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ABSTRACT Porcine epidemic diarrhea virus (PEDV) requires complete dependence on the metabolic system of the host cell to complete its life cycle. There is a strong link between efficient viral replication and cellular lipid synthesis. However, the mechanism by which PEDV interacts with host cells to hijack cellular lipid metabolism to promote its replication remains unclear. In this study, PEDV infection significantly enhanced the expression of lipid synthesis-related genes and increased cellular lipid accumulation. Furthermore, using liquid chromatography-tandem mass spectrometry, we identified heterogeneous nuclear ribonucleoprotein A3 (HNRNPA3) as the interacting molecule of PEDV NSP9. We demonstrated that the expression of HNRNPA3 was downregulated by PEDV-induced miR-218-5p through targeting its 3′ untranslated region. Interestingly, knocking down HNRNPA3 facilitated the PEDV replication by promoting cellular lipid synthesis. We next found that the knockdown of HNRNPA3 potentiated the transcriptional activity of sterol regulatory element-binding transcription factor 1 (SREBF1) through zinc finger protein 135 (ZNF135) as well as PI3K/AKT and JNK signaling pathways. In summary, we propose a model in which PEDV downregulates HNRNPA3 expression to promote the expression and activation of SREBF1 and increase cellular lipid accumulation, providing a novel mechanism by which PEDV interacts with the host to utilize cellular lipid metabolism to promote its replication.IMPORTANCEAs the major components and structural basis of the viral replication complexes of positive-stranded RNA viruses, lipids play an essential role in viral replication. However, how PEDV manipulates host cell lipid metabolism to promote viral replication by interacting with cell proteins remains poorly understood. Here, we found that SREBF1 promotes cellular lipid synthesis, which is essential for PEDV replication. Moreover, HNRNPA3 negatively regulates SREBF1 activation and specifically reduces lipid accumulation, ultimately inhibiting PEDV dsRNA synthesis. Our study provides new insight into the mechanisms by which PEDV hijacks cell lipid metabolism to benefit viral replication, which can offer a potential target for therapeutics against PEDV infection.

Numerous studies have documented that the interaction of viral and cellular proteins is essential in the viral life cycle. In our previous study, to screen cellular proteins that take part in the life cycle of JEV, cellular proteins that interacted with JEV NS3 were identified by Co-immunoprecipitation coupled with mass spectrometry analysis (Co-IP-MS), the results showed that ILF2, DnaJA1, DnaJA2, CKB, TUFM, and PABPC1 that putatively interact with NS3. Another candidate protein, DnaJA2, which interacted with JEV NS3 protein, was selected for further study. Overexpression of DnaJA2 increased JEV infection. Conversely, the knockdown of DnaJA2 suppressed JEV infection. Furthermore, DnaJA2 interacted with NS5 besides NS3 and colocalized with viral dsRNA. Additionally, the level of viral NS3 protein expression was higher in cells overexpressing DnaJA2 than in cells with empty vector expression, whereas DnaJA2 knockdown resulted in NS3 protein degradation, which was subsequently restored by MG132 treatment. Further analysis revealed that the C-terminal of DnaJA2 was a critical domain for interaction with NS3 and promoted JEV infection. Collectively, our study identified DnaJA2 as an essential host factor required for JEV infection, potentially representing a novel therapeutic target for the development of antiviral therapies against JEV.

