Intracellular Membrane Rearrangements Research Articles

Nonstructural protein 4 of human norovirus self-assembles into various membrane-bridging multimers

Single-stranded, positive-sense RNA ((+)RNA) viruses replicate their genomes in virus-induced intracellular membrane compartments. (+)RNA viruses dedicate a significant part of their small genomes (a few thousands to a few tens of thousands of bases) to the generation of these compartments by encoding membrane-interacting proteins and/or protein domains. Noroviruses are a very diverse genus of (+)RNA viruses including human and animal pathogens. Human noroviruses are the major cause of acute gastroenteritis worldwide, with genogroup II genotype 4 (GII.4) noroviruses accounting for the vast majority of infections. Three viral proteins encoded in the N-terminus of the viral replication polyprotein direct intracellular membrane rearrangements associated with norovirus replication. Of these three, nonstructural protein 4 (NS4) seems to be the most important, although its exact functions in replication organelle formation are unknown. Here we produce, purify and characterize GII.4 NS4. AlphaFold modeling combined with experimental data refine and correct our previous crude structural model of NS4. Using simple artificial liposomes, we report an extensive characterization of the membrane properties of NS4. We find that NS4 self-assembles and thereby bridges liposomes together. Cryo-EM, NMR and membrane flotation show formation of several distinct NS4 assemblies, at least two of them bridging pairs of membranes together in different fashions. Noroviruses belong to (+)RNA viruses whose replication compartment is extruded from the target endomembrane and generates double-membrane vesicles. Our data establish that the 21-kDa GII.4 human norovirus NS4 can, in the absence of any other factor, recapitulate in tubo several features, including membrane apposition, that occur in such processes.

Oligomeric assembly of the C-terminal and transmembrane region of SARS-CoV-2 nsp3.

Membrane remodeling is at the heart of intracellular replication for single-stranded, positive-sense RNA viruses. In the case of coronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), this leads to the formation of a network of double-membrane vesicles (DMVs). Targeting DMV biogenesis offers promising prospects for antiviral therapies. This requires a better understanding of the molecular mechanisms and proteins involved. Three non-structural proteins (nsp3, nsp4, and nsp6) direct the intracellular membrane rearrangements upon SARS-CoV-2 infection. All of them contain transmembrane helices. The nsp3 component, the largest and multi-functional protein of the virus, plays an essential role in this process. Aiming to understand its structural organization, we used a cell-free protein synthesis assay to produce and reconstitute the C-terminal part of nsp3 (nsp3C) including transmembrane domains into phospholipid nanodiscs. Our work reveals the oligomeric organization of one key player in the biogenesis of SARS-CoV-2 DMVs, providing basis for the design of future antiviral strategies.

PACSIN2 Orchestrates Actin Reorganization during Megakaryocyte Maturation

Thrombocytopenia, defined by a platelet count below 150,000/µL, poses significant mortality risks, primarily due to bleeding. Blood platelets are produced by bone marrow megakaryocytes (MKs) in unique cellular processes that require polyploidization and extensive intracellular membrane rearrangements leading to the formation of the demarcation membrane system (DMS), the surface-connected membrane reservoir for future platelets. The membrane-deforming F-BAR protein PACSIN2 is one of the most abundant and conserved BAR proteins in platelets, and exomechip analysis and genome-wide association studies have associated PACSIN2 single nucleotide variants with platelet count and mean platelet volume in humans. Our previous work has shown that PACSIN2 localizes with the initiating DMS in MKs in a process regulated by the cytoskeletal and scaffolding protein filamin A (FlnA). Recently, we have found that Pacsin2 -/- mice display mild thrombocytopenia with slightly enlarged platelets and severe thrombus formation defects due to increased activation of platelet integrin β1. Here we investigated the role of PACSIN2 in MK maturation and platelet production. Cultured fetal liver cell-derived Pacsin2 -/- MKs formed proplatelets normally compared to controls. Pacsin2 -/- bone marrow sections displayed MK count comparable to control mice, and freshly isolated bone marrow Pacsin2 -/- MKs had normal ploidy, indicating normal MK maturation. The observed effects of PACSIN2 deficiency led to a defective formation of the DMS within MKs. In PACSIN2 deficient MKs, the DMS exhibited a less distinct structure, making it challenging to delineate clear platelet territories. Interestingly, the lack of PACSIN2 also impacted the spatial distribution of GPIbα and integrin β1 membrane receptors. These receptors, typically dispersed within the cytoplasm in normal MKs, were concentrated at the cell periphery in Pacsin2 -/-MKs. A similar relocation was observed for polymerized actin. This finding underscores the pivotal role of PACSIN2 in actin reorganization within MKs, which in turn influences the rearrangement of GPIbα and integrin β1. The data imply that receptor and actin cytoskeleton rearrangements are critical to DMS formation and subsequent platelet production in vivo.

