Modulation of the Host Lipid Landscape to Promote RNA Virus Replication: The Picornavirus Encephalomyocarditis Virus Converges on the Pathway Used by Hepatitis C Virus

Cardioviruses, including encephalomyocarditis virus (EMCV) and the human Saffold virus, are small non-enveloped viruses belonging to the Picornaviridae, a large family of positive-sense RNA [(+)RNA] viruses. All (+)RNA viruses remodel intracellular membranes into unique structures for viral genome replication. Accumulating evidence suggests that picornaviruses from different genera use different strategies to generate viral replication organelles (ROs). For instance, enteroviruses (e.g. poliovirus, coxsackievirus, rhinovirus) rely on the Golgi-localized phosphatidylinositol 4-kinase III beta (PI4KB), while cardioviruses replicate independently of the kinase. By which mechanisms cardioviruses develop their ROs is currently unknown. Here we show that cardioviruses manipulate another PI4K, namely the ER-localized phosphatidylinositol 4-kinase III alpha (PI4KA), to generate PI4P-enriched ROs. By siRNA-mediated knockdown and pharmacological inhibition, we demonstrate that PI4KA is an essential host factor for EMCV genome replication. We reveal that the EMCV nonstructural protein 3A interacts with and is responsible for PI4KA recruitment to viral ROs. The ensuing phosphatidylinositol 4-phosphate (PI4P) proved important for the recruitment of oxysterol-binding protein (OSBP), which delivers cholesterol to EMCV ROs in a PI4P-dependent manner. PI4P lipids and cholesterol are shown to be required for the global organization of the ROs and for viral genome replication. Consistently, inhibition of OSBP expression or function efficiently blocked EMCV RNA replication. In conclusion, we describe for the first time a cellular pathway involved in the biogenesis of cardiovirus ROs. Remarkably, the same pathway was reported to promote formation of the replication sites of hepatitis C virus, a member of the Flaviviridae family, but not other picornaviruses or flaviviruses. Thus, our results highlight the convergent recruitment by distantly related (+)RNA viruses of a host lipid-modifying pathway underlying formation of viral replication sites.     

Structural insights into Acyl‐coenzyme A binding domain containing 3 (ACBD3) protein hijacking by picornaviruses

Many picornaviruses hijack the Golgi resident Acyl‐coenzyme A binding domain containing 3 (ACBD3) protein in order to recruit the phosphatidylinositol 4‐kinase B (PI4KB) to viral replication organelles (ROs). PI4KB, once recruited and activated by ACBD3 protein, produces the lipid phosphatidylinositol 4‐phosphate (PI4P), which is a key step in the biogenesis of viral ROs. To do so, picornaviruses use their small nonstructural protein 3A that binds the Golgi dynamics domain of the ACBD3 protein. Here, we present the analysis of the highly flexible ACBD3 proteins and the viral 3A protein in solution using small‐angle X‐ray scattering and computer simulations. Our analysis revealed that both the ACBD3 protein and the 3A:ACBD3 protein complex have an extended and flexible conformation in solution.     

Autophagy promotes the replication of encephalomyocarditis virus in host cells

A growing number of studies have demonstrated that autophagy has a diverse role in the infection process of different pathogens. However, to date, it is unknown whether autophagy is activated in encephalomyocarditis virus (EMCV)-infected host cells, and if so, what its role is in this process. In the present study, we first demonstrated that EMCV infection significantly increases the number of double- and single-membrane vesicles in the cytoplasm of host cells. It was then confirmed that these observed vesicles are indeed related to autophagy, and that EMCV replication is required for the induction of autophagy by examining LC3-I/-II conversion and p62/SQSTM1 degradation using immunoblotting. Next, we performed confocal immunofluorescence analysis and discovered that, during EMCV replication, both the nonstructural protein 3A and capsid protein VP1 colocalized with LC3. The colocalizations of both 3A and VP1 protein with autophagosome-like vesicles were further confirmed using immunoelectron microscopy, indicating that EMCV undergoes RNA replication on the membranes of these vesicles. Finally, we used pharmacological regulators and siRNAs to examine the role of autophagy in EMCV replication. Our results suggest that autophagy not only promotes the replication of EMCV in host cells, but it also provides a topological mechanism for releasing cytoplasmic viruses in a nonlytic manner. Noticeably, the autophagic pharmaceuticals we used had no significant effect on virus entry or cell viability, both of which may affect viral replication. To our knowledge, ours is the first strong evidence indicating that autophagy is involved in EMCV infection in host cells.     

