledge of mRNA Polyadenylation in Baculovirus Genes. 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J Gen Virol 70(Pt 9):2449–2459 Bartel DP (2009) MicroRNAs: target recognition and regulatory functions. Cell 136(2):215–233 Wahle E, Kuhn U (1997) The mechanism of 3' cleavage and polyadenylation of eukaryotic pre-mRNA. Prog Nucleic Acid Res Mol Biol 57:41–71 Proudfoot N (1991) Poly(A) signals. Cell 64(4):671–674 Colgan DF, Manley JL (1997) Mechanism and regulation of mRNA polyadenylation. Genes Dev 11(21):2755–2766 Mayr C, Bartel DP (2009) Widespread shortening of 3'UTRs by alternative cleavage and polyadenylation activates oncogenes in cancer cells. Cell 138(4):673–684 Graham SV (2008) Papillomavirus 3' UTR regulatory elements. Front Biosci 13:5646–5663 Schwartz S (2008) HPV-16 RNA processing. Front Biosci 13:5880–5891 McNally MT (2008) RNA processing control in avian retroviruses. Front Biosci 13:3869–3883 Qiu J, Pintel DJ (2004) Alternative polyadenylation of adeno-associated virus type 5 RNA within an internal intron is governed by the distance between the promoter and the intron and is inhibited by U1 small nuclear RNP binding to the intervening donor. J Biol Chem 279(15):14889–14898 X., J., et al., The role of alternative polyadenylation in the antiviral innate immune response, (2017) 8: p. 14605 Cai W, Ricci EP (2025) mRNA 3'UTR length matters: alternative polyadenylation shapes autophagy and inflammatory responses in macrophages. Cell Mol Immunol 22(3):336–338 Liang J et al (2025) Atractylenolide-Ⅲ binds non-structural protein-1 to suppress influenza A by modulating macrophage polarization and alternative polyadenylation. Phytomedicine 141:156704 Uhl LK, Fasina OO (2025) Parvovirus RNA Processing: Compact Genomic Organization and Unique Alternative mRNA Processing Mechanisms. Viruses, 17(7) Vogelstein B, Gillespie D (1979) Preparative and analytical purification of DNA from agarose. Proc Natl Acad Sci U S A 76(2):615–619 Wang L et al (2009) Characterization of a virion occlusion-defective Autographa californica multiple nucleopolyhedrovirus mutant lacking the p26, p10 and p74 genes. J Gen Virol 90(Pt 7):1641–1648 Lutz CS (2008) Alternative polyadenylation: a twist on mRNA 3' end formation. ACS Chem Biol 3(10):609–617 Tian B et al (2005) A large-scale analysis of mRNA polyadenylation of human and mouse genes. Nucleic Acids Res 33(1):201–212 Stark A et al (2005) Animal MicroRNAs confer robustness to gene expression and have a significant impact on 3'UTR evolution. Cell 123(6):1133–1146 Jin J, Dong W, Guarino LA (1998) The LEF-4 subunit of baculovirus RNA polymerase has RNA 5'-triphosphatase and ATPase activities. J Virol 72(12):10011–10019 Guarino LA et al (1998) A virus-encoded RNA polymerase purified from baculovirus-infected cells. J Virol 72(10):7985–7991 Salem TZ et al (2015) The Influence of SV40 polyA on Gene Expression of Baculovirus Expression Vector Systems. PLoS ONE 10(12):e0145019 Chen YR et al (2013) The transcriptome of the baculovirus Autographa californica multiple nucleopolyhedrovirus in Trichoplusia ni cells. J Virol 87(11):6391–6405 Schmit JD, Dill KA (2010) The stabilities of protein crystals. J Phys Chem B 114(11):4020–4027 Perez Canadillas JM, Varani G (2003) Recognition of GU-rich polyadenylation regulatory elements by human CstF-64 protein. EMBO J 22(11):2821–2830 Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Cheng","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzElEQVRIiWNgGAWjYDACCTBpw8AGJA8wNoBI4rSkka7lMIRDlBb+2c3HHn5tOx/Nx3468cDHHQxyfDcSCFhy51i6scyZ27ltPLkbDs48w2AsSUiLgUSOmbREBVALQ+6Gw7xtDIkbCGvJ/yYtYXAut43/LVhLPRFactgkP1QcyG2TgNiSYEDQLzfSzKQZziQDtbwF+qVNwnDmmQf4tfDPSH4m+bPNLnd+f+7mDx/bbOT5jhOwBQSYeZBsJawcBBh/EKduFIyCUTAKRioAAGhJSn+KQ7ATAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-9755-7926","institution":"Miami University","correspondingAuthor":true,"prefix":"","firstName":"Xiao-Wen","middleName":"","lastName":"Cheng","suffix":""},{"id":539210135,"identity":"0d490301-0e33-48c7-8c07-077f2cbc0e76","order_by":1,"name":"Jianli Xue","email":"","orcid":"","institution":"Miami University","correspondingAuthor":false,"prefix":"","firstName":"Jianli","middleName":"","lastName":"Xue","suffix":""},{"id":539210136,"identity":"88103b14-4bd9-4a40-8fab-90697d4f62ea","order_by":2,"name":"Hui Shang","email":"","orcid":"","institution":"Miami University","correspondingAuthor":false,"prefix":"","firstName":"Hui","middleName":"","lastName":"Shang","suffix":""}],"badges":[],"createdAt":"2025-10-30 21:57:55","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7992808/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7992808/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":95815032,"identity":"0213bb8b-0f28-4018-a3a3-df0e60376152","added_by":"auto","created_at":"2025-11-13 09:33:35","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":14345,"visible":true,"origin":"","legend":"","description":"","filename":"Primerlist.docx","url":"https://assets-eu.researchsquare.com/files/rs-7992808/v1/639f5273b1f8d29886fbb66f.docx"},{"id":95815036,"identity":"f26bdc20-cd6a-46de-80ed-f3728a4a3a7e","added_by":"auto","created_at":"2025-11-13 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09:33:35","extension":"xml","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":84361,"visible":true,"origin":"","legend":"","description":"","filename":"ARVID25008390structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7992808/v1/dc2b11673ca8dab75765e214.xml"},{"id":95815043,"identity":"abb5ce1c-8522-42fd-b063-e746ead589cf","added_by":"auto","created_at":"2025-11-13 09:33:35","extension":"html","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":95291,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7992808/v1/0dcd0de707a956882d91b279.html"},{"id":95819026,"identity":"add322b1-173e-4708-82e3-e4489f5cb35b","added_by":"auto","created_at":"2025-11-13 10:37:31","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":655202,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of 3’downstream sequence of AcMNPV \u003cem\u003egp37 \u003c/em\u003e(A), \u003cem\u003epolh\u003c/em\u003e (B) genes and the CFDEFNPV spindlin gene (C). All the downstream sequences start with the stop codon (TAA or TGA) and all the AATAAA sequences are bolded and underlined and the GT rich regions downstream of the AATAAA are underlined.\u003c/p\u003e","description":"","filename":"figure1A.