Plant viruses are responsible for worldwide production losses of numerous economically important crops. The most common plant RNA viruses are positive-sense single-stranded RNA viruses [(+)ss RNA viruses]. These viruses have small genomes that encode a limited number of proteins. The viruses depend on their host’s machinery for the replication of their RNA genome, assembly, movement, and attraction to the vectors for dispersal. Recently researchers have reported that chloroplast proteins are crucial for replicating (+)ss plant RNA viruses. Some chloroplast proteins, including translation initiation factor [eIF(iso)4E] and 75 DEAD-box RNA helicase RH8, help viruses fulfill their infection cycle in plants. In contrast, other chloroplast proteins such as PAP2.1, PSaC, and ATPsyn-α play active roles in plant defense against viruses. This is also consistent with the idea that reactive oxygen species, salicylic acid, jasmonic acid, and abscisic acid are produced in chloroplast. However, knowledge of molecular mechanisms and functions underlying these chloroplast host factors during the virus infection is still scarce and remains largely unknown. Our review briefly summarizes the latest knowledge regarding the possible role of chloroplast in plant virus replication, emphasizing chloroplast-related proteins. We have highlighted current advances regarding chloroplast-related proteins’ role in replicating plant (+)ss RNA viruses.

Human noroviruses (HuNVs) are the leading cause of gastroenteritis worldwide. NS1.2 is critical for HuNV pathogenesis, but the function is still unclear. The GII NS1.2 of HuNVs, unlike GI NS1.2, was localized to the endoplasmic reticulum (ER) and lipid droplets (LDs) and is accompanied by a distorted-filamentous ER morphology and aggregated-enlarged LDs. LC3 was recruited to the NS1.2-localized membrane through an autophagy-independent pathway. NS1.2, expressed from a cDNA clone of GII.4 norovirus, formed complexes with NTPase and NS4, which exhibited aggregated vesicle-like structures that were also colocalized with LC3 and LDs. NS1.2 is structurally divided into three domains from the N terminus: an inherently disordered region (IDR), a region that contains a putative hydrolase with the H-box/NC catalytic center (H-box/NC), and a C-terminal 251–330 a.a. region containing membrane-targeting domain. All three functional domains of NS1.2 were required for the induction of the filamentous ER. The IDR was essential for LC3 recruitment by NS1.2. Both the H-Box/NC and membrane-targeting domains are required for the induction of aggregated-enlarged LDs, NS1.2 self-assembly, and interaction with NTPase. The membrane-targeting domain was sufficient to interact with NS4. The study characterized the NS1.2 domain required for membrane targeting and protein–protein interactions, which are crucial for forming a viral replication complex. [ABSTRACT FROM AUTHOR]

The spread of mosquito-borne Zika virus (ZIKV), which causes neurological disorders and microcephaly, highlights the need for countermeasures against sudden viral epidemics. Here, we tested the concept that drugs targeting host proteostasis provide effective antivirals. We show that different cytosolic Hsp70 isoforms are recruited to ZIKV-induced compartments and are required for virus replication at pre- and post-entry steps. Drugs targeting Hsp70 significantly reduce replication of different ZIKV strains in human and mosquito cells, including human neural stem cells and a placental trophoblast cell line, at doses without appreciable toxicity to the host cell. By targeting several ZIKV functions, including entry, establishment of active replication complexes, and capsid assembly, Hsp70 inhibitors are refractory to the emergence of drug-resistant virus. Importantly, these drugs protected mouse models from ZIKV infection, reducing viremia, mortality, and disease symptoms. Hsp70 inhibitors are thus attractive candidates for ZIKV therapeutics with the added benefit of a broad spectrum of action.

Replication and movement are two critical steps in plant virus infection. Recent advances in the understanding of the architecture and subcellular localization of virus‐induced inclusions and the interactions between viral replication complex (VRC) and movement proteins (MPs) allow for the dissection of the intrinsic relationship between replication and movement, which has revealed that recruitment of VRCs to the plasmodesma (PD) via direct or indirect MP‐VRC interactions is a common strategy used for cell‐to‐cell movement by most plant RNA viruses. In this review, we summarize the recent advances in the understanding of virus‐induced inclusions and their roles in virus replication and cell‐to‐cell movement, analyze the advantages of such coreplicational movement from a viral point of view and discuss the possible mechanical force by which MPs drive the movement of virions or viral RNAs through the PD. Finally, we highlight the missing pieces of the puzzle of viral movement that are especially worth investigating in the near future. [ABSTRACT FROM AUTHOR]

