Full-Length Glycosylated Gag of Murine Leukemia Virus Can Associate with the Viral Envelope as a Type I Integral Membrane Protein

Abstract

The glycosylated Gag protein (gPr80) of murine leukemia viruses (MLVs) has been shown to exhibit multiple roles in facilitating retrovirus release, infection, and resistance to host-encoded retroviral restriction factors, such as APOBEC3, SERINC3, and SERINC5. One way in which gPr80 helps MLVs to escape host innate immune restriction is by increasing capsid stability, a feature that protects viral replication intermediates from being detected by cytosolic DNA sensors. gPr80 also increases the resistance of MLVs to deamination and restriction by mouse APOBEC3 (mA3). How the gPr80 accessory protein, with its three N-linked glycosylation sites, contributes to these resistance mechanisms is still not fully understood. Here we further characterized the function of gPr80 and, more specifically, revealed that the asparagines targeted for glycosylation in gPr80 also contribute to capsid stability through their parallel involvement in the Pr65 Gag structural polyprotein. In fact, we demonstrate that sensitivity to deamination by the mA3 and human A3 proteins is directly linked to capsid stability. We also show that full-length gPr80 is detected in purified viruses. However, our results suggest that gPr80 is inserted in the N_exoC_cyto orientation of a type I integral membrane protein. Additionally, our experiments have revealed the existence of a large population of Env-deficient virus-like particles (VLPs) harboring gPr80 inserted in the opposite (N_cytoC_exo) polarity, which is typical of type II integral membrane proteins. Overall this study provides new insight into the complex nature of the MLV gPr80 accessory protein.IMPORTANCE Viruses have evolved numerous strategies to infect, spread in, and persist in their hosts. Here we analyze the details of how the MLV-encoded glycosylated Gag (gPr80) protein protects the virus from being restricted by host innate immune defenses. gPr80 is a variant of the structural Pr65 Gag protein with an 88-amino-acid extended leader sequence that directs the protein for translation and glycosylation in the endoplasmic reticulum. This study dissects the specific contributions of gPr80 glycans and capsid stability in helping the virus to infect cells, spread, and counteract the effects of the host intrinsic restriction factor APOBEC3. Overall this study provides further insight into the elusive role of the gPr80 protein.

Keywords: APOBEC3; MLV; SERINC3; SERINC5; glyco-Gag.