Autophagy promotes the replication of encephalomyocarditis virus in host cells

A growing number of studies have demonstrated that autophagy has a diverse role in the infection process of different pathogens. However, to date, it is unknown whether autophagy is activated in encephalomyocarditis virus (EMCV)-infected host cells, and if so, what its role is in this process. In the present study, we first demonstrated that EMCV infection significantly increases the number of double- and single-membrane vesicles in the cytoplasm of host cells. It was then confirmed that these observed vesicles are indeed related to autophagy, and that EMCV replication is required for the induction of autophagy by examining LC3-I/-II conversion and p62/SQSTM1 degradation using immunoblotting. Next, we performed confocal immunofluorescence analysis and discovered that, during EMCV replication, both the nonstructural protein 3A and capsid protein VP1 colocalized with LC3. The colocalizations of both 3A and VP1 protein with autophagosome-like vesicles were further confirmed using immunoelectron microscopy, indicating that EMCV undergoes RNA replication on the membranes of these vesicles. Finally, we used pharmacological regulators and siRNAs to examine the role of autophagy in EMCV replication. Our results suggest that autophagy not only promotes the replication of EMCV in host cells, but it also provides a topological mechanism for releasing cytoplasmic viruses in a nonlytic manner. Noticeably, the autophagic pharmaceuticals we used had no significant effect on virus entry or cell viability, both of which may affect viral replication. To our knowledge, ours is the first strong evidence indicating that autophagy is involved in EMCV infection in host cells.     

Coxsackievirus mutants that can bypass host factor PI4KIIIβ and the need for high levels of PI4P lipids for replication

RNA viruses can rapidly mutate and acquire resistance to drugs that directly target viral enzymes, which poses serious problems in a clinical context. Therefore, there is a growing interest in the development of antiviral drugs that target host factors critical for viral replication, since they are unlikely to mutate in response to therapy. We recently demonstrated that phosphatidylinositol-4-kinase IIIβ (PI4KIIIβ) and its product phosphatidylinositol-4-phosphate (PI4P) are essential for replication of enteroviruses, a group of medically important RNA viruses including poliovirus (PV), coxsackievirus, rhinovirus, and enterovirus 71. Here, we show that enviroxime and GW5074 decreased PI4P levels at the Golgi complex by directly inhibiting PI4KIIIβ. Coxsackievirus mutants resistant to these inhibitors harbor single point mutations in the non-structural protein 3A. These 3A mutations did not confer compound-resistance by restoring the activity of PI4KIIIβ in the presence of the compounds. Instead, replication of the mutant viruses no longer depended on PI4KIIIβ, since their replication was insensitive to siRNA-mediated depletion of PI4KIIIβ. The mutant viruses also did not rely on other isoforms of PI4K. Consistently, no high level of PI4P could be detected at the replication sites induced by the mutant viruses in the presence of the compounds. Collectively, these findings indicate that through specific single point mutations in 3A, CVB3 can bypass an essential host factor and lipid for its propagation, which is a new example of RNA viruses acquiring resistance against antiviral compounds, even when they directly target host factors.     