Metabolic reprogramming by Zika virus provokes inflammation in human placenta

The recent outbreak of Zika virus (ZIKV) was associated with birth defects and pregnancy loss when maternal infection occurs in early pregnancy, but specific mechanisms driving placental insufficiency and subsequent ZIKV-mediated pathogenesis remain unclear. Here we show, using large scale metabolomics, that ZIKV infection reprograms placental lipidome by impairing the lipogenesis pathways. ZIKV-induced metabolic alterations provide building blocks for lipid droplet biogenesis and intracellular membrane rearrangements to support viral replication. Furthermore, lipidome reprogramming by ZIKV is paralleled by the mitochondrial dysfunction and inflammatory immune imbalance, which contribute to placental damage. In addition, we demonstrate the efficacy of a commercially available inhibitor in limiting ZIKV infection, provides a proof-of-concept for blocking congenital infection by targeting metabolic pathways. Collectively, our study provides mechanistic insights on how ZIKV targets essential hubs of the lipid metabolism that may lead to placental dysfunction and loss of barrier function.

Hepatitis C Virus Replication Depends on Endosomal Cholesterol Homeostasis.

Similar to other positive-strand RNA viruses, hepatitis C virus (HCV) causes massive rearrangements of intracellular membranes, resulting in a membranous web (MW) composed of predominantly double-membrane vesicles (DMVs), the presumed sites of RNA replication. DMVs are enriched for cholesterol, but mechanistic details on the source and recruitment of cholesterol to the viral replication organelle are only partially known. Here we focused on selected lipid transfer proteins implicated in direct lipid transfer at various endoplasmic reticulum (ER)-membrane contact sites. RNA interference (RNAi)-mediated knockdown identified several hitherto unknown HCV dependency factors, such as steroidogenic acute regulatory protein-related lipid transfer domain protein 3 (STARD3), oxysterol-binding protein-related protein 1A and -B (OSBPL1A and -B), and Niemann-Pick-type C1 (NPC1), all residing at late endosome and lysosome membranes and required for efficient HCV RNA replication but not for replication of the closely related dengue virus. Focusing on NPC1, we found that knockdown or pharmacological inhibition caused cholesterol entrapment in lysosomal vesicles concomitant with decreased cholesterol abundance at sites containing the viral replicase factor NS5A. In untreated HCV-infected cells, unesterified cholesterol accumulated at the perinuclear region, partially colocalizing with NS5A at DMVs, arguing for NPC1-mediated endosomal cholesterol transport to the viral replication organelle. Consistent with cholesterol being an important structural component of DMVs, reducing NPC1-dependent endosomal cholesterol transport impaired MW integrity. This suggests that HCV usurps lipid transfer proteins, such as NPC1, at ER-late endosome/lysosome membrane contact sites to recruit cholesterol to the viral replication organelle, where it contributes to MW functionality.IMPORTANCE A key feature of the replication of positive-strand RNA viruses is the rearrangement of the host cell endomembrane system to produce a membranous replication organelle (RO). The underlying mechanisms are far from being elucidated fully. In this report, we provide evidence that HCV RNA replication depends on functional lipid transport along the endosomal-lysosomal pathway that is mediated by several lipid transfer proteins, such as the Niemann-Pick type C1 (NPC1) protein. Pharmacological inhibition of NPC1 function reduced viral replication, impaired the transport of cholesterol to the viral replication organelle, and altered organelle morphology. Besides NPC1, our study reports the importance of additional endosomal and lysosomal lipid transfer proteins required for viral replication, thus contributing to our understanding of how HCV manipulates their function in order to generate a membranous replication organelle. These results might have implications for the biogenesis of replication organelles of other positive-strand RNA viruses.