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7992808/v1/e9e6a410393c83ced0e1f8f3.jpg"},{"id":95815029,"identity":"b330ede8-08ec-4ed3-a5de-c50703ba19e2","added_by":"auto","created_at":"2025-11-13 09:33:35","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":448135,"visible":true,"origin":"","legend":"\u003cp\u003eA schematic presentation of 6 constructed viruses: Acpolh-polhUTR and Acpolh-gp37UTR that have \u003cem\u003epolh\u003c/em\u003e ORF with polyhedrin UTR or gp37 UTR, Acgfp-polhUTR and Acgfp-gp37UTR that have GFP ORF with \u003cem\u003epolh\u003c/em\u003e UTR or gp37 UTR. Acluc-polhUTR and Acluc-polhUTR have GFP ORF with \u003cem\u003epolh\u003c/em\u003e UTR or gp37 UTR at the \u003cem\u003epolh\u003c/em\u003e locus. All constructs used the strong AcMNPV polyhedrin promoter to drive the expression of the reporter genes.\u003c/p\u003e","description":"","filename":"Figure2cropped.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7992808/v1/3ba66f02f0b6cd46c68a976f.jpg"},{"id":95818951,"identity":"1f4da124-b5b0-4e87-8fa4-5506dc3bd6aa","added_by":"auto","created_at":"2025-11-13 10:36:00","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":328276,"visible":true,"origin":"","legend":"\u003cp\u003eInsect cell infection with the constructed viruses. Images of Sf21 cells infected by Acpolh-polhUTR (a) or Acpolh-gp37UTR (b) at an MOI of 5 p.f.u./cell and observed at 4 d p. i. . White arrowheads point to cells with polyhedra in the nucleus. Bar markers represent 10 μm. Quantitative analysis of polyhedral production in Sf21 cells infected with Acpolh-polhUTR or Acpolh-gp37UTR with difference in polyhedron number (c) and polyhedral diameter (d). Statistical difference is indicated with by asterisk * (p\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"Fig3Sf21cropped.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7992808/v1/0f8d1a49cbbdcf0005d7d749.jpg"},{"id":95819073,"identity":"28b98c0a-bb62-4230-8f51-4a4579e5737c","added_by":"auto","created_at":"2025-11-13 10:37:52","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":349583,"visible":true,"origin":"","legend":"\u003cp\u003eInsect cell infection with the constructed viruses. Images of Hi5 cells were infected by Acpolh-polhUTR (a) or Acpolh-gp37UTR (b) at an MOI of 5 p.f.u./cell and observed at 4 d p. i. . White arrowheads point to cells with polyhedra in the nucleus. Scale bar = 10 μm. Quantitative analysis of polyhedral production in Hi5 cells infected with Acpolh-polhUTR or Acpolh-gp37UTR with difference in polyhedron number (c) and polyhedral diameter (d). Statistical difference is indicated with by asterisk * (p\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"Fig4Hi5cropped.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7992808/v1/c55d64ea3ce68576b5236099.jpg"},{"id":95818811,"identity":"822f8a2c-a8dd-4116-9f58-f5ae67c11cdd","added_by":"auto","created_at":"2025-11-13 10:33:46","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":624255,"visible":true,"origin":"","legend":"\u003cp\u003eProtein yield assay of Sf21 cells infected by a pair of Acpolh-polhUTR or Acpolh-gp37UTR, Acgfp-polhUTR or Acgfp-gp37UTR, and Acluc-polhUTR or Acluc-polhUTR.\u0026nbsp;\u0026nbsp; a, polyhedrin protein yield comparison between Acpolh-polhUTR or Acpolh-gp37UTR. Standard curve was constructed to estimate polyhedrin yields from different viral constructs using 0.2 mg/ml to 1.4 mg/ml BSA in Na\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e. The diluted dye reagent was added to each sample and the absorbance value was recorded at OD\u003csub\u003e595\u003c/sub\u003e. \u003cstrong\u003e\u0026nbsp;b\u003c/strong\u003e, GFP expression yield comparison between Acgfp-polhUTR and Acgfp-gp37UTR. \u003cstrong\u003ec\u003c/strong\u003e. Luciferase expression yield comparison between Acluc-polhUTR and Acluc-gp37UTR. \u0026nbsp;Statistical difference is indicated with by asterisk * (p\u0026lt;0.05).\u0026nbsp;\u0026nbsp;\u003c/p\u003e","description":"","filename":"Fig5proteincropped.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7992808/v1/b4aedfc28d47a3cfb56600bf.jpg"},{"id":97250213,"identity":"6751fda8-fa85-4eb0-8510-dc707c12fc05","added_by":"auto","created_at":"2025-12-02 13:14:06","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3068778,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7992808/v1/f4f41764-d62e-4c56-b868-520d88e584b8.pdf"}],"financialInterests":"","formattedTitle":"gp37 3’ UTR reduces protein production at the polyhedrin locus of Autographa californica multicapsid nucleopolyhedrovirus","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBaculoviruses are large, double-stranded DNA viruses widely utilized both as biological control agents against insect pests and vectors for protein expression in biotechnology applications [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The type species of the family \u003cem\u003eBaculoviridae\u003c/em\u003e, \u003cem\u003eAutographa californica\u003c/em\u003e multicapsid nucleopolyhedrovirus (AcMNPV), has served as a key model system for baculovirus genetic research due to the high permissiveness of serval insect cell lines to AcMNPV infection [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Notably, \u003cem\u003eAcMNPV\u003c/em\u003e was the first baculovirus to have its genome fully sequenced, revealing a compact 134-kilobase (kb) genome [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eDuring a productive infection cycle in permissive insect cells, AcMNPV enters the cells likely through receptor-mediated endocytosis. Once internalized, virion-containing endosomes are transported to the nucleus along actin filaments [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Fusion of the viral envelope with the endosomal membrane releases the nucleocapsids into the cytoplasm near the nucleus, after which they are imported into the nucleus through the nuclear pore complex. Inside the nucleus, the nucleocapsids uncoat, releasing the viral DNA genome to initiate transcription. Viral gene expression occurs in distinct temporal phases: early transcribed by host RNA polymerase II, late transcribed by virus-encoded RNA polymerase, and an undefined phase characterized by transcripts lacking a canonical 3\u0026prime; end processing signal [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eDuring the late phase of infection, the \u003cem\u003epolh\u003c/em\u003e gene is highly expressed, leading to the production of large polyhedrin protein complexes known