Isoforms of Human MARCH1 Differ in Ability to Restrict Influenza A Viruses Due to Differences in Their N Terminal Cytoplasmic Domain

MARCH1 and MARCH8 are closely related E3 ubiquitin ligases that ubiquitinate an overlapping spectrum of host proteins and restrict replication of certain viruses. While the antiviral activity of MARCH8 has been intensively studied, less is known regarding virus inhibition by MARCH1. Isoforms 1 and 2 of MARCH1 are very similar in overall structure but show major differences in their N-terminal cytoplasmic domain (N-CT). Herein, we used a doxycycline-inducible overexpression system to demonstrate that MARCH1.1 reduces titres of influenza A virus (IAV) released from infected cells whereas MARCH1.2 does not. The deletion of the entire N-CT of MARCH1.2 restored its ability to restrict IAV infectivity and sequential deletions mapped the restoration of IAV inhibition to delete the 16 N-terminal residues within the N-CT of MARCH1.2. While only MARCH1.1 mediated anti-IAV activity, qPCR demonstrated the preferential expression of MARCH1.2 over MARCH1.1 mRNA in unstimulated human peripheral blood mononuclear cells and also in monocyte-derived macrophages. Together, these studies describe the differential ability of MARCH1 isoforms to restrict IAV infectivity for the first time. Moreover, as published immunological, virological and biochemical studies examining the ability of MARCH1 to target particular ligands generally use only one of the two isoforms, these findings have broader implications for our understanding of how MARCH1 isoforms might differ in their ability to modulate particular host and/or viral proteins

Host Cell Restriction Factors that Limit Influenza A Infection

Viral infection of different cell types induces a unique spectrum of host defence genes, including interferon-stimulated genes (ISGs) and genes encoding other proteins with antiviral potential. Although hundreds of ISGs have been described, the vast majority have not been functionally characterised. Cellular proteins with putative antiviral activity (hereafter referred to as “restriction factors”) can target various steps in the virus life-cycle. In the context of influenza virus infection, restriction factors have been described that target virus entry, genomic replication, translation and virus release. Genome wide analyses, in combination with ectopic overexpression and/or gene silencing studies, have accelerated the identification of restriction factors that are active against influenza and other viruses, as well as providing important insights regarding mechanisms of antiviral activity. Herein, we review current knowledge regarding restriction factors that mediate anti-influenza virus activity and consider the viral countermeasures that are known to limit their impact. Moreover, we consider the strengths and limitations of experimental approaches to study restriction factors, discrepancies between in vitro and in vivo studies, and the potential to exploit restriction factors to limit disease caused by influenza and other respiratory viruses

Caveats of Using Overexpression Approaches to Screen Cellular Host IFITM Proteins for Antiviral Activity

Ectopic protein overexpression in immortalised cell lines is a commonly used method to screen host factors for their antiviral activity against different viruses. However, the question remains as to what extent such artificial protein overexpression recapitulates endogenous protein function. Previously, we used a doxycycline-inducible overexpression system, in conjunction with approaches to modulate the expression of endogenous protein, to demonstrate the antiviral activity of IFITM1, IFITM2, and IFITM3 against influenza A virus (IAV) but not parainfluenza virus-3 (PIV-3) in A549 cells. We now show that constitutive overexpression of the same IFITM constructs in A549 cells led to a significant restriction of PIV-3 infection by all three IFITM proteins. Variable IFITM mRNA and protein expression levels were detected in A549 cells with constitutive versus inducible overexpression of each IFITM. Our findings show that overexpression approaches can lead to levels of IFITM1, IFITM2, and IFITM3 that significantly exceed those achieved through interferon stimulation of endogenous protein. We propose that exceedingly high levels of overexpressed IFITMs may not accurately reflect the true function of endogenous protein, thus contributing to discrepancies when attributing the antiviral activity of individual IFITM proteins against different viruses. Our findings clearly highlight the caveats associated with overexpression approaches used to screen cellular host proteins for antiviral activity

Inhibition of the Hantavirus Fusion Process by Predicted Domain III and Stem Peptides from Glycoprotein Gc

Hantaviruses can cause hantavirus pulmonary syndrome or hemorrhagic fever with renal syndrome in humans. To enter cells, hantaviruses fuse their envelope membrane with host cell membranes. Previously, we have shown that the Gc envelope glycoprotein is the viral fusion protein sharing characteristics with class II fusion proteins. The ectodomain of class II fusion proteins is composed of three domains connected by a stem region to a transmembrane anchor in the viral envelope. These fusion proteins can be inhibited through exogenous fusion protein fragments spanning domain III (DIII) and the stem region. Such fragments are thought to interact with the core of the fusion protein trimer during the transition from its pre-fusion to its post-fusion conformation. Based on our previous homology model structure for Gc from Andes hantavirus (ANDV), here we predicted and generated recombinant DIII and stem peptides to test whether these fragments inhibit hantavirus membrane fusion and cell entry. Recombinant ANDV DIII was soluble, presented disulfide bridges and beta-sheet secondary structure, supporting the in silico model. Using DIII and the C-terminal part of the stem region, the infection of cells by ANDV was blocked up to 60% when fusion of ANDV occurred within the endosomal route, and up to 95% when fusion occurred with the plasma membrane. Furthermore, the fragments impaired ANDV glycoprotein-mediated cell-cell fusion, and cross-inhibited the fusion mediated by the glycoproteins from Puumala virus (PUUV). The Gc fragments interfered in ANDV cell entry by preventing membrane hemifusion and pore formation, retaining Gc in a non-resistant homotrimer stage, as described for DIII and stem peptide inhibitors of class II fusion proteins. Collectively, our results demonstrate that hantavirus Gc shares not only structural, but also mechanistic similarity with class II viral fusion proteins, and will hopefully help in developing novel therapeutic strategies against hantaviruses.CONICYT (Chile) through FONDECYT grant, Basal fundin

