Filamin B restricts vaccinia virus spread and is targeted by vaccinia virus protein C4.

Vaccinia virus (VACV) is a large DNA virus that encodes scores of proteins that modulate the host immune response. VACV protein C4 is one such immunomodulator known to inhibit the activation of both the NF-κB signaling cascade and the DNA-PK-mediated DNA sensing pathway. Here, we show that the N-terminal region of C4, which neither inhibits NF-κB nor mediates interaction with DNA-PK, still contributes to virus virulence. Furthermore, this domain interacts directly and with high affinity to the C-terminal domain of filamin B (FLNB). FLNB is a large actin-binding protein that stabilizes the F-actin network and is implicated in other cellular processes. Deletion of FLNB from cells results in larger VACV plaques and increased infectious viral yield, indicating that FLNB restricts VACV spread. These data demonstrate that C4 has a new function that contributes to virulence and engages the cytoskeleton. Furthermore, we show that the cytoskeleton performs further previously uncharacterized functions during VACV infection. IMPORTANCE: Vaccinia virus (VACV), the vaccine against smallpox and monkeypox, encodes many proteins to counteract the host immune response. Investigating these proteins provides insights into viral immune evasion mechanisms and thereby indicates how to engineer safer and more immunogenic VACV-based vaccines. Here, we report that the N-terminal domain of VACV protein C4 interacts directly with the cytoskeletal protein filamin B (FLNB), and this domain of C4 contributes to virus virulence. Furthermore, VACV replicates and spreads better in cells lacking FLNB, thus demonstrating that FLNB has antiviral activity. VACV utilizes the cytoskeleton for movement within and between cells; however, previous studies show no involvement of C4 in VACV replication or spread. Thus, C4 associates with FLNB for a different reason, suggesting that the cytoskeleton has further uncharacterized roles during virus infection.

Fungal Ice2p is in the same superfamily as SERINCs, restriction factors for HIV and other viruses.

Ice2p is an integral endoplasmic reticulum (ER) membrane protein in budding yeast S. cerevisiae named ICE because it is required for Inheritance of Cortical ER. Ice2p has also been reported to be involved in an ER metabolic branch-point that regulates the flux of lipid either to be stored in lipid droplets or to be used as membrane components. Alternately, Ice2p has been proposed to act as a tether that physically bridges the ER at contact sites with both lipid droplets and the plasma membrane via a long loop on the protein's cytoplasmic face that contains multiple predicted amphipathic helices. Here we carried out a bioinformatic analysis to increase understanding of Ice2p. First, regarding topology, we found that diverse members of the fungal Ice2 family have 10 transmembrane helices (TMHs), which places the long loop on the exofacial face of Ice2p, where it cannot form inter-organelle bridges. Second, we identified Ice2p as a full-length homolog of SERINC (serine incorporator), a family of proteins with 10 TMHs found universally in eukaryotes. Since SERINCs are potent restriction factors for HIV and other viruses, study of Ice2p may reveal functions or mechanisms that shed light on viral restriction by SERINCs.

RNA Exosome Complex Regulates Stability of the Hepatitis B Virus X-mRNA Transcript in a Non-stop-mediated (NSD) RNA Quality Control Mechanism.

Hepatitis B virus (HBV) is a stealth virus, minimally inducing the interferon system required for efficient induction of both innate and adaptive immune responses. However, 90% of acutely infected adults can clear the virus, suggesting the presence of other, interferon-independent pathways leading to viral clearance. Given the known ability of helicases to bind viral nucleic acids, we performed a functional screening assay to identify helicases that regulate HBV replication. We identified the superkiller viralicidic activity 2-like (SKIV2L) RNA helicase (a homolog of the Saccharomyces cerevisiae Ski2 protein) on the basis of its direct and preferential interaction with HBV X-mRNA. This interaction was essential for HBV X-mRNA degradation at the RNA exosome. The degradation of HBV X-mRNA at the RNA exosome was also mediated by HBS1L (HBS1-like translational GTPase) protein, a known component of the host RNA quality control system. We found that the redundant HBV-precore translation initiation site present at the 3'-end of HBV X-mRNA (3' precore) is translationally active. The initiation of translation from this site without a proper stop codon was identified by the non-stop-mediated RNA decay mechanism leading to its degradation. Although 3' precore is present in the five main HBV-RNA transcripts, only X-mRNA lacks the presence of an upstream start codons for large, middle, and small (L, M, and S) HBV surface proteins. These upstream codons are in-frame with 3' precore translation initiation site, blocking its translation from the other HBV-mRNA transcripts. To our knowledge, this is the first demonstration of the anti-viral function of the non-stop-mediated RNA decay mechanism.

Disruption of Transmembrane Phosphatidylserine Asymmetry by HIV-1 Incorporated SERINC5 Is Not Responsible for Virus Restriction.

Host restriction factor SERINC5 (SER5) incorporates into the HIV-1 membrane and inhibits infectivity by a poorly understood mechanism. Recently, SER5 was found to exhibit scramblase-like activity leading to the externalization of phosphatidylserine (PS) on the viral surface, which has been proposed to be responsible for SER5's antiviral activity. This and other reports that document modulation of HIV-1 infectivity by viral lipid composition prompted us to investigate the role of PS in regulating SER5-mediated HIV-1 restriction. First, we show that the level of SER5 incorporation into virions correlates with an increase in PS levels in the outer leaflet of the viral membrane. We developed an assay to estimate the PS distribution across the viral membrane and found that SER5, but not SER2, which lacks antiviral activity, abrogates PS asymmetry by externalizing this lipid. Second, SER5 incorporation diminished the infectivity of pseudoviruses produced from cells lacking a flippase subunit CDC50a and, therefore, exhibited a higher baseline level of surface-accessible PS. Finally, exogenous manipulation of the viral PS levels utilizing methyl-alpha-cyclodextrin revealed a lack of correlation between external PS and virion infectivity. Taken together, our study implies that the increased PS exposure to SER5-containing virions itself is not directly linked to HIV-1 restriction.

Dual role of the chromatin-binding factor PHF13 in the pre- and post-integration phases of HIV-1 replication.

Viruses interact with multiple host cell factors. Some of these are required to promote viral propagation, others have roles in inhibiting infection. Here, we delineate the function of the cellular factor PHF13 (or SPOC1), a putative HIV-1 restriction factor. Early in the HIV-1 replication cycle PHF13 increased the number of integrated proviral copies and the number of infected cells. However, after HIV-1 integration, high levels of PHF13 suppressed viral gene expression. The antiviral activity of PHF13 is counteracted by the viral accessory protein Vpr, which mediates PHF13 degradation. Altogether, the transcriptional master regulator and chromatin binding protein PHF13 does not have purely repressive effects on HIV-1 replication, but also promotes viral integration. By the functional characterization of the dual role of PHF13 during the HIV-1 replication cycle, we reveal a surprising and intricate mechanism through which HIV-1 might regulate the switch from integration to viral gene expression. Furthermore, we identify PHF13 as a cellular target specifically degraded by HIV-1 Vpr.