All intracellular pathogens critically depend on host cell organelles and metabolites for successful infection and replication. One hallmark of positive-strand RNA viruses is to induce alterations of the (endo)membrane system in order to shield their double-stranded RNA replication intermediates from detection by the host cell’s surveillance systems. This spatial seclusion also allows for accruing host and viral factors and building blocks required for efficient replication of the genome and prevents access of antiviral effectors. Even though the principle is iterated by almost all positive-strand RNA viruses infecting plants and animals, the specific structure and the organellar source of membranes differs. Here, we discuss the characteristic ultrastructural features of the virus-induced membranous replication organelles in plant and animal cells and the scientific progress gained by advanced microscopy methods.

Porcine epidemic diarrhea virus (PEDV) requires complete dependence on the metabolic system of the host cell to complete its life cycle. There is a strong link between efficient viral replication and cellular lipid synthesis. However, the mechanism by which PEDV interacts with host cells to hijack cellular lipid metabolism to promote its replication remains unclear. In this study, PEDV infection significantly enhanced the expression of lipid synthesis-related genes and increased cellular lipid accumulation. Furthermore, using liquid chromatography-tandem mass spectrometry, we identified heterogeneous nuclear ribonucleoprotein A3 (HNRNPA3) as the interacting molecule of PEDV NSP9. We demonstrated that the expression of HNRNPA3 was downregulated by PEDV-induced miR-218-5p through targeting its 3' untranslated region. Interestingly, knocking down HNRNPA3 facilitated the PEDV replication by promoting cellular lipid synthesis. We next found that the knockdown of HNRNPA3 potentiated the transcriptional activity of sterol regulatory element-binding transcription factor 1 (SREBF1) through zinc finger protein 135 (ZNF135) as well as PI3K/AKT and JNK signaling pathways. In summary, we propose a model in which PEDV downregulates HNRNPA3 expression to promote the expression and activation of SREBF1 and increase cellular lipid accumulation, providing a novel mechanism by which PEDV interacts with the host to utilize cellular lipid metabolism to promote its replication.IMPORTANCEAs the major components and structural basis of the viral replication complexes of positive-stranded RNA viruses, lipids play an essential role in viral replication. However, how PEDV manipulates host cell lipid metabolism to promote viral replication by interacting with cell proteins remains poorly understood. Here, we found that SREBF1 promotes cellular lipid synthesis, which is essential for PEDV replication. Moreover, HNRNPA3 negatively regulates SREBF1 activation and specifically reduces lipid accumulation, ultimately inhibiting PEDV dsRNA synthesis. Our study provides new insight into the mechanisms by which PEDV hijacks cell lipid metabolism to benefit viral replication, which can offer a potential target for therapeutics against PEDV infection., Competing Interests: The authors declare no conflict of interest.

Chikungunya virus (CHIKV) is a re-emerging, pathogenic alphavirus that is transmitted to humans by Aedes spp. mosquitoes—causing fever and debilitating joint pain, with frequent long-term health implications and high morbidity. The CHIKV lifecycle is poorly understood and specific antiviral therapeutics or vaccines are lacking. In this study, we investigated the role of host-cell chloride (Cl-) channels on CHIKV replication.We demonstrate that specific pharmacological Cl- channel inhibitors significantly inhibit CHIKV replication in a dose-dependent manner, suggesting that Cl-channels are pro-viral factors in human cells. Further analysis of the effect of the inhibitors on CHIKV attachment, entry, viral protein expression and replicon replication demonstrated that Cl- channels are specifically required for efficient CHIKV genome replication. This was conserved in mosquito cells, where CHIKV replication and genome copy number was significantly reduced following Cl- channel inhibition. siRNA silencing identified chloride intracellular channels 1 and 4 (CLIC1 and CLIC4, respectively) as required for efficient CHIKV replication and protein affinity chromatography showed low levels of CLIC1 in complex with CHIKV nsP3, an essential component of the viral replication machinery. In summary, for the first time we demonstrate that efficient replication of the CHIKV genome depends on cellular Cl- channels, in both human and mosquito cells and identifies CLIC1 and CLIC4 as agonists of CHIKV replication in human cells. We observe a modest interaction, either direct or indirect, between CLIC1 and nsP3 and hypothesize that CLIC1 may play a role in the formation/maintenance of CHIKV replication complexes. These findings advance our molecular understanding of CHIKV replication and identify potential druggable targets for the treatment and prevention of CHIKV mediated disease. [ABSTRACT FROM AUTHOR]

