Modulation of Zika virus replication via glycosphingolipids.

The enveloped positive-sense RNA viruses including Zika virus (ZIKV) need host lipids to successfully replicate. The nature of the lipids and the replication step(s) where lipids are utilized often vary amongst viruses. In this study, we demonstrate that ZIKV particle envelope is significantly enriched in distinct sphingolipid species. To determine the role of sphingolipids in ZIKV replication, we leveraged a panel of sphingolipid-deficient cell lines. Notably, knockout of glucosylceramide and lactosylceramide synthase encoding genes (GCSKO; B4G5KO) resulted in a marked decrease in ZIKV titers. GCSKO or pharmacological inhibition of GCS also led to a significant decrease in ZIKV genome replication. Further analysis indicated that GCSKO reduced intracellular virus titers but had minimal impact on ZIKV binding. Restoration of B4G5 expression in B4G5KO cells or supplementing PDMP-treated cells with glucosylceramide led to a significant rescue of ZIKV replication. Altogether, our findings suggest that ZIKV needs glycosphingolipids to facilitate virus replication.

Sphingolipids as Potential Therapeutic Targets against Enveloped Human RNA Viruses.

Several notable human diseases are caused by enveloped RNA viruses: influenza, AIDS, hepatitis C, dengue hemorrhagic fever, microcephaly, and Guillain-Barré Syndrome. Being enveloped, the life cycle of this group of viruses is critically dependent on host lipid biosynthesis. Viral binding and entry involve interactions between viral envelope glycoproteins and cellular receptors localized to lipid-rich regions of the plasma membrane. Subsequent infection by these viruses leads to reorganization of cellular membranes and lipid metabolism to support the production of new viral particles. Recent work has focused on defining the involvement of specific lipid classes in the entry, genome replication assembly, and viral particle formation of these viruses in hopes of identifying potential therapeutic targets for the treatment or prevention of disease. In this review, we will highlight the role of host sphingolipids in the lifecycle of several medically important enveloped RNA viruses.

Hepatitis C Virus NS4B Can Suppress STING Accumulation To Evade Innate Immune Responses.

UNLABELLED: The cyclic dinucleotide 2',3'-cGAMP can bind the adaptor protein STING (stimulator of interferon [IFN] genes) to activate the production of type I IFNs and proinflammatory cytokines. We found that cGAMP added to the culture medium could suppress the replication of the hepatitis C virus (HCV) genotype 1b strain Con1 subgenomic replicon in human hepatoma cells. Knockdown of STING expression diminished the inhibitory effect on replicon replication, while overexpression of STING enhanced the inhibitory effects of cGAMP. The addition of cGAMP into 1b/Con1 replicon cells significantly increased the expression of type I IFNs and antiviral interferon-stimulated genes. Unexpectedly, replication of the genotype 2a JFH1 replicon and infectious JFH1 virus was less sensitive to the inhibitory effect of cGAMP than was that of 1b/Con1 replicon. Using chimeric replicons, 2a NS4B was identified to confer resistance to cGAMP. Transient expression of 2a NS4B resulted in a pronounced inhibitory effect on STING-mediated beta IFN (IFN-ß) reporter activation compared to that of 1b NS4B. 2a NS4B was found to suppress STING accumulation in a dose-dependent manner. The predicted transmembrane domain of 2a NS4B was required to inhibit STING accumulation. These results demonstrate a novel genotype-specific inhibition of the STING-mediated host antiviral immune response. IMPORTANCE: The cyclic dinucleotide cGAMP was found to potently inhibit the replication of HCV genotype 1b Con1 replicon but was less effective for the 2a/JFH1 replicon and infectious JFH1 virus. The predicted transmembrane domain in 2a NS4B was shown to be responsible for the decreased sensitivity to cGAMP. The N terminus of NS4B has been reported to suppress STING-mediated signaling by disrupting the interaction of STING and TBK1 and/or MAVS. We show that 2a/JFH1 NS4B has an additional mechanism to evade STING signaling through suppressing STING accumulation.

Lipids and RNA virus replication.

Most viruses rely heavily on their host machinery to successfully replicate their genome and produce new virus particles. Recently, the interaction of positive-strand RNA viruses with the lipid biosynthetic and transport machinery has been the subject of intense investigation. In this review, we will discuss the contribution of various host lipids and related proteins in RNA virus replication and maturation.

Modulation of hepatitis C virus genome replication by glycosphingolipids and four-phosphate adaptor protein 2.

