Conserved residues of the immunosuppressive domain of MLV are essential for regulating the fusion-critical SU-TM disulfide bond

ABSTRACT The ENV protein of murine leukemia virus (MLV) is the prototype of a large clade of retroviral fusogens, collectively known as gamma-type Envs. Gamma-type ENVs are found in retroviruses and related endogenous retroviruses (ERV) representing a broad range of vertebrate hosts. All gamma-type Envs contain a highly conserved stretch of 26-residues in the transmembrane subunit (TM) comprising two motifs, a putative immunosuppressive domain (ISD) and a CX 6 CC motif. The extraordinary conservation of the ISD and its invariant association with the CX 6 CC suggests a fundamental contribution to Env function. To investigate function of the ISD, we characterized several mutants with single amino acid substitutions at conserved positions in the MLV ISD. A majority abolished infectivity, although we did not observe a corresponding loss in intrinsic ability to mediate membrane fusion. Ratios of the surface subunit (SU) to capsid protein (CA) in virions were diminished for a majority of the mutants, while TM/CA ratios were similar to wild type. Specific loss of SU reflected premature isomerization of the labile disulfide bond that links SU and TM prior to fusion and entry. Indeed, all non-infectious mutants displayed significantly lower disulfide stability than wild type MLV Env. These results reveal a role for residues at MLV ISD positions 2, 3, 4, 7, and 10 in regulating a late step in fusion, and suggest that the ISD is part of a larger domain, encompassing both the ISD and CX6CC motifs, that is critical for formation and regulation of the metastable, intersubunit disulfide bond. IMPORTANCE The gamma-type Env is an extremely prevalent viral fusogen, extensively found within retroviruses and endogenous loci across vertebrate species and are further found in filoviruses such as Ebola virus. The fusion mechanism of gamma-type Envs is unique from other Class I fusogens such as those of IAV and HIV-1. Gamma-type Envs contain a hallmark feature known as the immunosuppressive domain (ISD) that has been the subject of some controversy in the literature surrounding its putative immunosuppressive effects. Despite the distinctive conservation of the ISD, little has been done to investigate the role of this region for the function of this widespread fusogen. Our work demonstrates the importance of the ISD for the function of gamma-type Envs in infection, particularly in regulating the intermediate steps of fusion with the host membrane. Understanding the fusion mechanism of gamma-type Envs has broad implications for understanding entry of extant viruses and aspects of host biology connected to co-opted endogenous gamma-type Envs.