G3BP-1 and -2 (hereafter referred to as G3BP) are multifunctional RNA-binding proteins involved in stress granule (SG) assembly. Viruses from diverse families target G3BP for recruitment to replication or transcription complexes in order to block SG assembly but also to acquire pro-viral effects via other unknown functions of G3BP. The Old World alphaviruses, including Semliki Forest virus (SFV) and chikungunya virus (CHIKV) recruit G3BP into viral replication complexes, via an interaction between FGDF motifs in the C-terminus of the viral non-structural protein 3 (nsP3) and the NTF2-like domain of G3BP. To study potential proviral roles of G3BP, we used human osteosarcoma (U2OS) cell lines lacking endogenous G3BP generated using CRISPR-Cas9 and reconstituted with a panel of G3BP1 mutants and truncation variants. While SFV replicated with varying efficiency in all cell lines, CHIKV could only replicate in cells expressing G3BP1 variants containing both the NTF2-like and the RGG domains. The ability of SFV to replicate in the absence of G3BP allowed us to study effects of different domains of the protein. We used immunoprecipitation to demonstrate that that both NTF2-like and RGG domains are necessary for the formation a complex between nsP3, G3BP1 and the 40S ribosomal subunit. Electron microscopy of SFV-infected cells revealed that formation of nsP3:G3BP1 complexes via the NTF2-like domain was necessary for clustering of cytopathic vacuoles (CPVs) and that the presence of the RGG domain was necessary for accumulation of electron dense material containing G3BP1 and nsP3 surrounding the CPV clusters. Clustered CPVs also exhibited localised high levels of translation of viral mRNAs as detected by ribopuromycylation staining. These data confirm that G3BP is a ribosomal binding protein and reveal that alphaviral nsP3 uses G3BP to concentrate viral replication complexes and to recruit the translation initiation machinery, promoting the efficient translation of viral mRNAs. [ABSTRACT FROM AUTHOR]

Positive-strand RNA viruses generally assemble RNA replication complexes on rearranged host membranes. Alphaviruses, other members of the alpha-like virus superfamily, and many other positive-strand RNA viruses invaginate host membrane into vesicular RNA replication compartments, known as spherules, whose interior is connected to the cytoplasm. Brome mosaic virus (BMV) and its close relative, cowpea chlorotic mottle virus (CCMV), form spherules along the endoplasmic reticulum. BMV spherule formation and RNA replication can be fully reconstituted in S. cerevisiae, enabling many studies identifying host factors and viral interactions essential for these processes. To better define and understand the conserved, core pathways of bromovirus RNA replication, we tested the ability of CCMV to similarly support spherule formation and RNA replication in yeast. Paralleling BMV, we found that CCMV RNA replication protein 1a was the only viral factor necessary to induce spherule membrane rearrangements and to recruit the viral 2a polymerase (2apol) to the endoplasmic reticulum. CCMV 1a and 2apol also replicated CCMV and BMV genomic RNA2, demonstrating core functionality of CCMV 1a and 2apol in yeast. However, while BMV and CCMV 1a/2apol strongly replicate each others’ genomic RNA3 in plants, neither supported detectable CCMV RNA3 replication in yeast. Moreover, in contrast to plant cells, in yeast CCMV 1a/2apol supported only limited replication of BMV RNA3 (<5% of that by BMV 1a/2apol). In keeping with this, we found that in yeast CCMV 1a was significantly impaired in recruiting BMV or CCMV RNA3 to the replication complex. Overall, we show that many 1a and 2apol functions essential for replication complex assembly, and their ability to be reconstituted in yeast, are conserved between BMV and CCMV. However, restrictions of CCMV RNA replication in yeast reveal previously unknown 1a-linked, RNA-selective host contributions to the essential early process of recruiting viral RNA templates to the replication complex. [ABSTRACT FROM AUTHOR]