as occlusion bodies. These structures encapsulate or occlude enveloped virions [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The formation of occlusion bodies is believed to be an evolutionary adaptation that protects virions from environmental degradation, thereby enhancing baculovirus transmission within insect populations under natural condition [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe \u003cem\u003egp37\u003c/em\u003e gene of \u003cem\u003eAcMNPV\u003c/em\u003e, a homologue of the \u003cem\u003espindlin\u003c/em\u003e gene of \u003cem\u003eChoristoneura fumiferana\u003c/em\u003e defective nucleopolyhedrovirus (\u003cem\u003eCfDEFNPV\u003c/em\u003e), is classified as a late gene, but is not highly expressed during cell infection. In contrast, \u003cem\u003eCfDEFNPV\u003c/em\u003e expresses the \u003cem\u003espindlin\u003c/em\u003e gene at high levels in permissive cells, resulting in the formation of distinctive spindle-shaped inclusions in the cytoplasm. Similar spindle-shaped inclusions have also been reported in cells infected with other nucleopolyhedroviruses (NPVs), including \u003cem\u003eChoristoneura murinana\u003c/em\u003e NPV, \u003cem\u003eOrgyia pseudotsugata\u003c/em\u003e MNPV, \u003cem\u003eCadra cautella\u003c/em\u003e NPV, and \u003cem\u003eArchips cerasivoranus\u003c/em\u003e NPV [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eHowever, \u003cem\u003eAcMNPV\u003c/em\u003e-infected cells do not exhibit spindle-shaped inclusions, suggesting that either \u003cem\u003egp37\u003c/em\u003e is not expressed at sufficient levels, or it is expressed abundantly but fails to crystallize into visible structures. The exact function of \u003cem\u003eAcMNPV gp37\u003c/em\u003e remains unclear, though it has been shown to be non-essential for viral replication [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Interestingly, the \u003cem\u003egp37\u003c/em\u003e gene from \u003cem\u003eCydia pomonella\u003c/em\u003e granulovirus (\u003cem\u003eCpGV\u003c/em\u003e) was recently reported to encode a toxin that enhances baculovirus efficacy in insect control [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Transcriptional analysis of \u003cem\u003eAcMNPV gp37\u003c/em\u003e during infection of permissive Sf21 cells has revealed multiple transcript variants, differing in the length of their 3\u0026prime; untranslated regions (3\u0026prime; UTRs) [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], suggesting complex post-transcriptional regulation.\u003c/p\u003e\u003cp\u003eThe 3\u0026prime; untranslated regions (3\u0026prime; UTRs) of eukaryotic mRNAs play multiple regulatory roles, including directing the formation of the poly(A) tail, influencing mRNA stability, and providing binding sites for microRNAs [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Proper 3\u0026prime; end formation is crucial for mRNA maturation and gene expression. This process typically requires cleavage and polyadenylation signals, located near the end of the 3\u0026prime; UTR, which facilitate the addition of the poly(A) tail to most transcripts [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cb\u003eAlternative polyadenylation (APA)\u003c/b\u003e, the use of multiple polyadenylation signals within a single transcript, can significantly impact gene regulation. APA may result in the removal of large portions of the 3\u0026prime; UTR, thereby reducing the transcript's exposure to regulatory elements such as microRNA binding sites or AU-rich elements, which are often associated with decreased stability [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Depending on the polyadenylation site used, APA can produce transcripts with subtle changes, such as variable 3\u0026prime; UTR lengths, or more dramatic consequences, including altered RNA stability, localization, or even changes in the coding sequence that yield protein isoforms with distinct domains. Most alternative polyadenylation occurred in cellular organisms. Given the compact and highly efficient nature of viral genomes, APA has been observed in numerous viruses, including human papillomavirus type 16 (HPV-16) and avian retroviruses, where it contributes to the diversity and regulation of viral gene expression [\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Alternative polyadenylation has also been reported in viruses, such as adeno-associated virus type 5 RNA [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. More recently, alternative polyadenylation has been reported in other viruses. For example, alternative polyadenylation has been reported in vesicular stomatitis virus, macrophages, influenza A and parvovirus [\u003cspan additionalcitationids=\"CR23 CR24\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Therefore, alternative polyadenylation might be widely used by viruses to increase their coding capacity, considering in general, the virus genome is much smaller and more compact than cellular genomes.\u003c/p\u003e\u003cp\u003eThree transcript isoforms of the \u003cem\u003eAcMNPV gp37\u003c/em\u003e gene, differing in their 3\u0026prime; untranslated regions (3\u0026prime; UTRs), have been detected during infection [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. These transcripts terminate at three distinct positions, each associated with a canonical polyadenylation signal (AAUAAA) located downstream of the \u003cem\u003egp37\u003c/em\u003e coding sequence. All three transcript variants are detectable from 6 hours post-infection (h p.i.), but their relative abundances change over time. The two shorter transcripts dominate in the early stages of infection, whereas the longest transcript becomes increasingly abundant as the infection progresses [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. This shift from shorter to longer transcripts reflects a switch in polyadenylation site usage, resulting in alternative 3\u0026prime; UTRs for \u003cem\u003egp37\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eA similar pattern where shorter transcript forms are more prevalent in the early stages of infection has been observed for other baculovirus mRNAs that undergo alternative polyadenylation [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Interestingly, the \u003cem\u003espindlin\u003c/em\u003e gene of \u003cem\u003eCfDEFNPV\u003c/em\u003e, which shares 59.09% similarity with \u003cem\u003eAcMNPV gp37\u003c/em\u003e, contains only a single polyadenylation signal downstream of its stop codon [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Despite this homology, the expression level of \u003cem\u003eCfDEFNPV spindlin\u003c/em\u003e is markedly higher than that of \u003cem\u003eAcMNPV gp37\u003c/em\u003e in infected cells.