UNLABELLED: Hepatitis C virus (HCV) assembles its replication complex on cytosolic membrane vesicles often clustered in a membranous web (MW). During infection, HCV NS5A protein activates PI4KIIIα enzyme, causing massive production and redistribution of phosphatidylinositol 4-phosphate (PI4P) lipid to the replication complex. However, the role of PI4P in the HCV life cycle is not well understood. We postulated that PI4P recruits host effectors to modulate HCV genome replication or virus particle production. To test this hypothesis, we generated cell lines for doxycycline-inducible expression of short hairpin RNAs (shRNAs) targeting the PI4P effector, four-phosphate adaptor protein 2 (FAPP2). FAPP2 depletion attenuated HCV infectivity and impeded HCV RNA synthesis. Indeed, FAPP2 has two functional lipid-binding domains specific for PI4P and glycosphingolipids. While expression of the PI4P-binding mutant protein was expected to inhibit HCV replication, a marked drop in replication efficiency was observed unexpectedly with the glycosphingolipid-binding mutant protein. These data suggest that both domains are crucial for the role of FAPP2 in HCV genome replication. We also found that HCV significantly increases the level of some glycosphingolipids, whereas adding these lipids to FAPP2-depleted cells partially rescued replication, further arguing for the importance of glycosphingolipids in HCV RNA synthesis. Interestingly, FAPP2 is redistributed to the replication complex (RC) characterized by HCV NS5A, NS4B, or double-stranded RNA (dsRNA) foci. Additionally, FAPP2 depletion disrupts the RC and alters the colocalization of HCV replicase proteins. Altogether, our study implies that HCV coopts FAPP2 for virus genome replication via PI4P binding and glycosphingolipid transport to the HCV RC. IMPORTANCE: Like most viruses with a positive-sense RNA genome, HCV replicates its RNA on remodeled host membranes composed of lipids hijacked from various internal membrane compartments. During infection, HCV induces massive production and retargeting of the PI4P lipid to its replication complex. However, the role of PI4P in HCV replication is not well understood. In this study, we have shown that FAPP2, a PI4P effector and glycosphingolipid-binding protein, is recruited to the HCV replication complex and is required for HCV genome replication and replication complex formation. More importantly, this study demonstrates, for the first time, the crucial role of glycosphingolipids in the HCV life cycle and suggests a link between PI4P and glycosphingolipids in HCV genome replication.

A human proteome microarray identifies that the heterogeneous nuclear ribonucleoprotein K (hnRNP K) recognizes the 5' terminal sequence of the hepatitis C virus RNA.

Stem-loop I (SL1) located in the 5' untranslated region of the hepatitis C virus (HCV) genome initiates binding to miR-122, a microRNA required for hepatitis HCV replication. However, proteins that bind SL1 remain elusive. In this study, we employed a human proteome microarray, comprised of ∼17,000 individually purified human proteins in full-length, and identified 313 proteins that recognize HCV SL1. Eighty-three of the identified proteins were annotated as liver-expressing proteins, and twelve of which were known to be associated with hepatitis virus. siRNA-induced silencing of eight out of 12 candidate genes led to at least 25% decrease in HCV replication efficiency. In particular, knockdown of heterogeneous nuclear ribonucleoprotein K (hnRNP K) reduced HCV replication in a concentration-dependent manner. Ultra-violet-crosslinking assay also showed that hnRNP K, which functions in pre-mRNA processing and transport, showed the strongest binding to the HCV SL1. We observed that hnRNP K, a nuclear protein, is relocated in the cytoplasm in HCV-expressing cells. Immunoprecipitation of the hnRNP K from Huh7.5 cells stably expressing HCV replicon resulted in the co-immunoprecipitation of SL1. This work identifies a cellular protein that could have an important role in the regulation of HCV RNA gene expression and metabolism.

Modulation of hepatitis C virus genome encapsidation by nonstructural protein 4B.

