Influence of GlycoGag on the Incorporation of Host Membrane Proteins Into the Envelope of the Moloney Murine Leukemia Virus

Mariam Maltseva,Marc-André Langlois

doi:10.3389/fviro.2021.747253

Mariam Maltseva, Marc-André Langlois

Open Access

https://doi.org/10.3389/fviro.2021.747253

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Abstract

Analysis of viral particle heterogeneity produced from infected cells has been limited by the inefficiency of traditional analytical methods to characterize large populations of viruses at an individual particle level. Flow virometry (FVM) is an emerging technique based on flow cytometry principles that enables a high throughput, multiparametric, and phenotypic characterization of viruses at a single particle resolution. Here, we performed FVM to analyze surface markers found on Murine Leukemia Virus (MLV) and glycosylated Gag-deficient (glycoGag) MLV. The glycoGag viral accessory protein has several roles in the MLV viral infection cycle including directing retroviral assembly and particle release at lipid rafts. Based on previous studies, we hypothesize that glycoGag modulates host protein incorporation into the viral envelope during viral assembly and budding. Here, by using FVM, we reveal that glycoGag is associated with an increased incorporation of the host-derived tetraspanins CD81 and CD63 along with the lipid raft marker and immune antigen Thy1.2 during the assembly and release of viral particles from infected NIH 3T3, EL4, and primary CD4+ T cells. Moreover, we show differences in the uptake of host proteins by viruses that are released from the two cell lines and primary T lymphocytes. Additionally, at the individual viral particle level, we observed a degree of expression heterogeneity of host-derived antigens within the viral population. Finally, certain cellular antigens can show either enrichment or exclusion from the viral envelope depending on whether glycoGag is expressed by the virus. This suggests that glycoGag is involved in a mechanism of selective host protein incorporation into the viral envelope.

Highlights

Retroviruses are a diverse group of enveloped, single-stranded, RNA viruses that infect a wide variety of vertebrates
We show that the glycoGag accessory protein is associated with a selective increase or decrease in the incorporation of certain host-derived proteins into the viral envelope
Analysis of Murine Leukemia Virus (MLV) virions showed a highly monodisperse GFP+ population above a nonfluorescent population composed of EVs and background noise that is mostly visible when compared to the overlaid signal of supernatant collected from uninfected cells (Figures 1E–G)

Summary

Introduction

Retroviruses are a diverse group of enveloped, single-stranded, RNA viruses that infect a wide variety of vertebrates. MLV is a simple enveloped retrovirus, initially presumed to code for three essential genes that are required for the retroviral replication cycle: Group Specific Antigen (Gag), envelope glycoprotein (Env), and viral RNA-dependent DNA polymerase and integrase (Pol). It has been shown that the MLV genome can code for an additional glycosylated variant of Gag known as glycoGag which harbors an additional 88 amino acids in its N-terminus (Figure 1A) [1,2,3]. GlycoGag is cleaved into two subunits with the amino-terminal cleavage product of the protein becoming membrane-associated and subsequently inserted into the viral envelope of budding progeny virions in the NexoCcyto orientation of a type I integral membrane protein [1, 4,5,6]

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