\u003c/p\u003e\u003cp\u003eGiven the regulatory significance of alternative polyadenylation in modulating transcript stability, localization, and translation, we hypothesize that the different 3\u0026prime; UTR architecture of \u003cem\u003egp37\u003c/em\u003e contributes to its lower protein expression levels of AcMNPV \u003cem\u003egp37\u003c/em\u003e compared to \u003cem\u003eCfDEFNPV spindlin\u003c/em\u003e. Specifically, the use of multiple polyadenylation sites and resulting 3\u0026prime; UTR heterogeneity may negatively impact \u003cem\u003egp37\u003c/em\u003e transcript stability or translation efficiency, thereby reducing overall protein expression.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cb\u003eCell lines and viruses.\u003c/b\u003e The insect cell lines Sf21 and Hi5 were maintained at 27\u0026deg;C in Grace\u0026rsquo;s medium supplemented with 0.33% yeastolate, 0.33% lactalbumin hydrolysate and 10% fetal bovine serum. AcMNPV Genomic DNA was used as the template in PCR amplification to clone the open reading frames (ORFs) of 3\u0026rsquo; UTR of the \u003cem\u003epolh and\u003c/em\u003e gp37 genes.\u003c/p\u003e\u003cp\u003e\u003cb\u003eComparison of 3\u0026rsquo; downstream sequences of AcMNPV\u003c/b\u003e \u003cb\u003egp37\u003c/b\u003e, \u003cb\u003epolyhedrin gene and CfDEFNPV spindlin gene.\u003c/b\u003e \u003cem\u003eAcMNPV gp37\u003c/em\u003e, \u003cem\u003eAcMNPV polh\u003c/em\u003e, and \u003cem\u003eCfDEFNPV spindlin\u003c/em\u003e were retrieved from GenBank. To identify putative polyadenylation signals, the DNASTAR software was used to search for the canonical hexamer motif AATAAA within the 3\u0026prime; downstream sequences. For \u003cem\u003eAcMNPV gp37\u003c/em\u003e and \u003cem\u003epolh\u003c/em\u003e, where transcript ends have been previously mapped, the search was limited to the known 3\u0026prime; untranslated regions (3\u0026prime; UTRs) [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. For \u003cem\u003eCfDEFNPV spindlin\u003c/em\u003e, whose transcript boundaries are unknown, the search was conducted within 1,000 nucleotides downstream of the stop codon (TAA or TGA). Additionally, GT-rich downstream elements, which are typically located 10\u0026ndash;30 nucleotides downstream of polyadenylation signals and are known to facilitate cleavage and polyadenylation, were visually inspected and compared the three genes.\u003c/p\u003e\u003cp\u003e\u003cb\u003eConstruction of viruses.\u003c/b\u003e Specific primers were designed to amplify the \u003cem\u003epolh\u003c/em\u003e ORF and its 3\u0026prime; downstream sequence (DS) from AcMNPV DNA. PCR products were verified by agarose gel electrophoresis, excised, and purified using the glassmilk method [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. These fragments were first cloned into the pGEM-T Easy vector (Promega) and confirmed via DNA sequencing, then subcloned into the pFastBac1 vector (Invitrogen) to generate pFastBacpolh-polhUTR. To construct a second recombinant, primers containing restriction enzyme (REN) sites were designed to separately amplify the polyhedrin ORF and the gp37 3\u0026prime; DS from AcMNPV DNA. After PCR amplification and agarose gel verification, the fragments were purified (glassmilk method) and cloned into pGEM-T Easy, resulting in pGEMT-polh and pGEMT-gp37UTR. Sequencing confirmed the integrity of both inserts. Using restriction enzymes EcoRI/XbaI (for polyhedrin ORF) and XbaI/XhoI (for \u003cem\u003egp37\u003c/em\u003e DS), the two fragments were excised and ligated into the pFastBac1 vector in a three-piece ligation, yielding pFastBacpolh-gp37UTR.\u003c/p\u003e\u003cp\u003eTo produce a construct for green fluorescent protein (\u003cem\u003egfp\u003c/em\u003e) expression, specific primers including REN sites were designed to amplify the GFP ORF using pBlueGFP (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] as the template and to amplify the polyhedrin 3\u0026rsquo; DS using AcMNPV DNA as the template and verified by agarose gel electrophoresis. The amplified PCR products were gel-extracted by the glassmilk method (Vogelstein and Gillespie, 1979), then cloned to the pGEM-T Easy vector to construct pGEMT-gfp, which has the GFP ORF and pGEMT -polhUTR, which has the polyhedrin 3\u0026rsquo; UTR. The GFP ORF and polyhedrin 3\u0026rsquo;UTR were retrieved from the plasmids pGEMT-gfp and pGEMT-polhUTR; then these two fragments were ligated to pFastBac1 in a three-piece DNA ligation reaction to construct pFastBacGFP-polhUTR. The GFP ORF and \u003cem\u003egp37\u003c/em\u003e 3\u0026rsquo;UTR fragments were retrieved from the plasmids pGEMT-gfp and pGEMT-gp37UTR; then the two fragments were ligated to pFastBac1 in a three-piece DNA ligation reaction to produce plasmid pFastBacGFP-gp37UTR.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eA primer list in this project\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"2\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGFP-F-EcoRI\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5\u0026rsquo;-GAATTCATGGTGAGCAAGGGCGAG-3\u0026rsquo;\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGFP-R-BamHI\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5\u0026rsquo;-GGATCCGGAACCACCACCACCCTTGTACAGCTCGTCCATG-3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAc-gp37UTR-XbaI\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5\u0026rsquo;-TCT AGA TAA AAC AAA CAA AAT TTT AAT TAC ATA TTA TAT TTA GCA AGA AG-3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAc-gp37UTR-XhoI\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5\u0026rsquo;-CTC GAG GAC GCA ATG GAG GCG TTG-3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAc-Polh-F-NotI\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5\u0026rsquo;-GCG GCC GCG TCT ATC AAT ATA TAG TTG CTG-3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAc-Polh-R-BclI\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5\u0026rsquo;-TGA TCA TAA CAC GCC CGA TGT TAA A-3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eIn addition to using GFP as a reporter, we also used luciferase (\u003cem\u003eluc\u003c/em\u003e) as a reporter gene to further support our discovery on UTR regulating gene expression. To construct a plasmid with the luciferase gene, we retrieved the luciferase gene from pGL-basic vector (Promega) by digestion with restriction enzyme BglII/XbaI. This REN fragment was purified by agarose gel electrophoresis using the glassmilk method. This fragment was cloned together with gp37UTR or polhUTR fragment to pFastBac1 similar to the GFP construct to produce pFastBacluc-polhUTR and pFastBacluc-gp37UTR.