Positive-strand RNA viruses universally remodel host intracellular membranes to form membrane-bound viral replication complexes, where viral offspring RNAs are synthesized. In the majority of cases, viral replication proteins are targeted to and play critical roles in the modulation of the designated organelle membranes. Many viral replication proteins do not have transmembrane domains, but contain single or multiple amphipathic alpha-helices. It has been conventionally recognized that these helices serve as an anchor for viral replication protein to be associated with membranes. We report here that a peptide representing the amphipathic α-helix at the N-terminus of the poliovirus 2C protein not only binds to liposomes, but also remodels spherical liposomes into tubules. The membrane remodeling ability of this amphipathic alpha-helix is similar to that recognized in other amphipathic alpha-helices from cellular proteins involved in membrane remodeling, such as BAR domain proteins. Mutations affecting the hydrophobic face of the amphipathic alpha-helix severely compromised membrane remodeling of vesicles with physiologically relevant phospholipid composition. These mutations also affected the ability of poliovirus to form plaques indicative of reduced viral replication, further underscoring the importance of membrane remodeling by the amphipathic alpha-helix in possible relation to the formation of viral replication complexes.

• DnaJA2 interacts with NS3 protein of JEV via its C-terminal. • Overexpression of DnaJA2 promotes JEV infection. • Knockdown of DnaJA2 suppresses JEV infection. • C-terminal retains the function of full-length protein to promote JEV infection. Numerous studies have documented that the interaction of viral and cellular proteins is essential in the viral life cycle. In our previous study, to screen cellular proteins that take part in the life cycle of JEV, cellular proteins that interacted with JEV NS3 were identified by Co-immunoprecipitation coupled with mass spectrometry analysis (Co-IP-MS), the results showed that ILF2, DnaJA1, DnaJA2, CKB, TUFM, and PABPC1 that putatively interact with NS3. Another candidate protein, DnaJA2, which interacted with JEV NS3 protein, was selected for further study. Overexpression of DnaJA2 increased JEV infection. Conversely, the knockdown of DnaJA2 suppressed JEV infection. Furthermore, DnaJA2 interacted with NS5 besides NS3 and colocalized with viral dsRNA. Additionally, the level of viral NS3 protein expression was higher in cells overexpressing DnaJA2 than in cells with empty vector expression, whereas DnaJA2 knockdown resulted in NS3 protein degradation, which was subsequently restored by MG132 treatment. Further analysis revealed that the C-terminal of DnaJA2 was a critical domain for interaction with NS3 and promoted JEV infection. Collectively, our study identified DnaJA2 as an essential host factor required for JEV infection, potentially representing a novel therapeutic target for the development of antiviral therapies against JEV. [ABSTRACT FROM AUTHOR]

Genome replication in flavivirus requires (—) strand RNA synthesis, (+) strand RNA synthesis, and 5’-RNA capping and methylation. To carry out viral genome replication, flavivirus assembles a replication complex, consisting of both viral and host proteins, on the cytoplasmic side of the endoplasmic reticulum (ER) membrane. Two major components of the replication complex are the viral non-structural (NS) proteins NS3 and NS5. Together they possess all the enzymatic activities required for genome replication, yet how these activities are coordinated during genome replication is not clear. We provide an overview of the flaviviral genome replication process, the membrane-bound replication complex, and recent crystal structures of full-length NS5. We propose a model of how NS3 and NS5 coordinate their activities in the individual steps of (—) RNA synthesis, (+) RNA synthesis, and 5’-RNA capping and methylation.