Hepatitis C Virus (HCV) NS4B protein has many roles in HCV genome replication. Recently, our laboratory (Q. Han, J. Aligo, D. Manna, K. Belton, S. V. Chintapalli, Y. Hong, R. L. Patterson, D. B. van Rossum, and K. V. Konan, J. Virol. 85:6464-6479, 2011) and others (D. M. Jones, A. H. Patel, P. Targett-Adams, and J. McLauchlan, J. Virol. 83:2163-2177, 2009; D. Paul, I. Romero-Brey, J. Gouttenoire, S. Stoitsova, J. Krijnse-Locker, D. Moradpour, and R. Bartenschlager, J. Virol. 85:6963-6976, 2011) have also reported NS4B's function in postreplication steps. Indeed, replacement of the NS4B C-terminal domain (CTD) in the HCV JFH1 (genotype 2a [G2a]) genome with sequences from Con1 (G1b) or H77 (G1a) had a negligible impact on JFH1 genome replication but attenuated virus production. Since NS4B interacts weakly with the HCV genome, we postulated that NS4B regulates the function of host or virus proteins directly involved in HCV production. In this study, we demonstrate that the integrity of the JFH1 NS4B CTD is crucial for efficient JFH1 genome encapsidation. Further, two adaptive mutations (NS4B N216S and NS5A C465S) were identified, and introduction of these mutations into the chimera rescued virus production to various levels, suggesting a genetic interaction between the NS4B and NS5A proteins. Interestingly, cells infected with chimeric viruses displayed a markedly decreased NS5A hyperphosphorylation state (NS5A p58) relative to JFH1, and the adaptive mutations differentially rescued NS5A p58 formation. However, immunofluorescence staining indicated that the decrease in NS5A p58 did not alter NS5A colocalization with the core around lipid droplets (LDs), the site of JFH1 assembly, suggesting that NS5A fails to facilitate the transfer of HCV RNA to the capsid protein on LDs. Alternatively, NS4B's function in HCV genome encapsidation may entail more than its regulation of the NS5A phosphorylation state.

Conserved GXXXG- and S/T-like motifs in the transmembrane domains of NS4B protein are required for hepatitis C virus replication.

Hepatitis C virus (HCV) nonstructural protein 4B (NS4B) is an integral membrane protein, which plays an important role in the organization and function of the HCV replication complex (RC). Although much is understood about its amphipathic N-terminal and C-terminal domains, we know very little about the role of the transmembrane domains (TMDs) in NS4B function. We hypothesized that in addition to anchoring NS4B into host membranes, the TMDs are engaged in intra- and intermolecular interactions required for NS4B structure/function. To test this hypothesis, we have engineered a chimeric JFH1 genome containing the Con1 NS4B TMD region. The resulting virus titers were greatly reduced from those of JFH1, and further analysis indicated a defect in genome replication. We have mapped this incompatibility to NS4B TMD1 and TMD2 sequences, and we have defined putative TMD dimerization motifs (GXXXG in TMD2 and TMD3; the S/T cluster in TMD1) as key structural/functional determinants. Mutations in each of the putative motifs led to significant decreases in JFH1 replication. Like most of the NS4B chimeras, mutant proteins had no negative impact on NS4B membrane association. However, some mutations led to disruption of NS4B foci, implying that the TMDs play a role in HCV RC formation. Further examination indicated that the loss of NS4B foci correlates with the destabilization of NS4B protein. Finally, we have identified an adaptive mutation in the NS4B TMD2 sequence that has compensatory effects on JFH1 chimera replication. Taken together, these data underscore the functional importance of NS4B TMDs in the HCV life cycle.

Autophagy protein ATG5 interacts transiently with the hepatitis C virus RNA polymerase (NS5B) early during infection.

Autophagy is an important cellular process by which ATG5 initiates the formation of double membrane vesicles (DMVs). Upon infection, DMVs have been shown to harbor the replicase complex of positive-strand RNA viruses such as MHV, poliovirus, and equine arteritis virus. Recently, it has been shown that autophagy proteins are proviral factors that favor initiation of hepatitis C virus (HCV) infection. Here, we identified ATG5 as an interacting protein for the HCV NS5B. ATG5/NS5B interaction was confirmed by co-IP and metabolic labeling studies. Furthermore, ATG5 protein colocalizes with NS4B, a constituent of the membranous web. Importantly, immunofluorescence staining demonstrated a strong colocalization of ATG5 and NS5B within perinuclear regions of infected cells at 2 days postinfection. However, colocalization was completely lacking at 5DPI, suggesting that HCV utilizes ATG5 as a proviral factor during the onset of viral infection. Finally, inhibition of autophagy through ATG5 silencing blocks HCV replication.

Endocytic Rab proteins are required for hepatitis C virus replication complex formation.