\u003c/p\u003e\u003cp\u003eThe plasmids pFastBac-polhUTR and pFastBac-gp37UTR, pFastBacgfp-polhUTR and pFastBacgfp-gp37UTR as well as pFastBacluc-polhUTR and pFastBacluc-gp37UTR were used to transform competent DH10Bac \u003cem\u003eE. coli\u003c/em\u003e cells (Invitrogen) following the protocol from the kit manufacturer. White colonies from each transformation were picked and confirmed by PCR with specific primers. DNAs were extracted from the bacteria that contain the right insertion in the bacmid and then used to transfect Sf21 cells to produce the viruses AcBacpolh-polhUTR, AcBacpolh-gp37UTR, AcBacgfp-polyhUTR, AcBacgfp-gp37UTR, AcBacluc-polhUTR and AcBacluc-gp37UTR. The newly constructed viruses were confirmed by polyhedra formation or by showing green fluorescence post-infection, or luciferase activity before propagation and tittered (O'Reilly et al., 1992).\u003c/p\u003e\u003cp\u003e\u003cb\u003eInfection and polyhedra production assay\u003c/b\u003e. Sf21 or Hi5 insect cells (3\u0026times;10⁶ cells/flask) were infected with either Acpolh-polhUTR or Acpolh-gp37UTR recombinant baculoviruses at a multiplicity of infection \u003cb\u003e(\u003c/b\u003eMOI\u003cb\u003e)\u003c/b\u003e of 5 plaque-forming units (p.f.u.) per cell in 25 cm\u0026sup2; tissue culture flasks. Infections were conducted at 27\u0026deg;C for 96 hours. Cell images were acquired using a SPOT Insight digital camera mounted on a Nikon Eclipse TE2000-U inverted microscope. After imaging, media were removed, and 1 ml of 0.1% SDS (w/v) was added to lyse the cells and release polyhedra. Flasks were gently rocked at 27\u0026deg;C for 1 hour. Cell lysates were transferred to 1.5 ml centrifuge tubes, \u003cb\u003eand\u003c/b\u003e 10 \u0026micro;l aliquots were taken for polyhedra yield estimation using a hemocytometer under a microscope.\u003c/p\u003e\u003cp\u003ePolyhedra size measurement and comparison. To assess differences in polyhedron size, 50 polyhedra were randomly selected and measured from Sf21 cells infected with either Acpolh-polhUTR \u003cb\u003eor\u003c/b\u003e Acpolh-gp37UTR. Measurements were performed using a Nikon Eclipse TE2000-U inverted microscope equipped with an ocular micrometer in one of the ocular lens. The same procedure was carried out on Hi5 cells infected with the same recombinant viruses. The purpose of these tests was to evaluate the effect of 3\u0026prime; downstream sequences on polyhedron size in different insect cell lines.\u003c/p\u003e\u003cp\u003e\u003cb\u003eProtein yield assay\u003c/b\u003e. Polyhedrin protein yields were estimated using the Bio-Rad protein assay kit \u003cb\u003ebased on the\u003c/b\u003e Bradford method. A standard curve was generated using bovine serum albumin (BSA) standards ranging from 0.2 mg/ml to 1.4 mg/ml in 0.1 M Na₂CO₃ (pH 10.5). The 5\u0026times; dye reagent was diluted to \u003cb\u003e1\u0026times;\u003c/b\u003e, and then added to each BSA dilution. The absorbance at 595 nm (OD₅₉₅) was measured using a spectrophotometer, with triplicate readings taken for each sample to ensure accuracy.\u003c/p\u003e\u003cp\u003eFor sample analysis, 50 \u0026micro;l of purified polyhedra from Sf21 cells infected with either Acpolh-polhUTR or Acpolh-gp37UTR were mixed with 50 \u0026micro;l of 0.1 M Na₂CO₃, followed by the addition of the diluted dye reagent. The absorbance at OD₅₉₅ was recorded, and polyhedrin concentrations were determined using the previously constructed standard curve. Estimated polyhedrin yields from Acpolh\u003cb\u003e-\u003c/b\u003epolhUTR- and Acgpolh\u003cb\u003e-\u003c/b\u003ep37UTR-derived polyhedra were \u003cb\u003estatistically analyzed\u003c/b\u003e to assess differences in protein production between the two constructs by Microsoft Excel.\u003c/p\u003e\u003cp\u003e\u003cb\u003eEstimation of reporter protein expression levels\u003c/b\u003e. Since polyhedra may not be completely dissolved and this may affect protein yield measurement accuracy, we then measured GFP expression levels using a spectrofluorometer. Sf21 cells infected with either Acgfp-polhUTR or Acgfp-gp37UTR followed the same infection protocol used for polyhedrin yield experiments, except that cells were processed for fluorescence analysis instead of protein quantification. After 96 hours of incubation, infected cells were dislodged from the tissue culture (TC) flasks by jet-flushing with media using a Pasteur pipette. The cell suspensions were centrifuged at 500 \u0026times; g for 5 minutes, and the supernatants were discarded. Residual media were carefully blotted from the cell pellets. Cells were lysed in 500 \u0026micro;l of 0.1% SDS, and the resulting lysates were used to measure GFP fluorescence using a Shimadzu RF-5301PC spectrofluorometer (excitation at 488 nm, emission at 507 nm). Fluorescence readings were taken in triplicate \u003cb\u003ef\u003c/b\u003eor each sample. Emission values were used to quantify and compare GFP expression levels between the two viral constructs (Acgfp-polhUTR vs Acgfpg-gp37UTR\u003cb\u003e)\u003c/b\u003e using a two-tailed Student's t-test performed in Microsoft Excel. To complement GFP measurement, we also used luciferase as a reporter gene for protein yield comparison between the two viral constructs (AcBacluc-polhUTR and AcBacluc-gp37UTR.).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e3\u0026rsquo; UTR sequence analysis. The 3\u0026prime; downstream sequences (DS) of the AcMNPV gp37 and polh genes, as well as the \u003cem\u003espindlin\u003c/em\u003e gene from CfDEFNPV, were analyzed for polyadenylation signal\u003cb\u003es\u003c/b\u003e. The 3\u0026prime; DS of AcMNPV \u003cem\u003egp37\u003c/em\u003e contains three potential polyadenylation \u003cb\u003esites\u003c/b\u003e, including a canonical AATAAA signal and associated GT-rich downstream sequences (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA,). In contrast, the spindlin gene, a \u003cem\u003egp37\u003c/em\u003e homologue from CfDEFNPV, has only a single polyadenylation site \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). The 3\u0026prime; DS of the AcMNPV polyhedrin gene contains two AATAAA motifs; however, only the second polyadenylation signal is followed by GT-rich downstream sequences, indicating that it may serve as the functional polyadenylation site (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). A summary comparison of the UTR regions of the three baculovirus genes were presented in Table D, E, and F.