Investigating the ability of mammalian Membrane Associated RING-CH proteins to modulate respiratory virus infections

Abstract

Respiratory viruses generally infect epithelial cells lining the upper and lower airways, however subsets of airway immune cells are also susceptible to infection. Of interest, airway epithelial cells (AEC) and airway macrophages (AM) are both susceptible to influenza virus infection, but only AEC support productive virus replication. Constitutive expression and/or induction of intracellular host proteins to limit or block virus replication is a well-known antiviral mechanism, however few such “restriction factors” have been well characterized and the antiviral activity of many putative restriction factors against respiratory viruses has not been reported. A recent RNA-seq study performed in our laboratory compared gene expression between AEC and AM in the presence or absence of influenza infection, allowing us to identify protein families with differential expression between these two cell types in steady-state and/or following virus infection. We hypothesized that amongst genes differentially expressed between AEC and AM would be those that represent novel restriction factors and these might contribute to the abortive versus productive phenotype observed in AM and AEC, respectively, in regard to influenza replication. One of the protein families identified were the membrane-associated RING-CH (MARCH) family proteins.

MARCH proteins are E3 ubiquitin ligases involved in the final step of the ubiquitination process. Amongst their functions, MARCH proteins modulate host immune responses by virtue of their ability to control the turnover of multiple immune molecules, including major histocompatibility complex (MHC) proteins. More recent evidence indicates that they can also modulate virus infection, acting as restriction factors against viruses such as human immunodeficiency virus (HIV)-I, or as proviral factors for viruses such as hepatitis C virus (HCV). Based on these findings, this thesis aimed to investigate the ability of MARCH family proteins to modulate infections caused by influenza and other respiratory viruses.

First, we generated stable cell lines with doxycycline (DOX)-inducible expression of several human MARCH proteins (MARCH1/2/3/5/6/7/8/9) and evaluated their ability to modulate either the early or late stages of infections caused by influenza A viruses (IAV), respiratory syncytial virus (RSV) or human metapneumovirus (hMPV). Although none of the MARCH proteins tested affected the early stages of IAV infection (as measured by flow cytometry for newly-synthesized viral proteins), inducible expression of MARCH8 was associated with a significant reduction in titres of infectious IAV released from infected cells. Moreover, MARCH8 expression was also associated with a reduced percentage of RSV-infected cells, consistent with its ability to restrict at an early stage of the RSV replication cycle, although this was not explored further in this thesis. We did not observe any significant differences in the ability of any MARCH protein tested to modulate early or late stages of hMPV infection. Subsequent studies have focussed on understanding MARCH8-mediated restriction of influenza virus infections.

Next, we characterized the antiviral activity of MARCH8 against influenza viruses. MARCH8 was shown to mediate antiviral activity against a range of influenza viruses relevant to human health, including A/H1N1, A/H1N1pdm09 and A/H3N2 viruses, as well as against an influenza B virus. We demonstrated that expression of MARCH8, but not the closely related MARCH1, was associated with a reduction in the specific infectivity of virus particles released from IAV-infected cells. Moreover, virus particles released from IAV-infected cells in the presence of MARCH8 exhibited altered protein composition and virion morphology, and MARCH8 was incorporated into the nascent virions. Of interest, MARCH8 expression did not alter cell-surface expression of the viral proteins HA, NA and M2, indicating no major defect in their synthesis and transport during viral replication. Overall, these findings are consistent with MARCH8 acting to block a step late in the virus replication cycle, possibly during virus assembly and/or budding from the cell surface.

Studies described in this thesis also attempted to determine features of MARCH8 and IAV relevant to MARCH8-mediated restriction of IAV. We used site-directed mutagenesis and reverse genetics to modify lysine residues in the cytoplasmic tails of the viral hemagglutinin (HA), neuraminidase (NA) and matrix protein (M)2 proteins, either alone or in combination. However, none of these substitutions were sufficient to overcome MARCH8-mediated restriction of IAV. We also modified several lysine residues in the viral M1 protein but again did not identify specific residues to abrogate MARCH8-mediated restriction. Currently, the identity of the particular viral and/or host proteins targeted by MARCH8 which result in inhibition of late stage IAV replication are not known.

To study MARCH8 itself, we generated mutants lacking E3 ligase activity and confirmed that this was essential for its anti-IAV activity. Furthermore, given that MARCH8, but not the closely related MARCH1, mediated potent anti-IAV activity against some strains of IAV, we generated cell lines with DOX-inducible expression of MARCH1-MARCH8 chimeric proteins in an effort to determine particular domains of MARCH8 critical to its anti-IAV activity. Chimeric proteins contained one or more domains/regions of MARCH1 substituted into the MARCH8 backbone. While all chimeric proteins retained activity against a common target protein (CD86), all of them also retained ability to restrict IAV replication. Based on these studies, we were unable to identify critical domains of MARCH8 that were essential for its antiviral activity against IAV.

Finally, we used a MARCH8 knockout (KO) mouse to evaluate the role of endogenous MARCH8 in modulating IAV infection in vivo. Following intranasal infection, MARCH8 KO mice exhibited enhanced weight loss and viral replication in the lungs at day 5 post- infection, although no major differences in virus titres or virus clearance were observed at later time-points. Moreover, we did not observe major differences in soluble inflammatory mediators or inflammatory cells at day 7 or 10 post-infection. These findings are consistent with a role for endogenous MARCH8 in controlling the early stages of IAV infection in vivo but suggest that it does not have a major impact on virus clearance or the development of adaptive immunity in this model. Of interest, siRNA knockdown of endogenous MARCH8 in a human epithelial cell line also resulted in a modest, but significant, reduction in titres of IAV released from infected cells in vitro.

Together, the studies presented in this thesis describe and characterize the antiviral activity of MARCH8 against influenza viruses. In addition, they provide preliminary data to indicate that MARCH8 can also mediate antiviral activity against the pneumovirus RSV, albeit by a distinct mechanism. Overall, these findings contribute to a growing body of evidence that MARCH8 plays an important role in modulating infections caused by a range of different viruses with relevance to human health.