Snake Kolmiovirus Encodes a Single Form of Delta Antigen and Shows No Evidence of Translation from Open Reading Frame 2

ABSTRACT Hepatitis D virus (HDV) is a satellite virus that utilizes hepatitis B virus (HBV) as a helper for infectious particle formation. HDV was originally identified as a novel antigen in liver biopsies of HBV patients, and later studies showed the “delta” antigen (DAg) to be the sole protein encoded by HDV. Until the discovery of HDV-like agents in birds and snakes in 2018, HDV was a unique example of animal satellite viruses. We identified Swiss snake colony virus 1 (SwSCV-1) in the brain of a Boa constrictor , and through comparison we found the genome organization of SwSCV-1 to resemble that of HDV. However, in addition to the DAg open reading frame (ORF), the genome of SwSCV-1 includes another >500 nt ORF, “ORF2”. To study whether the putative ORF2-encoded protein plays a role in the SwSCV-1 life cycle, we established an infectious clone of the virus with a point mutation in the methionine initiation codon of ORF2. The mutation did not significantly affect initiation of replication, establishment of persistent infection, or infectious particle formation upon superinfection with a helper virus. Using additional methods, we gathered further evidence confirming that ORF2 is not actively translated in boa constrictor cells. We further showed that unlike HDV, SwSCV-1 expresses a single form of the DAg. Although the proteins encoded by SwSCV-1 and HDV only include one and two forms of the DAg, respectively, whether other kolmioviruses express additional forms of DAg or related proteins in some cell types or host species merits further research. IMPORTANCE Approximately 40 years after the discovery of hepatitis D virus (HDV), satellite viruses with similar genome organization were found in various animals, thereby giving rise to family Kolmioviridae . HDV encodes a single protein, the delta antigen (DAg), which comes in small and approximately 20 amino acids longer large form. The genome of some HDV species and many of the newly found kolmioviruses contains additional open reading frames (ORFs), potentially enabling protein expression. Here, we studied the viral proteins expressed during Swiss snake colony virus 1 (SwSCV-1) infection of boa constrictor cells. Our findings show that unlike HDV, SwSCV-1 encodes only a single form of DAg. In addition, our study suggests that, like in HDV, the additional ORF in SwSCV-1 genome does not give rise to a protein. Although we could not demonstrate expression of additional viral proteins during SwSCV-1 infection, it is important to study the proteome of other kolmioviruses.