Hepatitis C virus (HCV) infection is known to induce autophagosome accumulation as observed by the typical punctate cytoplasmic distribution of LC3B-II in infected cells. Previously, we showed that viral RNA-dependent RNA polymerase (NS5B) interacts with ATG5, a major component of the autophagy elongation complex that is involved in the formation of double-membrane vesicles (DMV), and demonstrated that the autophagy elongation complex (ATG5-12/16L1) but not LC3B is required for proper membranous web formation. In this study, the colocalization and in situ interaction of all HCV replicase components with the constituent of the autophagy elongation complex and LC3B were analyzed. The results clearly show the recruitment of the elongation complex to the site of viral replication. Using in situ proximity ligation assay, we show that ATG5, but not ATG16L1, interacts with several HCV replicase components suggesting that the recruitment is directed via the ATG5-12 conjugate. Interestingly, no E3-like conjugation activity of ATG5-12/16L1 can be detected at the at HCV replication site since LC3B-II is not found along with the elongation complex at the site of viral replication. In agreement with this result, no sign of in situ interaction of LC3B with the replicase components is observed. Finally, using dominant negative forms of ATG proteins, we demonstrate that ATG5-12 conjugate, but not LC3-II formation, is critical for viral replication. Altogether, these findings suggest that although HCV needs the elongation complex for its replication, it has developed a mechanism to avoid canonical LC3-II accumulation at viral replication site. [ABSTRACT FROM AUTHOR]

Positive-sense RNA virus intracellular replication is intimately associated with membrane platforms that are derived from host organelles and comprised of distinct lipid composition. For flaviviruses, such as West Nile virus strain Kunjin virus (WNVKUN) we have observed that these membrane platforms are derived from the endoplasmic reticulum and are rich in (at least) cholesterol. To extend these studies and identify the cellular lipids critical for WNVKUN replication we utilized a whole cell lipidomics approach and revealed an elevation in phospholipase A2 (PLA2) activity to produce lyso-phosphatidylcholine (lyso-PChol). We observed that the PLA2 enzyme family is activated in WNVKUN-infected cells and the generated lyso-PChol lipid moieties are sequestered to the subcellular sites of viral replication. The requirement for lyso-PChol was confirmed using chemical inhibition of PLA2, where WNVKUN replication and production of infectious virus was duly affected in the presence of the inhibitors. Importantly, we could rescue chemical-induced inhibition with the exogenous addition of lyso-PChol species. Additionally, electron microscopy results indicate that lyso-PChol appears to contribute to the formation of the WNVKUN membranous replication complex (RC); particularly affecting the morphology and membrane curvature of vesicles comprising the RC. These results extend our current understanding of how flaviviruses manipulate lipid homeostasis to favour their own intracellular replication. [ABSTRACT FROM AUTHOR]

• The MET domain of PlAMV replicase interacts with NbDRP2 in Nicotiana benthamiana. • PlAMV infection induced NbDRP2 gene expression. • The accumulation level of PlAMV was decreased in the NbDRP2 knockdown plants. • PlAMV accumulation was suppressed in protoplasts treated with dynamin inhibitor. • NbDRP2 plays a proviral role during PlAMV replication. Positive-strand RNA viruses replicate their RNA in the viral replication complex, a spherical structure formed by remodeling of host intracellular membranes. This process also requires the interaction between viral membrane-associated replication proteins and host factors. We previously identified the membrane-associated determinant of the replicase of plantago asiatica mosaic virus (PlAMV), a positive-strand RNA virus of the genus Potexvirus , in its methyltransferase (MET) domain, and suggested that its interaction with host factors is required to establish viral replication. Here we identified Nicotiana benthamiana dynamin-related protein 2 (NbDRP2) as an interactor of the MET domain of the PlAMV replicase by co-immunoprecipitation (Co-IP) and mass spectrometry analysis. NbDRP2 is closely related to the DRP2 subfamily proteins in Arabidopsis thaliana , AtDRP2A and AtDRP2B. Confocal microscopy observation and Co-IP confirmed the interaction between the MET domain and NbDRP2. Also, the expression of NbDRP2 was induced by PlAMV infection. PlAMV accumulation was reduced when the expression of NbDRP2 gene was suppressed by virus-induced gene silencing. In addition, PlAMV accumulation was reduced in protoplasts treated with dynamin inhibitor. These results indicate a proviral role of the interaction of NbDRP2 with the MET domain in PlAMV replication. [ABSTRACT FROM AUTHOR]