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eConstruction of three sets of viruses.\u003c/b\u003e In order to understand the regulatory effect of the alternative polyadenylation signals in the 3\u0026rsquo; DS of AcMNPV \u003cem\u003egp37\u003c/em\u003e, the 3\u0026rsquo; DS of \u003cem\u003egp37\u003c/em\u003e was fused to the downstream of three reporter genes, the \u003cem\u003epolh\u003c/em\u003e gene, the \u003cem\u003egfp\u003c/em\u003e gene and \u003cem\u003eluc\u003c/em\u003e gene. The 3\u0026rsquo; DS of the \u003cem\u003epolh\u003c/em\u003e gene was used as a strong polyadenylation signal with high protein expression to compare with the \u003cem\u003egp37\u003c/em\u003e 3\u0026rsquo; DS. The reporter genes and the 3\u0026rsquo; DSs were under the control of a strong \u003cem\u003epolh\u003c/em\u003e gene promoter. The Acpolh-polhUTR had the \u003cem\u003epolh\u003c/em\u003e ORF followed by 3\u0026rsquo; DS of \u003cem\u003epolh\u003c/em\u003e whereas the Acpolh-gp37UTR had \u003cem\u003epolh\u003c/em\u003e ORF followed by the 3\u0026rsquo; DS of \u003cem\u003egp37\u003c/em\u003e at the \u003cem\u003epolh\u003c/em\u003e locus. Since these two viruses were constructed using the AcMNPV bacmid, the endogenous \u003cem\u003epolh\u003c/em\u003e gene is not present. The Acgfp-polhUTR and Acgfp-gp37UTR viruses had the \u003cem\u003egfp\u003c/em\u003e gene followed by the 3\u0026rsquo; DS of \u003cem\u003epolh\u003c/em\u003e or the 3\u0026rsquo; DS of \u003cem\u003egp37\u003c/em\u003e at the \u003cem\u003epolh\u003c/em\u003e locus, respectively. At the same time we also successfully constructed two viruses (Acluc-polhUTR and Acluc-gp37UTR) that express luciferase to further support our discovery (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003ePolyhedron production and size comparisons between Acpolh-polhUTR and Acpolh-gp37UTR.\u003c/b\u003e The 3\u0026rsquo; DS of \u003cem\u003egp37\u003c/em\u003e increased polyhedron sizes but reduced polyhedron yields. In the \u003cem\u003epolh\u003c/em\u003e virus set, the \u003cem\u003epolh\u003c/em\u003e gene was used as the reporter gene. Polyhedra are formed in the nuclei of infected cells by baculovirus at the late stage of infection, which give a good visible tracking of the dynamics of polyhedrin expression under the microscope. The Sf21cells infected with Acpolh\u003cb\u003e-\u003c/b\u003egp37UTR showed much larger but fewer polyhedra in the nuclei of infected cells compared to Acpolh-polhUTR (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea and b). When the polyhedral numbers produced in the Sf21 cells infected with Acpolh\u003cb\u003e-\u003c/b\u003epolhUTR and Acpolh-gp37UTR were compared, production of polyhedra by Ac\u003cb\u003epolh-\u003c/b\u003epolhUTR showed twice as much as that produced by Acpolh-UTR (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). When the sizes of polyhedra formed by Acpolh-polhUTR and Acpolh-gp37UTR in Sf21 cells were measured and compared, polyhedral size formed by Acpolh-gp37UTR is 1.63 times larger in diameter than that of Acpolh-polhUTR (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). Therefore we see in inverse relationship between polyhedral number and size. Then we further compared polyhedrin protein yield form Sf21 cells infected by the two viruses. It is unknown if the size and number difference is cell type dependence or not, we further confirmed your discovery in Sf21 in Hi5 cells.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo further confirm the reduction of polyhedra number and increase of polyhedron size from Acpolh-gp37UTR infected cells, Hi5 cells were infected by Acpolh-polhUTR or Acpolh-gp37UTR for polyhedron production comparison in terms of polyhedron number and size. Similar phenomena were observed in Hi5 cells infection as in Sf21 cells infection (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea and b). To quantify our observation, the infection of Hi5 cells by Acpolh-gp37UTR produced 1.44-fold larger polyhedra in diameter than Acpolh-polhUTR (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec) and the reduction of total polyhedron production is about 6-fold (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). Therefore, infections of both Sf21 cells and Hi5 cells by the two viruses confirmed that the gp37 3\u0026rsquo; DS reduces polyhedron yield but increases polyhedron size compared to the \u003cem\u003epolh\u003c/em\u003e 3\u0026rsquo; DS, an inverse relationship. The next question we asked is if the total protein production is affected by the UTR regions.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eComparison of protein production\u003c/b\u003e. Although polyhedron yields were reduced when the \u003cem\u003egp37\u003c/em\u003e DS replaced the \u003cem\u003epolh\u003c/em\u003e 3\u0026rsquo; DS, the total protein yield was not affected. This conclusion is drawn from two independent assays. The Bio-Rad protein assay was used to compare the total protein expression levels of \u003cem\u003epolh\u003c/em\u003e from Sf21 cells infected with Acpolh-gp37UTR and Acpolh-polhUTR. It was found that the total \u003cem\u003epolh\u003c/em\u003e amounts from Sf21 cells infected with Acpolh-gp37UTR and Sf21 cells infected with Acpolh-polhUTR were at the same level (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). This result is supported by comparing GFP expression levels in Sf21 cells infected by Acgfp-polhUTR and Acgfp-gp37UTR. GFP expression by the two viral constructs in Sf21 cells showed no statistic difference, although it appeared different when the images of Sf21 cells infected with the two viruses were compared (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed). Furthermore, luciferase assay also did not show difference between AcBacluc-gp37UTR and AcBacluc-gp37UTR. This result further confirms that the total protein production levels either by \u003cem\u003epolh\u003c/em\u003e or GFP or luciferase were not affected by the 3\u0026rsquo; DS of gp37.