Inhibition of Cytosolic Phospholipase A2α Impairs an Early Step of Coronavirus Replication in Cell Culture.

Coronavirus replication is associated with intracellular membrane rearrangements in infected cells, resulting in the formation of double-membrane vesicles (DMVs) and other membranous structures that are referred to as replicative organelles (ROs). The latter provide a structural scaffold for viral replication/transcription complexes (RTCs) and help to sequester RTC components from recognition by cellular factors involved in antiviral host responses. There is increasing evidence that plus-strand RNA (+RNA) virus replication, including RO formation and virion morphogenesis, affects cellular lipid metabolism and critically depends on enzymes involved in lipid synthesis and processing. Here, we investigated the role of cytosolic phospholipase A2α (cPLA2α) in coronavirus replication using a low-molecular-weight nonpeptidic inhibitor, pyrrolidine-2 (Py-2). The inhibition of cPLA2α activity, which produces lysophospholipids (LPLs) by cleaving at the sn-2 position of phospholipids, had profound effects on viral RNA and protein accumulation in human coronavirus 229E-infected Huh-7 cells. Transmission electron microscopy revealed that DMV formation in infected cells was significantly reduced in the presence of the inhibitor. Furthermore, we found that (i) viral RTCs colocalized with LPL-containing membranes, (ii) cellular LPL concentrations were increased in coronavirus-infected cells, and (iii) this increase was diminished in the presence of the cPLA2α inhibitor Py-2. Py-2 also displayed antiviral activities against other viruses representing the Coronaviridae and Togaviridae families, while members of the Picornaviridae were not affected. Taken together, the study provides evidence that cPLA2α activity is critically involved in the replication of various +RNA virus families and may thus represent a candidate target for broad-spectrum antiviral drug development.IMPORTANCE Examples of highly conserved RNA virus proteins that qualify as drug targets for broad-spectrum antivirals remain scarce, resulting in increased efforts to identify and specifically inhibit cellular functions that are essential for the replication of RNA viruses belonging to different genera and families. The present study supports and extends previous conclusions that enzymes involved in cellular lipid metabolism may be tractable targets for broad-spectrum antivirals. We obtained evidence to show that a cellular phospholipase, cPLA2α, which releases fatty acid from the sn-2 position of membrane-associated glycerophospholipids, is critically involved in coronavirus replication, most likely by producing lysophospholipids that are required to form the specialized membrane compartments in which viral RNA synthesis takes place. The importance of this enzyme in coronavirus replication and DMV formation is supported by several lines of evidence, including confocal and electron microscopy, viral replication, and lipidomics studies of coronavirus-infected cells treated with a highly specific cPLA2α inhibitor.

The autophagy elongation complex (ATG5-12/16L1) positively regulates HCV replication and is required for wild-type membranous web formation

Hepatitis C virus (HCV) infection induces intracellular membrane rearrangements, thus forming a membranous web (MW) in which HCV replication and assembly occur. The HCV-induced MW is primarily composed of double membrane vesicles (DMVs) transfused by multi-membrane vesicles. The autophagy machinery has been proposed to participate in the formation of such vesicles. However, no clear evidence has been found linking autophagy to the formation of these DMVs. In this study, we evaluated the role of the autophagy elongation complex (ATG5-12/16L1) in HCV replication and MW formation. Using a dominant negative form of ATG12 and an siRNA approach, we demonstrated that the ATG5-12 conjugate, but not LC3-II formation, is crucial for efficient viral replication. Furthermore, purification of HCV MW revealed the presence of ATG5-12 and ATG16L1 along with HCV nonstructural proteins. Interestingly, LC3 was not recruited along with the elongation complex to the site of viral replication. Finally, inhibition of the elongation complex, but not LC3, greatly impaired the formation of the wild-type MW phenotype. To our knowledge, this study provides the first evidence of the involvement of autophagy proteins in the formation of wild-type MWs.