During infection, hepatitis C virus (HCV) NS4B protein remodels host membranes to form HCV replication complexes (RC) which appear as foci under fluorescence microscopy (FM). To understand the role of Rab proteins in forming NS4B foci, cells expressing the HCV replicon were examined biochemically and via FM. First, we show that an isolated NS4B-bound subcellular fraction is competent for HCV RNA synthesis. Further, this fraction is differentially enriched in Rab1, 2, 5, 6 and 7. However, when examined via FM, NS4B foci appear to be selectively associated with Rab5 and Rab7 proteins. Additionally, dominant negative (DN) Rab6 expression impairs Rab5 recruitment into NS4B foci. Further, silencing of Rab5 or Rab7 resulted in a significant decrease in HCV genome replication. Finally, expression of DN Rab5 or Rab7 led to a reticular NS4B subcellular distribution, suggesting that endocytic proteins Rab5 and Rab7, but not Rab11, may facilitate NS4B foci formation.

Bovine viral diarrhea virus NS4B protein is an integral membrane protein associated with Golgi markers and rearranged host membranes.

BACKGROUND: Very little is known about BVDV NS4B, a protein of approximately 38 kDa. However, a missense mutation in NS4B has been implicated in changing BVDV from a cytopathic to noncytopathic virus, suggesting that NS4B might play a role in BVDV pathogenesis. Though this is one possible function, it is also likely that NS4B plays a role in BVDV genome replication. For example, BVDV NS4B interacts with NS3 and NS5A, implying that NS4B is part of a complex, which contains BVDV replicase proteins. Other possible BVDV NS4B functions can be inferred by analogy to hepatitis C virus (HCV) NS4B protein. For instance, HCV NS4B remodels host membranes to form the so-called membranous web, the site for HCV genome replication. Finally, HCV NS4B is membrane-associated, implying that HCV NS4B may anchor the virus replication complex to the membranous web structure. Unlike its HCV counterpart, we know little about the subcellular distribution of BVDV NS4B protein. Further, it is not clear whether NS4B is localized to host membrane alterations associated with BVDV infection. RESULTS: We show first that release of infectious BVDV correlates with the kinetics of BVDV genome replication in infected cells. Secondly, we found that NS4B subcellular distribution changes over the course of BVDV infection. Further, BVDV NS4B is an integral membrane protein, which colocalizes mainly with the Golgi compartment when expressed alone or in the context of BVDV infection. Additionally, BVDV induces host membrane rearrangement and these membranes contain BVDV NS4B protein. Finally, NS4B colocalizes with replicase proteins NS5A and NS5B proteins, raising the possibility that NS4B is a component of the BVDV replication complex. Interestingly, NS4B was found to colocalize with mitochondria suggesting that this organelle might play a role in BVDV genome replication or cytopathogenicity. CONCLUSION: These results show that BVDV NS4B is an integral membrane protein associated with the Golgi apparatus and virus-induced membranes, the putative site for BVDV genome replication. On the basis of NS4B Colocalization with NS5A and NS5B, we conclude that NS4B protein is an integral component of the BVDV replication complex.

Formation and function of hepatitis C virus replication complexes require residues in the carboxy-terminal domain of NS4B protein.

During replication, hepatitis C virus (HCV) NS4B protein rearranges intracellular membranes to form foci, or the web, the putative site for HCV replication. To understand the role of the C-terminal domain (CTD) in NS4B function, mutations were introduced into NS4B alone or in the context of HCV polyprotein. First, we show that the CTD is required for NS4B-induced web structure, but it is not sufficient to form the web nor is it required for NS4B membrane association. Interestingly, all the mutations introduced into the CTD impeded HCV genome replication, but only two resulted in a disruption of NS4B foci. Further, we found that NS4B interacts with NS3 and NS5A, and that mutations causing NS4B mislocalization have a similar effect on these proteins. Finally, we show that the redistribution of Rab5 to NS4B foci requires an intact CTD, suggesting that Rab5 facilitates NS4B foci formation through interaction with the CTD.

Participation of rab5, an early endosome protein, in hepatitis C virus RNA replication machinery.