Rewiring of Cellular Membrane Homeostasis by Picornaviruses

Viruses are obligatory intracellular parasites and utilize host elements to support key viral processes, including penetration of the plasma membrane, initiation of infection, replication, and suppression of the host''s antiviral defenses. In this review, we focus on picornaviruses, a family of positive-strand RNA viruses, and discuss the mechanisms by which these viruses hijack the cellular machinery to form and operate membranous replication complexes. Studies aimed at revealing factors required for the establishment of viral replication structures identified several cellular-membrane-remodeling proteins and led to the development of models in which the virus used a preexisting cellular-membrane-shaping pathway “as is” for generating its replication organelles. However, as more data accumulate, this view is being increasingly questioned, and it is becoming clearer that viruses may utilize cellular factors in ways that are distinct from the normal functions of these proteins in uninfected cells. In addition, the proteincentric view is being supplemented by important new studies showing a previously unappreciated deep remodeling of lipid homeostasis, including extreme changes to phospholipid biosynthesis and cholesterol trafficking. The data on viral modifications of lipid biosynthetic pathways are still rudimentary, but it appears once again that the viruses may rewire existing pathways to generate novel functions. Despite remarkable progress, our understanding of how a handful of viral proteins can completely overrun the multilayered, complex mechanisms that control the membrane organization of a eukaryotic cell remains very limited.     

The NLRP3 inflammasome detects encephalomyocarditis virus and vesicular stomatitis virus infection

Inflammasomes are cytosolic protein complexes that regulate caspase-1 activation and the secretion of interleukin-1β (IL-1β) and IL-18. Several different inflammasome complexes have been identified, but the NLRP3 inflammasome is particularly notable because of its central role in diseases of inflammation. Recent work has demonstrated an essential role for the NLRP3 inflammasome in host defense against influenza virus. We show here that two other RNA viruses, encephalomyocarditis virus (EMCV) and vesicular stomatitis virus (VSV), activate the NLRP3 inflammasome in dendritic cells and macrophages through a mechanism requiring viral replication. Inflammasome activation in response to both viruses does not require MDA5 or RIG-I signaling. Despite the ability of the NLRP3 inflammasome to detect EMCV and VSV, wild-type and caspase-1-deficient mice were equally susceptible to infection with both viruses. These findings indicate that the NLRP3 inflammasome may be a common pathway for RNA virus detection, but its precise role in the host response may be variable.     

Influenza A Virus Encoding Secreted Gaussia Luciferase as Useful Tool to Analyze Viral Replication and Its Inhibition by Antiviral Compounds and Cellular Proteins

Reporter genes inserted into viral genomes enable the easy and rapid quantification of virus replication, which is instrumental to efficient in vitro screening of antiviral compounds or in vivo analysis of viral spread and pathogenesis. Based on a published design, we have generated several replication competent influenza A viruses carrying either fluorescent proteins or Gaussia luciferase. Reporter activity could be readily quantified in infected cultures, but the virus encoding Gaussia luciferase was more stable than viruses bearing fluorescent proteins and was therefore analyzed in detail. Quantification of Gaussia luciferase activity in the supernatants of infected culture allowed the convenient and highly sensitive detection of viral spread, and enzymatic activity correlated with the number of infectious particles released from infected cells. Furthermore, the Gaussia luciferase encoding virus allowed the sensitive quantification of the antiviral activity of the neuraminidase inhibitor (NAI) zanamivir and the host cell interferon-inducible transmembrane (IFITM) proteins 1–3, which are known to inhibit influenza virus entry. Finally, the virus was used to demonstrate that influenza A virus infection is sensitive to a modulator of endosomal cholesterol, in keeping with the concept that IFITMs inhibit viral entry by altering cholesterol levels in the endosomal membrane. In sum, we report the characterization of a novel influenza A reporter virus, which allows fast and sensitive detection of viral spread and its inhibition, and we show that influenza A virus entry is sensitive to alterations of endosomal cholesterol levels.     

The tyrosine kinase p56lck is essential in coxsackievirus B3-mediated heart disease

Infections are thought to be important in the pathogenesis of many heart diseases. Coxsackievirus B3 (CVB3) has been linked to chronic dilated cardiomyopathy, a common cause of progressive heart disease, heart failure and sudden death. We show here that the sarcoma (Src) family kinase Lck (p56lck) is required for efficient CVB3 replication in T-cell lines and for viral replication and persistence in vivo. Whereas infection of wild-type mice with human pathogenic CVB3 caused acute and very severe myocarditis, meningitis, hepatitis, pancreatitis and dilated cardiomyopathy, mice lacking the p56lck gene were completely protected from CVB3-induced acute pathogenicity and chronic heart disease. These data identify a previously unknown function of Src family kinases and indicate that p56lck is the essential host factor that controls the replication and pathogenicity of CVB3.     