Dynamin 2 (DNM2) and PACSIN2 Regulate Megakaryocyte Demarcation Membrane System Formation and Platelet Production in Concert

Blood platelets are produced in the bone marrow by megakaryocytes (MKs) in a process that requires extensive intracellular membrane rearrangements. These include the formation of the demarcation membrane system (DMS), the surface-connected membrane extension that invaginates into the cell body and further develops to provide membranes for future platelets. The precise molecular mechanisms responsible for these unique membrane rearrangements remain poorly understood. We have recently shown that Dnm2fl/fl Pf4-Cre mice specifically lacking the large GTPase dynamin 2 (DNM2) in MKs develop severe macrothrombocytopenia due to impaired receptor-mediated endocytosis (RME) (Bender, Giannini et al. Blood. 2015;125(6):1014-1024). Specifically, Dnm2fl/fl Pf4-Cre MKs accumulate arrested endocytic clathrin-coated vesicles that obstruct DMS formation. The actin nucleating factor Arp2/3 complex and polymerized actin clustered with clathrin at sites of impaired RME in Dnm2fl/fl Pf4-Cre MKs. We hypothesized that a DNM2 partner recruits actin-regulatory proteins at sites of RME and investigated the contribution of the F-BAR protein PACSIN2, an internal component of the initiating DMS (Jurak Begonja, Pluthero et al. Blood. 2015;126(1):80-88), in DMS formation and platelet production, as PACSIN2 interacts with DNM2 and actin-regulatory proteins such as N-WASP and filamin A (FlnA). Pacsin2-/- mice developed mild thrombocytopenia with slightly enlarged and shallow platelets. The DMS appeared less well defined and platelet territories were not readily visualized in Pacsin2-/- MKs. Pacsin2-/- Dnm2fl/fl Pf4-Cre mice lacking both PACSIN2 and DNM2 in MKs were further generated to determine the contribution of PACSIN2 in clathrin and actin clustering in Dnm2fl/fl Pf4-Cre MKs. Strikingly, PACSIN2 genetic deletion significantly improved the severe thrombocytopenia of Dnm2fl/fl Pf4-Cre mice. Specifically, PACSIN2 deletion abrogated the accumulation of clathrin and actin clusters, thereby unclogging DMS formation, which appeared as elongated maze-like membrane tubules in Pacsin2-/- Dnm2fl/fl Pf4-Cre MKs. Our results show that DNM2 terminates PACSIN2-dependent actin polymerization that accompanies RME, thereby allowing membrane rearrangements required for DMS formation. DisclosuresNo relevant conflicts of interest to declare.

Morphological and Biochemical Characterization of the Membranous Hepatitis C Virus Replication Compartment

Like all other positive-strand RNA viruses, hepatitis C virus (HCV) induces rearrangements of intracellular membranes that are thought to serve as a scaffold for the assembly of the viral replicase machinery. The most prominent membranous structures present in HCV-infected cells are double-membrane vesicles (DMVs). However, their composition and role in the HCV replication cycle are poorly understood. To gain further insights into the biochemcial properties of HCV-induced membrane alterations, we generated a functional replicon containing a hemagglutinin (HA) affinity tag in nonstructural protein 4B (NS4B), the supposed scaffold protein of the viral replication complex. By using HA-specific affinity purification we isolated NS4B-containing membranes from stable replicon cells. Complementing biochemical and electron microscopy analyses of purified membranes revealed predominantly DMVs, which contained viral proteins NS3 and NS5A as well as enzymatically active viral replicase capable of de novo synthesis of HCV RNA. In addition to viral factors, co-opted cellular proteins, such as vesicle-associated membrane protein-associated protein A (VAP-A) and VAP-B, that are crucial for viral RNA replication, as well as cholesterol, a major structural lipid of detergent-resistant membranes, are highly enriched in DMVs. Here we describe the first isolation and biochemical characterization of HCV-induced DMVs. The results obtained underline their central role in the HCV replication cycle and suggest that DMVs are sites of viral RNA replication. The experimental approach described here is a powerful tool to more precisely define the molecular composition of membranous replication factories induced by other positive-strand RNA viruses, such as picorna-, arteri- and coronaviruses.