Like most positive-strand RNA viruses, hepatitis C virus (HCV) is believed to replicate its genome on the surface of rearranged membranes. We have shown previously that HCV NS4AB, but not the product NS4B, inhibits endoplasmic reticulum (ER)-to-Golgi protein traffic (K. V. Konan, T. H. Giddings, Jr., M. Ikeda, K. Li, S. M. Lemon, and K. Kirkegaard, J. Virol. 77:7843-7855). However, both NS4AB and NS4B can induce "membranous web" formation, first reported by Egger et al. (D. B Egger, R. Gosert, L. Bianchi, H. E. Blum, D. Moradpour, and K. Bienz, J. Virol. 76:5974-5984), which is also observed in HCV-infected cells (Y. Rouille, F. Helle, D. Delgrange, P. Roingeard, C. Voisset, E. Blanchard, S. Belouzard, J. McKeating, A. H. Patel, G. Maertens, T. Wakita, C. Wychowski, and J. Dubuisson, J. Virol. 80:2832-2841) and cells that bear a subgenomic NS5A-green fluorescent protein (GFP) replicon (D. Moradpour, M. J. Evans, R. Gosert, Z. Yuan, H. E. Blum, S. P. Goff, B. D. Lindenbach, and C. M. Rice, J. Virol. 78:7400-7409). To determine the intracellular origin of the web, we examined NS4B colocalization with endogenous cellular markers in the context of the full-length or subgenomic replicon. We found that, in addition to ER markers, early endosome (EE) proteins, including Rab5, were associated with web-inducing protein NS4B. Furthermore, an immunoisolated fraction containing NS4B was found to contain both ER and EE proteins. Using fluorescence microscopy, we showed that wild-type and constitutively active Rab5 proteins were associated with NS4B. Interestingly, expression of dominant-negative Rab5 resulted in significant loss of GFP fluorescence in NS5A-GFP replicon cells. We also found that a small reduction in Rab5 protein expression decreased HCV RNA synthesis significantly. Furthermore, transfection of labeled Rab5 small interfering RNAs into NS5A-GFP replicon cells resulted in a significant decrease in GFP fluorescence. Finally, Rab5 protein was found to coimmunoprecipitate with HCV NS4B. These studies suggest that EE proteins, including Rab5, may play a role in HCV genome replication or web formation.

Influenza virus infection increases p53 activity: role of p53 in cell death and viral replication.

The induction of apoptotic cell death is a hallmark of influenza virus infection. Although a variety of cellular and viral proteins have been implicated in this process, to date no conserved cellular pathway has been identified. In this study, we report that the tumor suppressor protein p53 is essential for the induction of cell death in influenza virus-infected cells. In primary human lung cells, influenza virus increased p53 protein levels. This was also noted in the human lung cell line A549, along with the up-regulation of p53-dependent gene transcription. Reduction of p53 activity in A549 cells inhibited influenza virus-induced cell death as measured by trypan blue exclusion and caspase activity. These findings were not cell type specific. Influenza virus-induced cell death was absent in mouse embryo fibroblasts isolated from p53 knockout mice, which was not the case in wild-type mouse embryo fibroblasts, suggesting that p53 is a common cellular pathway leading to influenza virus-induced cell death. Surprisingly, inhibiting p53 activity led to elevated virus replication. Mechanistically, this may be due to the decrease in interferon signaling in p53-deficient cells, suggesting that functional p53 is involved in the interferon response to influenza infection. To our knowledge, these are the first studies demonstrating that p53 is involved in influenza virus-induced cell death and that inhibiting p53 leads to increased viral titers, potentially through modulation of the interferon response.

Nonstructural protein precursor NS4A/B from hepatitis C virus alters function and ultrastructure of host secretory apparatus.

The nonstructural proteins of hepatitis C virus (HCV) have been shown previously to localize to the endoplasmic reticulum (ER) when expressed singly or in the context of other HCV proteins. To determine whether the expression of HCV nonstructural proteins alters ER function, we tested the effect of expression of NS2/3/4A, NS4A, NS4B, NS4A/B, NS4B/5A, NS5A, and NS5B from genotype 1b HCV on anterograde traffic from the ER to the Golgi apparatus. Only the nominal precursor protein NS4A/B affected the rate of ER-to-Golgi traffic, slowing the rate of Golgi-specific modification of the vesicular stomatitis virus G protein expressed by transfection by approximately threefold. This inhibition of ER-to-Golgi traffic was not observed upon expression of the processed proteins NS4A and NS4B, singly or in combination. To determine whether secretion of other cargo proteins was inhibited by NS4A/B expression, we monitored the appearance of newly synthesized proteins on the cell surface in the presence and absence of NS4A/B expression; levels of all were reduced in the presence of NS4A/B. This reduction is also seen in cells that contain genome length HCV replicons: the rate of appearance of major histocompatibility complex class I (MHC-I) on the cell surface was reduced by three- to fivefold compared to that for a cured cell line. The inhibition of protein secretion caused by NS4A/B does not correlate with the ultrastructural changes leading to the formation a "membranous web" (D. Egger et al., J. Virol. 76:5974-5984, 2002), which can be caused by expression of NS4B alone. Inhibition of global ER-to-Golgi traffic could, by reducing cytokine secretion, MHC-I presentation, and transport of labile membrane proteins to the cell surface, have significant effects on the host immune response to HCV infection.