Cleavage and degradation of EDEM1 promotes coxsackievirus B3 replication via ATF6a‐mediated unfolded protein response signalling

Our previous study of coxsackievirus B3 (CVB3)‐induced unfolded protein responses (UPR) found that overexpression of ATF6a enhances CVB3 VP1 capsid protein production and increases viral particle formation. These findings implicate that ATF6a signalling benefits CVB3 replication. However, the mechanism by which ATF6a signalling is transduced to promote virus replication is unclear. In this study, using a Tet‐On inducible ATF6a HeLa cell line, we found that ATF6a signalling downregulated the protein expression of the endoplasmic reticulum (ER) degradation‐enhancing α‐mannosidase‐like protein 1 (EDEM1), resulting in accumulation of CVB3 VP1 protein; in contrast, expression of a dominant negative ATF6a had the opposite effect. Furthermore, we found that EDEM1 was cleaved by both CVB3 protease 3C and virus‐activated caspase and subsequently degraded via the ubiquitin‐proteasome pathway. However, overexpression of EDEM1 caused VP1 degradation, likely via a glycosylation‐independent and ubiquitin‐lysosome pathway. Finally, we demonstrated that CRISPR/Cas9‐mediated knockout of EDEM1 increased VP1 accumulation and thus CVB3 replication. This is the first study to report the ER protein quality control of non‐enveloped RNA virus and reveals a novel mechanism by which CVB3 evades host ER quality control pathways through cleavage and degradation of the UPR target gene EDEM1, to ultimately benefit its own replication.     

Duck hepatitis B virus requires cholesterol for endosomal escape during virus entry

The identity and functionality of biological membranes are determined by cooperative interaction between their lipid and protein constituents. Cholesterol is an important structural lipid that modulates fluidity of biological membranes favoring the formation of detergent-resistant microdomains. In the present study, we evaluated the functional role of cholesterol and lipid rafts for entry of hepatitis B viruses into hepatocytes. We show that the duck hepatitis B virus (DHBV) attaches predominantly to detergent-soluble domains on the plasma membrane. Cholesterol depletion from host membranes and thus disruption of rafts does not affect DHBV infection. In contrast, depletion of cholesterol from the envelope of both DHBV and human HBV strongly reduces virus infectivity. Cholesterol depletion increases the density of viral particles and leads to changes in the ultrastructural appearance of the virus envelope. However, the dual topology of the viral envelope protein L is not significantly impaired. Infectivity and density of viral particles are partially restored upon cholesterol replenishment. Binding and entry of cholesterol-deficient DHBV into hepatocytes are not significantly impaired, in contrast to their release from endosomes. We therefore conclude that viral but not host cholesterol is required for endosomal escape of DHBV.     

Interactions between enteroviruses and autophagy in vivo

Autophagy plays a protective role during many viral and bacterial infections. Predictably, evolution has led to several viruses developing mechanisms by which to evade the inhibitory effects of the pathway. However, one family of viruses, the picornaviruses, has gone one step further, by actively exploiting autophagy. Using mice in which Atg5 has been conditionally deleted in pancreatic acinar cells, we have studied the outcome of infection by coxsackievirus B3 (CVB3), a member of the enterovirus genus and picornavirus family. Two key findings emerged: disruption of autophagy (1) dramatically compromised virus replication in vivo, and (2) significantly limited pancreatic disease.     

Interactions between enteroviruses and autophagy in vivo

Autophagy plays a protective role during many viral and bacterial infections. Predictably, evolution has led to several viruses developing mechanisms by which to evade the inhibitory effects of the pathway. However, one family of viruses, the picornaviruses, has gone one step further, by actively exploiting autophagy. Using mice in which Atg5 has been conditionally deleted in pancreatic acinar cells, we have studied the outcome of infection by coxsackievirus B3 (CVB3), a member of the enterovirus genus and picornavirus family. Two key findings emerged: disruption of autophagy (1) dramatically compromised virus replication in vivo, and (2) significantly limited pancreatic disease.     