Phosphoinositides in the Hepatitis C Virus Life Cycle

Eukaryotes possess seven different phosphoinositides (PIPs) that help form the unique signatures of various intracellular membranes. PIPs serve as docking sites for the recruitment of specific proteins to mediate membrane alterations and integrate various signaling cascades. The spatio-temporal regulation of PI kinases and phosphatases generates distinct intracellular hubs of PIP signaling. Hepatitis C virus (HCV), like other plus-strand RNA viruses, promotes the rearrangement of intracellular membranes to assemble viral replication complexes. HCV stimulates enrichment of phosphatidylinositol 4-phosphate (PI4P) pools near endoplasmic reticulum (ER) sites by activating PI4KIIIα, the kinase responsible for generation of ER-specific PI4P pools. Inhibition of PI4KIIIα abrogates HCV replication. PI4P, the most abundant phosphoinositide, predominantly localizes to the Golgi and plays central roles in Golgi secretory functions by recruiting effector proteins involved in transport vesicle generation. The PI4P effector proteins also include the lipid-transfer and structural proteins such as ceramide transfer protein (CERT), oxysterol binding protein (OSBP) and Golgi phosphoprotein 3 (GOLPH3) that help maintain Golgi-membrane composition and structure. Depletion of Golgi-specific PI4P pools by silencing PI4KIIIβ, expression of dominant negative CERT and OSBP mutants, or silencing GOLPH3 perturb HCV secretion. In this review we highlight the role of PIPs and specifically PI4P in the HCV life cycle.

Early Dengue Virus Protein Synthesis Induces Extensive Rearrangement of the Endoplasmic Reticulum Independent of the UPR and SREBP-2 Pathway

The rearrangement of intracellular membranes has been long reported to be a common feature in diseased cells. In this study, we used dengue virus (DENV) to study the role of the unfolded protein response (UPR) and sterol-regulatory-element-binding-protein-2 (SREBP-2) pathway in the rearrangement and expansion of the endoplasmic reticulum (ER) early after infection. Using laser scanning confocal and differential interference contrast microscopy, we demonstrate that rearrangement and expansion of the ER occurs early after DENV-2 infection. Through the use of mouse embryonic fibroblast cells deficient in XBP1 and ATF6, we show that ER rearrangement early after DENV infection is independent of the UPR. We then demonstrate that enlargement of the ER is independent of the SREBP-2 activation and upregulation of 3-hydroxy-3-methylglutaryl-Coenzyme-A reductase, the rate-limiting enzyme in the cholesterol biosynthesis pathway. We further show that this ER rearrangement is not inhibited by the treatment of DENV-infected cells with the cholesterol-inhibiting drug lovastatin. Using the transcription inhibitor actinomycin D and the translation elongation inhibitor cycloheximide, we show that de novo viral protein synthesis but not host transcription is necessary for expansion and rearrangement of the ER. Lastly, we demonstrate that viral infection induces the reabsorption of lipid droplets into the ER. Together, these results demonstrate that modulation of intracellular membrane architecture of the cell early after DENV-2 infection is driven by viral protein expression and does not require the induction of the UPR and SREBP-2 pathways. This work paves the way for further study of virally-induced membrane rearrangements and formation of cubic membranes.