Localization of predicted DIII and stem fragments in ANDV Gc.

<p>(A) Molecular model for ANDV Gc in its pre-fusion conformation [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004799#pntd.0004799.ref023" target="_blank">23</a>] based on the TBEV E protein (PDB: 1SVB) [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004799#pntd.0004799.ref031" target="_blank">31</a>]. The predicted DIII is indicated in blue and the predicted domains I-II are shown in grey. (B) Scheme of the ANDV Gc primary structure indicating predicted residues for DIII (blue) and the predicted stem region (green). S indicates the stem region; TM indicates the transmembrane region; Ct indicates the cytoplasmic tail. (C) Sequence logo representation of a multiple sequence alignment including DIII and stem regions of the genus <i>Hantavirus</i> built with WebLogo [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004799#pntd.0004799.ref065" target="_blank">65</a>]. The sequence conservation at each position is represented by the overall height of the stack at that position, while the conservation of a specific residue at that position is given by the height of the symbols. Acid residues are indicated in red, basic residues in blue, apolar residues in black and polar residues in green. For the multiple sequence alignment, glycoprotein sequences were retrieved from GenBank representing the four hantavirus phylogroups [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004799#pntd.0004799.ref066" target="_blank">66</a>] from different reservoirs, including rodent-borne hantaviruses from the subfamiliies <i>Avicolinae</i> (PUUV; accession no. NP_941983.1), <i>Murinae</i> (HTNV; accession no. NP_941978.1), <i>Sigmodontinae</i> (ANDV; accession no. AAO86638.1), and also hantaviruses harbored in shrews, moles and bats from the families <i>Soricidae</i> (Thottapalayam virus; accession no. ABU82619.1), <i>Talpidae</i> (Asama virus; accession no. ACI28508.1), and <i>Rhinolophidae</i> (Longquan virus; accession no. AGI62348.1). The regions spanned by recombinant domains and peptides are indicated schematically below the logo representation by DIII, R1, R2, R2.1 and R2.2. (D) Sequence logo representation of a sequence alignment from the partial stem region of ANDV and PUUV Gc.</p

Characterization of recombinant DIII proteins.

<p>(A) Analysis of purified ANDV DIII by size-exclusion chromatography. The figure depicts the elution profile at 280 nm, and indicates the void volume and the elution volume of molecular size markers. The insert shows the analysis of an aliquot of the fraction corresponding to the chromatography peak, through SDS-PAGE followed by Coomassie staining. (B) Comparison of DIII proteins left untreated (-) or reduced (+) with 50 mM dithiothreitol followed by alkylation and western blot analysis. (C) Circular dichroism spectrum of ANDV DIII. The spectrum was acquired from 5 scans between 190 and 260 nm.</p

Exogenous DIII and stem fragments block the virus-cell membrane fusion process.

<p>(A) Incubation for 1 h of Vero E6 cells with exogenous DIII and stem fragments at 10 and 20 μM, respectively, followed by washing and subsequent inoculation with ANDV. (B) Inhibition of the fusion of ANDV with the plasma membrane by exogenous DIII and stem fragments. The Gc fragments or controls were added during the 5 min low pH incubation step. The statistical evaluation of each data point was performed in relation to the Mock treatment: ***, P < 0.00025; **, P < 0.0025; *, P < 0.025; ns, not significant.</p

Exogenous DIII and stem fragments interfere with the formation of a stable post-fusion Gc trimer.

<p>(A) Low pH-induced multimerization changes of ANDV Gc in the presence or absence of exogenous DIII assessed by sucrose gradient sedimentation. ANDV was incubated for 30 min at pH 7.4 or pH 5.5, with or without ANDV DIII (6 μM). The presence of Gc in each fraction of the gradient was assessed by western blot analysis. The top panel indicates the sedimentation of molecular mass markers. (B) Gc homotrimer stability in the presence or absence of exogenous DIII and stem fragment R2. ANDV VLPs were incubated at the indicated pH and DIII (6 μM) or R2 (20 μM) added before or after acidification. After back-neutralization, the VLPs were treated with trypsin for 30 min and the digestion of Gc was assessed by western blot analysis. The trypsin resistance of Gc was quantified by densitometry (n = 3) and the statistical evaluation performed in relation to the input:***, P < 0.00025; **, P < 0.0025; *, P < 0.025; ns, not significant.</p