Mechanism of Viral Glycoprotein Targeting by Membrane-Associated RING-CH Proteins

Abstract

An emerging class of cellular inhibitory proteins has been identified that targets viral glycoproteins. These include the membrane-associated RING-CH (MARCH) family of E3 ubiquitin ligases that, among other functions, downregulate cell surface proteins involved in adaptive immunity. The RING-CH domain of MARCH proteins is thought to function by catalyzing the ubiquitination of the cytoplasmic tails (CTs) of target proteins, leading to their degradation. MARCH proteins have recently been reported to target retroviral envelope glycoproteins (Env) and vesicular stomatitis virus G glycoprotein (VSV-G). However, the mechanism of antiviral activity remains poorly defined. Here we show that MARCH8 antagonizes the full-length forms of HIV-1 Env, VSV-G, Ebola virus glycoprotein (EboV-GP), and the spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), thereby impairing the infectivity of virions pseudotyped with these viral glycoproteins. This MARCH8-mediated targeting of viral glycoproteins requires the E3 ubiquitin ligase activity of the RING-CH domain. We observe that MARCH8 protein antagonism of VSV-G is CT dependent. In contrast, MARCH8-mediated targeting of HIV-1 Env, EboV-GP, and SARS-CoV-2 S protein by MARCH8 does not require the CT, suggesting a novel mechanism of MARCH-mediated antagonism of these viral glycoproteins. Confocal microscopy data demonstrate that MARCH8 traps the viral glycoproteins in an intracellular compartment. We observe that the endogenous expression of MARCH8 in several relevant human cell types is rapidly inducible by type I interferon. These results help to inform the mechanism by which MARCH proteins exert their antiviral activity and provide insights into the role of cellular inhibitory factors in antagonizing the biogenesis, trafficking, and virion incorporation of viral glycoproteins. IMPORTANCE Viral envelope glycoproteins are an important structural component on the surfaces of enveloped viruses that direct virus binding and entry and also serve as targets for the host adaptive immune response. In this study, we investigate the mechanism of action of the MARCH family of cellular proteins that disrupt the trafficking and virion incorporation of viral glycoproteins across several virus families. This research provides novel insights into how host cell factors antagonize viral replication, perhaps opening new avenues for therapeutic intervention in the replication of a diverse group of highly pathogenic enveloped viruses.

MARCH8 Restricts Influenza A Virus Infectivity but Does Not Downregulate Viral Glycoprotein Expression at the Surface of Infected Cells

“…Next, we showed that MARCH8 mRNA expression in 293T cells ( Fig. 4A ) was reduced between 2 and 24 h after (i) exposure to type I interferon (IFN) or (ii) infection with IAV, whereas IFN-regulated gene IFITM3 was strongly upregulated by both type I IFN and by IAV infection; it should be noted that previous studies reported rapid (0.5 h) induction of MARCH8 by type I IFNs in 293T cells, which returned to baseline levels within 2 to 4 h ( 8 ). Similar results were seen using human monocyte-derived macrophages (hMDMs) ( Fig.…”

“…Differences in the relative proportion or the absolute amount of specific viral proteins in a packaged virion could contribute to overall reduction in IAV infectivity. Reduced incorporation of Env into HIV-1 ( 14 ) or VSV-G, EboV-GP, and SARS-CoV-2 S glycoproteins into pseudotyped particles ( 8 ) correlated with reduced infectivity of virions released from MARCH8-expressing cells. As seen in Fig.…”

“…Human ( 8 , 14 , 16 ) and mouse ( 9 ) MARCH8 reduce the expression of a range of viral glycoproteins from the surface of infected cells, thereby reducing their incorporation into virions. Surprisingly, we detected no significant differences in the cell surface expression of HA, NA, or M2 proteins in IAV-infected cells in presence or absence of inducible MARCH8 at 8 hpi, a relatively early time point in the IAV replication cycle ( Fig.…”

“…Tada et al first reported that human MARCH8 reduced the infectivity of HIV-1 and vesicular stomatitis virus glycoprotein (VSV-G)-pseudotyped viruses by downregulating envelope glycoproteins at the cell surface, resulting in reduced incorporation into nascent virions. Although this antiviral activity was E3 ligase dependent, subsequent studies demonstrated that MARCH8 targeted lysine (K) residues in the cytoplasmic tail (CT) of VSV-G, whereas HIV-1 Env downregulation occurred even if its entire CT was deleted ( 8 – 10 ). Similarly, CT deletion mutants of Ebola virus glycoprotein (EboV-GP) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) glycoproteins remained sensitive to human MARCH8 ( 8 ).…”

“…Although this antiviral activity was E3 ligase dependent, subsequent studies demonstrated that MARCH8 targeted lysine (K) residues in the cytoplasmic tail (CT) of VSV-G, whereas HIV-1 Env downregulation occurred even if its entire CT was deleted ( 8 – 10 ). Similarly, CT deletion mutants of Ebola virus glycoprotein (EboV-GP) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) glycoproteins remained sensitive to human MARCH8 ( 8 ). In addition, mouse MARCH1 and -8 were recently shown to target and downregulate the p15E envelope glycoprotein subunit of mouse leukemia virus (MLV) ( 9 ) and human MARCH1 and -2 both mediate antiviral activity against HIV-1 and VSV-G-pseudotyped viruses ( 9 , 11 ).…”