The F-BAR Protein PACSIN 2 Specifically Coats the Anastomosing Intracellular Membrane Systems of Platelets and Megakaryocytes,

Abstract 3261Megakaryocyte (MK) maturation and platelet formation are associated with extensive intracellular membrane rearrangements. However, the cellular proteins and mechanisms responsible for the formation of the platelet open canalicular system (OCS) are unknown. PACSIN 1–3 belong to the novel family of F-BAR domain-containing proteins, which bind and deform membranes, promoting tubular invaginations. Blood platelets and MKs contain the ubiquitously expressed PACSIN 2, but not PACSIN 1 or 3, as evidenced by intracellular flow cytometry and immunoblot analysis. Spinning disk laser fluorescence confocal microscopy revealed a specific association between PACSIN 2 and tubular structures in human platelets. PACSIN 2 did not colocalize with granule or lysosome markers, i.e. CD62P, CD63 and LAMP1, suggesting that it coats the OCS. In mouse MKs, PACSIN 2 was distributed through the cell body and colocalized with the membrane marker, CD61. Immunoprecipitation of PACSIN 2 from human platelet lysates pulled down filamin A (FlnA), a major platelet cytoskeletal protein. The interaction required PACSIN 2 F-BAR domain, as evidenced by in vitro binding assays using recombinant proteins. Mice lacking FlnA in the MK lineage have a severe thrombocytopenia with large platelets (Falet et al, J Exp Med 2010; Jurak Begonja et al, Blood 2011). In contrast to wild type platelets, FlnA-null platelets had diffuse PACSIN 2 staining and abnormal intracellular membrane structures, suggesting that the PACSIN 2-FlnA interaction is necessary for the integrity of the platelet intracellular membrane systems. PACSIN 2-null mice had a mild thrombocytopenia, enlarged spleens and increased spleen megakaryopoiesis. Together, the data shows that PACSIN 2 is expressed in platelets and MKs, where it specifically coats the anastomosing intracellular membrane systems in cooperation with FlnA. Disclosures:No relevant conflicts of interest to declare.

West Nile Virus Replication Requires Fatty Acid Synthesis but Is Independent on Phosphatidylinositol-4-Phosphate Lipids

West Nile virus (WNV) is a neurovirulent mosquito-borne flavivirus, which main natural hosts are birds but it also infects equines and humans, among other mammals. As in the case of other plus-stranded RNA viruses, WNV replication is associated to intracellular membrane rearrangements. Based on results obtained with a variety of viruses, different cellular processes have been shown to play important roles on these membrane rearrangements for efficient viral replication. As these processes are related to lipid metabolism, fatty acid synthesis, as well as generation of a specific lipid microenvironment enriched in phosphatidylinositol-4-phosphate (PI4P), has been associated to it in other viral models. In this study, intracellular membrane rearrangements following infection with a highly neurovirulent strain of WNV were addressed by means of electron and confocal microscopy. Infection of WNV, and specifically viral RNA replication, were dependent on fatty acid synthesis, as revealed by the inhibitory effect of cerulenin and C75, two pharmacological inhibitors of fatty acid synthase, a key enzyme of this process. However, WNV infection did not induce redistribution of PI4P lipids, and PI4P did not localize at viral replication complex. Even more, WNV multiplication was not inhibited by the use of the phosphatidylinositol-4-kinase inhibitor PIK93, while infection by the enterovirus Coxsackievirus B5 was reduced. Similar features were found when infection by other flavivirus, the Usutu virus (USUV), was analyzed. These features of WNV replication could help to design specific antiviral approaches against WNV and other related flaviviruses.

Foot-and-mouth disease virus utilizes an autophagic pathway during viral replication

Foot-and-mouth disease virus (FMDV) is the type species of the Aphthovirus genus within the Picornaviridae family. Infection of cells with positive-strand RNA viruses results in a rearrangement of intracellular membranes into viral replication complexes. The origin of these membranes remains unknown; however induction of the cellular process of autophagy is beneficial for the replication of poliovirus, suggesting that it might be advantageous for other picornaviruses. By using confocal microscopy we showed in FMDV-infected cells co-localization of non-structural viral proteins 2B, 2C and 3A with LC3 (an autophagosome marker) and viral structural protein VP1 with Atg5 (autophagy-related protein), and LC3 with LAMP-1. Importantly, treatment of FMDV-infected cell with autophagy inducer rapamycin, increased viral yield, and inhibition of autophagosomal pathway by 3-methyladenine or small-interfering RNAs, decreased viral replication. Altogether, these studies strongly suggest that autophagy may play an important role during the replication of FMDV.