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003emRNA processing plays a major regulatory role in eukaryotic cells. Alternative polyadenylation has been recently identified as an important contributor to gene regulation [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Alternative polyadenylation is known to regulate gene expression in various ways [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Other than producing different proteins, alternative polyadenylation can be used to produce the same coding region of an mRNA but with different 3\u0026rsquo; UTRs [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. It has only been recently appreciated that nearly all genes have additional polyadenylation signals in their 3\u0026rsquo; UTRs, and more than half of the human genes are alternatively polyadenylated [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Many alternative polyadenylation signals are evolutionarily conserved [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The use of alternative polyadenylation signals can protect the transcript from the stronger regulatory potential of longer 3\u0026rsquo; UTRs. For example, the shorter 3\u0026rsquo; UTR can lose microRNA (miRNA) complementary sites [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. It was reported that alternative polyadenylation can activate oncogenes in cancer cells by shortening the 3\u0026rsquo; UTRs of oncogenes; these shorter isoforms of oncogenes have higher stability and produce more proteins in part through the loss of miRNA-mediated repression [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The alternative polyadenylated isoforms of the same gene can also have different translation efficiency [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn this study, we demonstrated that 3\u0026rsquo; DS of the \u003cem\u003espindlin\u003c/em\u003e gene from CfDEFNPV has one putative polyadenylation site whereas the 3\u0026rsquo; DS of AcMNPV\u003cem\u003egp37\u003c/em\u003e has multiple polyadenylation sites (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). It was also reported that the AcMNPV \u003cem\u003egp37\u003c/em\u003e transcripts have different lengths and the different 3\u0026rsquo; end of the transcripts match well with the three polyadenylation signals in the 3\u0026rsquo; UTR of \u003cem\u003egp37\u003c/em\u003e. The percentage of the largest transcript decreases during later times post-infection while the percentage of the smaller transcripts increases [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eWhether baculovirus late gene late gene mRNA 3\u0026rsquo;end processing follows cellular gene using AAUAAA as the polyadenylation positioning signal and G/U rich region for stimulation of polyadenylation is not certain. Early studies suggest baculovirus does not use AAUAAA and G/U signal for polyadenylation for cleavage before polyadenylation, and it was reported that a stretch of seven Us (UUUUUUU) is the signal for termination [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. However, these were performed using a combination of 4 AcMNPV RNA polymerase subunits \u003cem\u003ein vitro\u003c/em\u003e [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Previously, we showed a stretch of seven Us was found in SV40 polyA [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. A more recent transcriptome analysis of AcMNPV \u003cem\u003ein vivo\u003c/em\u003e showed that 82.5% gene is associated with the cellular canonical polyadenylation signals; only about 13% carry the T-rich (U-rich in RNA) sequence as suggested by [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Another search for seven U in AcMNPV \u003cem\u003epolh\u003c/em\u003e, \u003cem\u003egp37\u003c/em\u003e, and CfDEFNPV \u003cem\u003espindlin\u003c/em\u003e did not yield any stretch of seven U (in RNA) or T in DNA. AcMNPV RNA polymerase (LEF 4, LEf 8, LEf 9 and P47) for late gene transcription may not represent what is really happening in the AcMNPV transcription \u003cem\u003ein vivo\u003c/em\u003e. There might be other proteins joining the AcMNPV polymerase for AcMNPV late gene transcript 3\u0026rsquo; processing, considering cellular transcription and AcMNPV gene transcription are all in the nucleus. These proteins might be either viral or cellar origin. A more recent AcMNPV transcriptome analysis in fact supports the idea of cellular factors involved in the 3\u0026rsquo; end processing of AcMNPV later transcripts by cleavage similar to the cellular mRNA 3\u0026rsquo; processing [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. In addition, a search in the AcMNPV genome did find a polyA polymerase, the enzyme to add a stretch of adenine. Of course this is not conclusive since there are many genes in the AcMNPV genome without gene function assignment.\u003c/p\u003e\u003cp\u003eIn order to test the regulatory function of the \u003cem\u003egp37\u003c/em\u003egene 3\u0026rsquo; DS, the 3\u0026rsquo; DS of \u003cem\u003epolh\u003c/em\u003e from AcMNPV, which is similar to 3\u0026rsquo; DS of the \u003cem\u003espindlin\u003c/em\u003e gene and has a high expression level, was used for comparison. When the \u003cem\u003epolh\u003c/em\u003e gene from AcMNPV was used as the marker gene, the virus Acpolh-gp37UTR that has \u003cem\u003egp37\u003c/em\u003e 3\u0026rsquo; DS makes fewer, but larger polyhedra in the nuclei of both infected Sf21 and Hi5 cells than does Acpolh-polhUTR, which has the \u003cem\u003epolh\u003c/em\u003e 3\u0026rsquo; DS. In Sf21 cells the reduction is 2-fold, and in Hi5 cells the reduction is 6-fold, both of which are significant. Surprisingly the total \u003cem\u003epolh\u003c/em\u003e protein levels from Acpolh-polhUTR- and Acpolh-gp37UTR-infected Sf21 cells are the same. This may reflect the size difference between polyhedra in Acpolh-polhUTR and Acpolh-gp37UTR infected cells. When GFP was used as the reporter gene instead of \u003cem\u003epolh\u003c/em\u003e in order to confirm that there is no difference at the final protein amount, the fluorescence of the cells infected with virus were very similar, which confirmed the observation that there is no difference in the final protein amount. The different size and number of polyhedra from the two virus infections indicates that there is a regulatory effect of the \u003cem\u003egp37\u003c/em\u003e 3\u0026rsquo; UTR since the coding regions of the two viruses are identical, the only difference being in the 3\u0026rsquo; DS of the reporter gene, \u003cem\u003epolh\u003c/em\u003e. But the regulation is not at the level of total protein production.