Abstract
ABSTRACTMembrane-associated RING-CH8 (MARCH8) impairs the cell surface expression of envelope glycoproteins from different viruses, reducing their incorporation into virions. Using stable cell lines with inducible MARCH8 expression, we show that MARCH8 did not alter susceptibility to influenza A virus (IAV) infection, but virions released from infected cells were markedly less infectious. Knockdown of endogenous MARCH8 confirmed its effect on IAV infectivity. The expression of MARCH8 impaired the infectivity of both H3N2 and H1N1 strains and was dependent on its E3 ligase activity. Although virions released in the presence of MARCH8 expressed smaller amounts of viral hemagglutinin (HA) and neuraminidase (NA) proteins, there was no impact on levels of the viral HA, NA, or matrix 2 (M2) proteins detected on the surface of infected cells. Moreover, mutation of lysine residues in the cytoplasmic tails of HA, NA, and/or M2, or in the viral M1 protein, did not abrogate MARCH8-mediated restriction. While MARCH1 and -8 target similar immunological ligands and both restrict HIV-1, only MARCH8 inhibited IAV infectivity. Deletion of the N-terminal cytoplasmic (N-CT) domain of MARCH8 confirmed it to be a critical determinant of IAV inhibition. Of interest, deletion of the MARCH1 N-CT or its replacement with the MARCH8 N-CT resulted in acquisition of IAV restriction. Together, these data demonstrate that MARCH8 restricts a late stage in IAV replication by a mechanism distinct to its reported activity against other viruses. Moreover, we show that the N-CT of MARCH8 is essential for anti-IAV activity, whereas the MARCH1 N-CT inhibits its ability to restrict IAV.
Highlights
IMPORTANCE The antiviral activity of Membrane-associated RING-CH8 (MARCH8) has been associated with the downregulation of envelope glycoproteins from a range of different viruses, resulting in reduced incorporation into nascent virions
Western blots were probed with anti-FLAG monoclonal antibody (MAb) to detect MARCH1 and -8. (D) Cells were transfected with a plasmid encoding cluster of differentiation 86 (CD86) and ZsGreen fluorescent protein
For HIV-1 and viruses pseudotyped with other viral glycoproteins, the reduced expression of viral glycoproteins at the cell surface is a key determinant of MARCH8-mediated antiviral activity [8, 9, 14, 16]
Summary
IMPORTANCE The antiviral activity of MARCH8 has been associated with the downregulation of envelope glycoproteins from a range of different viruses, resulting in reduced incorporation into nascent virions. 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 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 HIV1 Env downregulation occurred even if its entire CT was deleted [8,9,10]. 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] Together, these studies demonstrate that MARCHmediated restriction is associated with downregulation of a broad range of viral envelope glycoproteins. These data indicate that MARCH8 mediates antiviral activity against IAV by a mechanism distinct from its reported activity against other viruses and identify the critical role of the N-CT domain for MARCH8-mediated restriction of IAV
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