The intricate interactions between viruses and hosts include exploitation of host cells for viral replication by using many cellular resources, metabolites and energy. Tomato bushy stunt virus (TBSV), similar to other (+)RNA viruses, induces major changes in infected cells that lead to the formation of large replication compartments consisting of aggregated peroxisomal and ER membranes. Yet, it is not known how TBSV obtains the energy to fuel these energy-consuming processes. In the current work, the authors discovered that TBSV co-opts the glycolytic ATP-generating Pgk1 phosphoglycerate kinase to facilitate the assembly of new viral replicase complexes. The recruitment of Pgk1 into the viral replication compartment is through direct interaction with the viral replication proteins. Altogether, we provide evidence that the ATP generated locally within the replication compartment by the co-opted Pgk1 is used to fuel the ATP-requirement of the co-opted heat shock protein 70 (Hsp70) chaperone, which is essential for the assembly of new viral replicase complexes and the activation of functional viral RNA-dependent RNA polymerase. The advantage of direct recruitment of Pgk1 into the virus replication compartment could be that the virus replicase assembly does not need to intensively compete with cellular processes for access to ATP. In addition, local production of ATP within the replication compartment could greatly facilitate the efficiency of Hsp70-driven replicase assembly by providing high ATP concentration within the replication compartment. [ABSTRACT FROM AUTHOR]

miR-122, a liver-specific microRNA, is one of the determinants for liver tropism of hepatitis C virus (HCV) infection. Although miR-122 is required for efficient propagation of HCV, we have previously shown that HCV replicates at a low rate in miR-122-deficient cells, suggesting that HCV-RNA is capable of propagating in an miR-122-independent manner. We herein investigated the roles of miR-122 in both the replication of HCV-RNA and the production of infectious particles by using miR-122-knockout Huh7 (Huh7-122KO) cells. A slight increase of intracellular HCV-RNA levels and infectious titers in the culture supernatants was observed in Huh7-122KO cells upon infection with HCV. Moreover, after serial passages of HCV in miR-122-knockout Huh7.5.1 cells, we obtained an adaptive mutant, HCV122KO, possessing G28A substitution in the 5’UTR of the HCV genotype 2a JFH1 genome, and this mutant may help to enhance replication complex formation, a possibility supported by polysome analysis. We also found the introduction of adaptive mutation around miR-122 binding site in the genotype 1b/2a chimeric virus, which originally had an adenine at the nucleotide position 29. HCV122KO exhibited efficient RNA replication in miR-122-knockout cells and non-hepatic cells without exogenous expression of miR-122. Competition assay revealed that the G28A mutant was dominant in the absence of miR-122, but its effects were equivalent to those of the wild type in the presence of miR-122, suggesting that the G28A mutation does not confer an advantage for propagation in miR-122-rich hepatocytes. These observations may explain the clinical finding that the positive rate of G28A mutation was higher in miR-122-deficient PBMCs than in the patient serum, which mainly included the hepatocyte-derived virus from HCV-genotype-2a patients. These results suggest that the emergence of HCV mutants that can propagate in non-hepatic cells in an miR-122-independent manner may participate in the induction of extrahepatic manifestations in chronic hepatitis C patients. [ABSTRACT FROM AUTHOR]