\u003c/p\u003e\u003cp\u003ePolyhedra are crystals and the formation of the crystals has two components, nucleation and growth. High protein concentration will lead to the supersaturating of the solution and drive the nucleation. The growth is favored by then reducing the protein concentration since the reduced protein concentration can prevent further nucleation from competing with the growth of established nuclei [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. A possible explanation for the fewer, larger polyhedra from Acpolh-gp37UTR-infected cells is that the 3\u0026rsquo; UTR of \u003cem\u003egp37\u003c/em\u003e changes the protein synthesis kinetics. During the early stage of the infection, Acpolh-polhUTR may have higher \u003cem\u003epolh\u003c/em\u003e expression than Acpolh-gp37UTR, making it possible for Acpolh-polhUTR to produce more polyhedra. At later stages of the infection, Acpolh-gp37UTR catches up in \u003cem\u003epolh\u003c/em\u003e expression but can only build on the established polyhedra. It will be interesting to test whether the different lengths of \u003cem\u003egp37\u003c/em\u003e transcripts have different RNA stability and translation efficiency.\u003c/p\u003e\u003cp\u003eHow the alternative polyadenylation occurs in \u003cem\u003egp37\u003c/em\u003e transcripts and regulates gene expression is unknown. It was reported that the efficiency of using different polyadenylation sites is controlled by the interaction of the regulatory cis-acting RNA elements and the trans-acting protein factors. Among the factors, the cleavage and stimulation factor CstF-64 binds to the GU-rich region downstream of the AAUAAA signal and then recruits other factors to assemble the polyadenylation complex. One possible explanation for the regulatory effect of \u003cem\u003egp37\u003c/em\u003e 3\u0026rsquo; UTR is that the downstream GU-rich regions of each AAUAAA bind to CstF-64 weakly, allowing the alternative polyadenylation to take place. It was reported the sequences in the GU-rich region controls the binding [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], so by controlling the sequences in the GU-rich region downstream of AAUAAA, AcMNPV can regulate the \u003cem\u003egp37\u003c/em\u003e expression without requirement of additional regulatory proteins.\u003c/p\u003e\u003cp\u003eIt was reported that multiple genes have different 3\u0026rsquo; end sites and the proportion of the various forms of these mRNAs may vary temporally, although often the shortest forms are more common in the earlier stages of transcription [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. It has been well known that viruses have very compact genomes and use their genetic information very efficiently, so alternative polyadenylation should be commonly employed in many viral genomes. The results in this study show that alternative polyadenylation regulates the polyhedra formation without changing the total amount of the protein expressed, suggesting a possible impact on changing protein expression kinetics. This study can serve as an example of the regulatory effect of alternative polyadenylation in baculovirus and other viruses.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eConflicts of interest\u003c/h2\u003e\u003cp\u003eThe authors declare no financial or commercial conflict of interest.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eAuthor contributions\u003c/h2\u003e\u003cp\u003eX-W C, HS and JL Xue planned experiments; X-W C and JL Xue wrote the manuscript\u003c/p\u003e\u003ch2\u003eACKNOWLEDGEMENTS\u003c/h2\u003e\u003cp\u003eWe would like to thank Miami University CBFG for help in using the equipment in luciferase assay. Dr. Gary R. Janssen is credited for proofreading this manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eDoerfler W (1986) The Molecular Biology of Baculoviruses. 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EMBO J 22(11):2821\u0026ndash;2830\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e "}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Gene transcription, gene regulation, protein expression","lastPublishedDoi":"10.21203/rs.3.rs-7992808/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7992808/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBaculovirus is a double-stranded DNA virus widely used in agriculture for the biological control of insect pests and in the pharmaceutical industry for vaccine production, due to its safety in humans. The type species, \u003cem\u003eAutographa californica\u003c/em\u003e multiple nucleopolyhedrovirus (AcMNPV), has a broad insect host range and is the most genetically characterized baculovirus. During infection of insect cells, early genes are transcribed by the host RNA polymerase II, while late and very late genes are transcribed by a virus-encoded RNA polymerase. One such late gene, \u003cem\u003egp37\u003c/em\u003e, is considered non-essential for viral replication, though its biological function remains unclear. In this study, we constructed three pairs of recombinant viruses, each containing complementary reporter genes, to investigate the regulatory role of the gp37 3' untranslated region (3' UTR). Our results show that the \u003cem\u003egp37\u003c/em\u003e 3' UTR modulates the expression of polyhedrin, green fluorescent protein and luciferase genes even when driven by the same promoter. This suggests a post-transcriptional regulatory role for the \u003cem\u003egp37\u003c/em\u003e 3' UTR in gene expression.\u003c/p\u003e","manuscriptTitle":"gp37 3’ UTR reduces protein production at the polyhedrin locus of Autographa californica multicapsid nucleopolyhedrovirus","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-13 09:33:30","doi":"10.21203/rs.3.rs-7992808/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6f5e72d8-c1ed-416f-aa2a-c6218c8eeb53","owner":[],"postedDate":"November 13th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-12-02T06:45:39+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-13 09:33:30","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7992808","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7992808","identity":"rs-7992808","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}