Feline calicivirus p32, p39 and p30 proteins localize to the endoplasmic reticulum to initiate replication complex formation

In common with other positive-strand RNA viruses, replication of feline calicivirus (FCV) results in rearrangement of intracellular membranes and production of numerous membrane-bound vesicular structures on which viral genome replication is thought to occur. In this study, bioinformatics approaches have identified three of the FCV non-structural proteins, namely p32, p39 and p30, as potential transmembrane proteins. These proteins were able to target enhanced cyan fluorescent protein to membrane fractions where they behaved as integral membrane proteins. Immunofluorescence microscopy of these proteins expressed in cells showed co-localization with endoplasmic reticulum (ER) markers. Further electron microscopy analysis of cells co-expressing FCV p39 or p30 with a horseradish peroxidase protein containing the KDEL ER retention motif demonstrated gross morphological changes to the ER. Similar reorganization patterns, especially for those produced by p30, were observed in naturally infected Crandel–Rees feline kidney cells. Together, the data demonstrate that the p32, p39 and p30 proteins of FCV locate to the ER and lead to reorganization of ER membranes. This suggests that they may play a role in the generation of FCV replication complexes and that the endoplasmic reticulum may represent the potential source of the membrane vesicles induced during FCV infection.

Potential roles for cellular cofactors in hepatitis C virus replication complex formation

Over 130 million people world-wide are chronically infected with hepatitis C virus (HCV). New antiviral treatment strategies are needed due to limitations with current therapy. The identification of cellular cofactors of infection has the potential to broadly expand our therapeutic targets. We recently reported an RNA interference screen of host membrane trafficking genes in HCV infection and replication and identified several cellular co-factors for viral replication. Phosphatidylinositol 4-kinase III alpha (PI4K-IIIα) was found to be essential for HCV replication. PI4K-IIIα co-localized with viral replication markers. Silencing of PI4K-IIIα by siRNAs prior to HCV infection prevented rearrangement of intracellular membranes associated with viral replication complexes, termed the membranous web. Our data suggest that PI4K-IIIα is involved in establishing HCV replication complexes, however the mechanism is unknown. From our analysis, along with several other studies that have identified cellular cofactors for HCV replication, we propose that PI4K-IIIα may nucleate replication complex formation by facilitating the interaction of viral and/or cellular proteins with cellular membrane-associated phospholipids.

Three-Dimensional Analysis of a Viral RNA Replication Complex Reveals a Virus-Induced Mini-Organelle

Positive-strand RNA viruses are the largest genetic class of viruses and include many serious human pathogens. All positive-strand RNA viruses replicate their genomes in association with intracellular membrane rearrangements such as single- or double-membrane vesicles. However, the exact sites of RNA synthesis and crucial topological relationships between relevant membranes, vesicle interiors, surrounding lumens, and cytoplasm generally are poorly defined. We applied electron microscope tomography and complementary approaches to flock house virus (FHV)–infected Drosophila cells to provide the first 3-D analysis of such replication complexes. The sole FHV RNA replication factor, protein A, and FHV-specific 5-bromouridine 5'-triphosphate incorporation localized between inner and outer mitochondrial membranes inside ∼50-nm vesicles (spherules), which thus are FHV-induced compartments for viral RNA synthesis. All such FHV spherules were outer mitochondrial membrane invaginations with interiors connected to the cytoplasm by a necked channel of ∼10-nm diameter, which is sufficient for ribonucleotide import and product RNA export. Tomographic, biochemical, and other results imply that FHV spherules contain, on average, three RNA replication intermediates and an interior shell of ∼100 membrane-spanning, self-interacting protein As. The results identify spherules as the site of protein A and nascent RNA accumulation and define spherule topology, dimensions, and stoichiometry to reveal the nature and many details of the organization and function of the FHV RNA replication complex. The resulting insights appear relevant to many other positive-strand RNA viruses and support recently proposed structural and likely evolutionary parallels with retrovirus and double-stranded RNA virus virions.