Modulation of membrane traffic between endoplasmic reticulum, ERGIC and Golgi to generate compartments for the replication of bacteria and viruses

Several bacteria and viruses remodel cellular membranes to form compartments specialised for replication. Bacteria replicate within inclusions which recruit membrane vesicles from the secretory pathway to provide nutrients for microbial growth and division. Viruses generate densely packed membrane vesicles called viroplasm which provide a platform to recruit host and viral proteins necessary for replication. This review describes examples where both intracellular bacteria (Salmonella, Chlamydia and Legionella) and viruses (picornaviruses and hepatitis C) recruit membrane vesicles to sites of replication by modulating proteins that control the secretory pathway. In many cases this involves modulation of Rab and Arf GTPases.     

Alteration of encephalomyocarditis virus pathogenicity due to a mutation at position 100 of VP1

Encephalomyocarditis virus (EMCV) infection leads to many diseases including encephalitis, myocarditis and diabetes in its natural host, the mouse. In this study, we generated four cDNA clones with a point mutation at position 100 of VP1. The amino acids isoleucine, alanine, serine and proline were substituted with threonine in the four different clones of EMCV strain BJC3 by site-specific mutagenesis, and viable viruses were rescued. Although all mutants and wild-type viruses display different plaque morphologies, they replicate comparably in BHK-21 cells. The pathogenicity of the mutated viruses was systematically analyzed to investigate the importance of this amino acid in the viral pathogenicity and disease phenotype of EMCV infection in mice. The results showed that the isoleucine- (T1100I) and proline-mutated viruses (T1100P) exhibited a reduced mortality, lower cerebral virus loads and alleviated brain damage while the viruses with serine (T1100S) and alanine (T1100A) substitutions displayed similar properties as the wild-type virus. These findings indicate that the amino acid at position 100 of VP1 is important for EMCV in vivo infection, and its mutation alters the pathogenicity of viral infection in mice.     

Virus entry, assembly, budding, and membrane rafts.

As intracellular parasites, viruses rely heavily on the use of numerous cellular machineries for completion of their replication cycle. The recent discovery of the heterogeneous distribution of the various lipids within cell membranes has led to the proposal that sphingolipids and cholesterol tend to segregate in microdomains called membrane rafts. The involvement of membrane rafts in biosynthetic traffic, signal transduction, and endocytosis has suggested that viruses may also take advantage of rafts for completion of some steps of their replication cycle, such as entry into their cell host, assembly, and budding. In this review, we have attempted to delineate all the reliable data sustaining this hypothesis and to build some models of how rafts are used as platforms for assembly of some viruses. Indeed, if in many cases a formal proof of raft involvement in a virus replication cycle is still lacking, one can reasonably suggest that, owing to their ability to specifically attract some proteins, lipid microdomains provide a particular milieu suitable for increasing the efficiency of many protein-protein interactions which are crucial for virus infection and growth.     

Virus Entry, Assembly, Budding, and Membrane Rafts

As intracellular parasites, viruses rely heavily on the use of numerous cellular machineries for completion of their replication cycle. The recent discovery of the heterogeneous distribution of the various lipids within cell membranes has led to the proposal that sphingolipids and cholesterol tend to segregate in microdomains called membrane rafts. The involvement of membrane rafts in biosynthetic traffic, signal transduction, and endocytosis has suggested that viruses may also take advantage of rafts for completion of some steps of their replication cycle, such as entry into their cell host, assembly, and budding. In this review, we have attempted to delineate all the reliable data sustaining this hypothesis and to build some models of how rafts are used as platforms for assembly of some viruses. Indeed, if in many cases a formal proof of raft involvement in a virus replication cycle is still lacking, one can reasonably suggest that, owing to their ability to specifically attract some proteins, lipid microdomains provide a particular milieu suitable for increasing the efficiency of many protein-protein interactions which are crucial for virus infection and growth.     