Replication of Norovirus in Cell Culture Reveals a Tropism for Dendritic Cells and Macrophages

Noroviruses are understudied because these important enteric pathogens have not been cultured to date. We found that the norovirus murine norovirus 1 (MNV-1) infects macrophage-like cells in vivo and replicates in cultured primary dendritic cells and macrophages. MNV-1 growth was inhibited by the interferon-αβ receptor and STAT-1, and was associated with extensive rearrangements of intracellular membranes. An amino acid substitution in the capsid protein of serially passaged MNV-1 was associated with virulence attenuation in vivo. This is the first report of replication of a norovirus in cell culture. The capacity of MNV-1 to replicate in a STAT-1-regulated fashion and the unexpected tropism of a norovirus for cells of the hematopoietic lineage provide important insights into norovirus biology.

Norwalk virus N-terminal nonstructural protein is associated with disassembly of the Golgi complex in transfected cells.

Norwalk virus is the prototype strain for members of the genus Norovirus in the family Caliciviridae, which are associated with epidemic gastroenteritis in humans. The nonstructural protein encoded in the N-terminal region of the first open reading frame (ORF1) of the Norwalk virus genome is analogous in gene order to proteins 2A and 2B of the picornaviruses; the latter is known for its membrane-associated activities. Confocal microscopy imaging of cells transfected with a vector plasmid that provided expression of the entire Norwalk virus N-terminal protein (amino acids 1 to 398 of the ORF1 polyprotein) showed colocalization of this protein with cellular proteins of the Golgi apparatus. Furthermore, this colocalization was characteristically associated with a visible disassembly of the Golgi complex into discrete aggregates. Deletion of a predicted hydrophobic region (amino acids 360 to 379) in a potential 2B-like (2BL) region (amino acids 301 to 398) near the C terminus of the Norwalk virus N-terminal protein reduced Golgi colocalization and disassembly. Confocal imaging was conducted to examine the expression characteristics of fusion proteins in which the 2BL region from the N-terminal protein of Norwalk virus (a genogroup I norovirus) or MD145 (a genogroup II norovirus) was fused to the C terminus of enhanced green fluorescent protein. Expression of each fusion protein in cells showed evidence for its colocalization with the Golgi apparatus. These data indicate that the N-terminal protein of Norwalk virus interacts with the Golgi apparatus and may play a 2BL role in the induction of intracellular membrane rearrangements associated with positive-strand RNA virus replication in cells.

Cowpea mosaic virus 32- and 60-kilodalton replication proteins target and change the morphology of endoplasmic reticulum membranes.

Cowpea mosaic virus (CPMV) replicates in close association with small membranous vesicles that are formed by rearrangements of intracellular membranes. To determine which of the viral proteins are responsible for the rearrangements of membranes and the attachment of the replication complex, we have expressed individual CPMV proteins encoded by RNA1 in cowpea protoplasts by transient expression and in Nicotiana benthamiana plants by using the tobacco rattle virus (TRV) expression vector. The 32-kDa protein (32K) and 60K, when expressed individually, accumulate in only low amounts but are found associated with membranes mainly derived from the endoplasmic reticulum (ER). 24K and 110K are freely soluble and accumulate to high levels. With the TRV vector, expression of 32K and 60K results in rearrangement of ER membranes. Besides, expression of 32K and 60K results in necrosis of the inoculated N. benthamiana leaves, suggesting that 32K and 60K are cytotoxic proteins. On the other hand, during CPMV infection 32K and 60K accumulate to high levels without causing necrosis.