Differential requirements for COPI coats in formation of replication complexes among three genera of Picornaviridae

Picornavirus RNA replication requires the formation of replication complexes (RCs) consisting of virus-induced vesicles associated with viral nonstructural proteins and RNA. Brefeldin A (BFA) has been shown to strongly inhibit RNA replication of poliovirus but not of encephalomyocarditis virus (EMCV). Here, we demonstrate that the replication of parechovirus 1 (ParV1) is partly resistant to BFA, whereas echovirus 11 (EV11) replication is strongly inhibited. Since BFA inhibits COPI-dependent steps in endoplasmic reticulum (ER)-Golgi transport, we tested a hypothesis that different picornaviruses may have differential requirements for COPI in the formation of their RCs. Using immunofluorescence and cryo-immunoelectron microscopy we examined the association of a COPI component, beta-COP, with the RCs of EMCV, ParV1, and EV11. EMCV RCs did not contain beta-COP. In contrast, beta-COP appeared to be specifically distributed to the RCs of EV11. In ParV1-infected cells beta-COP was largely dispersed throughout the cytoplasm, with some being present in the RCs. These results suggest that there are differences in the involvement of COPI in the formation of the RCs of various picornaviruses, corresponding to their differential sensitivity to BFA. EMCV RCs are likely to be formed immediately after vesicle budding from the ER, prior to COPI association with membranes. ParV1 RCs are formed from COPI-containing membranes but COPI is unlikely to be directly involved in their formation, whereas formation of EV11 RCs appears to be dependent on COPI association with membranes.     

IFITM3 inhibits influenza A virus infection by preventing cytosolic entry

To replicate, viruses must gain access to the host cell's resources. Interferon (IFN) regulates the actions of a large complement of interferon effector genes (IEGs) that prevent viral replication. The interferon inducible transmembrane protein family members, IFITM1, 2 and 3, are IEGs required for inhibition of influenza A virus, dengue virus, and West Nile virus replication in vitro. Here we report that IFN prevents emergence of viral genomes from the endosomal pathway, and that IFITM3 is both necessary and sufficient for this function. Notably, viral pseudoparticles were inhibited from transferring their contents into the host cell cytosol by IFN, and IFITM3 was required and sufficient for this action. We further demonstrate that IFN expands Rab7 and LAMP1-containing structures, and that IFITM3 overexpression is sufficient for this phenotype. Moreover, IFITM3 partially resides in late endosomal and lysosomal structures, placing it in the path of invading viruses. Collectively our data are consistent with the prediction that viruses that fuse in the late endosomes or lysosomes are vulnerable to IFITM3's actions, while viruses that enter at the cell surface or in the early endosomes may avoid inhibition. Multiple viruses enter host cells through the late endocytic pathway, and many of these invaders are attenuated by IFN. Therefore these findings are likely to have significance for the intrinsic immune system's neutralization of a diverse array of threats.     

The crystal structure of coxsackievirus B3 RNA-dependent RNA polymerase in complex with its protein primer VPg confirms the existence of a second VPg binding site on Picornaviridae polymerases

The RNA-dependent RNA polymerase (RdRp) is a central piece in the replication machinery of RNA viruses. In picornaviruses this essential RdRp activity also uridylates the VPg peptide, which then serves as a primer for RNA synthesis. Previous genetic, binding, and biochemical data have identified a VPg binding site on poliovirus RdRp and have shown that is was implicated in VPg uridylation. More recent structural studies have identified a topologically distinct site on the closely related foot-and-mouth disease virus RdRp supposed to be the actual VPg-primer-binding site. Here, we report the crystal structure at 2.5-Å resolution of active coxsackievirus B3 RdRp (also named 3D^pol) in a complex with VPg and a pyrophosphate. The pyrophosphate is situated in the active-site cavity, occupying a putative binding site either for the coproduct of the reaction or an incoming NTP. VPg is bound at the base of the thumb subdomain, providing first structural evidence for the VPg binding site previously identified by genetic and biochemical methods. The binding mode of VPg to CVB3 3D^pol at this site excludes its uridylation by the carrier 3D^pol. We suggest that VPg at this position is either uridylated by another 3D^pol molecule or that it plays a stabilizing role within the uridylation complex. The CVB3 3D^pol/VPg complex structure is expected to contribute to the understanding of the multicomponent VPg-uridylation complex essential for the initiation of genome replication of picornaviruses.