Dengue is the most common mosquito-borne viral disease in humans. Changes of lipid-related metabolites in endoplasmic reticulum of dengue virus (DENV) infected cells have been associated with replicative complexes formation. Previously, we reported that DENV infection inhibits HMGCR phosphorylation generating a cholesterol-enriched cellular environment in order to favor viral replication. In this work, using enzymatic assays, ELISA, and WB we found a significant higher activity of HMGCR in DENV infected cells, associated with the inactivation of AMPK. AMPK activation by metformin declined the HMGCR activity suggesting that AMPK inactivation mediates the enhanced activity of HMGCR. A reduction on AMPK phosphorylation activity was observed in DENV infected cells at 12 and 24 hpi. HMGCR and cholesterol co-localized with viral proteins NS3, NS4A and E, suggesting a role for HMGCR and AMPK activity in the formation of DENV replicative complexes. Furthermore, metformin and lovastatin (HMGCR inhibitor) altered this co-localization as well as replicative complexes formation supporting that active HMGCR is required for replicative complexes formation. In agreement, metformin prompted a significant dose-dependent antiviral effect in DENV infected cells, while compound C (AMPK inhibitor) augmented the viral genome copies and the percentage of infected cells. The PP2A activity, the main modulating phosphatase of HMGCR, was not affected by DENV infection. These data demonstrate that the elevated activity of HMGCR observed in DENV infected cells is mediated through AMPK inhibition and not by increase in PP2A activity. Interestingly, the inhibition of this phosphatase showed an antiviral effect in an HMGCR-independent manner. These results suggest that DENV infection increases HMGCR activity through AMPK inactivation leading to higher cholesterol levels in endoplasmic reticulum necessary for replicative complexes formation. This work provides new information about the mechanisms involved in host lipid metabolism during DENV replicative cycle and identifies new potential antiviral targets for DENV replication. [ABSTRACT FROM AUTHOR]

The viruses of the family Flaviviridae possess a positive-strand RNA genome and express a single polyprotein which is processed into functional proteins. Initially, the nonstructural (NS) proteins, which are not part of the virions, form complexes capable of genome replication. Later on, the NS proteins also play a critical role in virion formation. The molecular basis to understand how the same proteins form different complexes required in both processes is so far unknown. For pestiviruses, uncleaved NS2-3 is essential for virion morphogenesis while NS3 is required for RNA replication but is not functional in viral assembly. Recently, we identified two gain of function mutations, located in the C-terminal region of NS2 and in the serine protease domain of NS3 (NS3 residue 132), which allow NS2 and NS3 to substitute for uncleaved NS2-3 in particle assembly. We report here the crystal structure of pestivirus NS3-4A showing that the NS3 residue 132 maps to a surface patch interacting with the C-terminal region of NS4A (NS4A-kink region) and suggesting a critical role of this contact in virion morphogenesis. We show that destabilization of this interaction, either by alanine exchanges at residue 132 of NS3 or at neighboring residues in NS4A, led to a gain of function of the NS3/4A complex in particle formation. In contrast, RNA replication and thus replicase assembly requires a stable association between NS3 and the NS4A-kink region. Thus, we propose that two variants of NS3/4A complexes exist in pestivirus infected cells each representing a basic building block required for either RNA replication or virion morphogenesis. This could be further corroborated by trans-complementation studies with a replication-defective NS3/4A double mutant that was still functional in viral assembly. Our observations illustrate the presence of alternative overlapping surfaces providing different contacts between the same proteins, allowing the switch from RNA replication to virion formation. [ABSTRACT FROM AUTHOR]

The definition of the precise molecular composition of membranous replication compartments is a key to understanding the mechanisms of virus multiplication. Here, we set out to investigate the protein composition of the potyviral replication complexes. We purified the potyviral 6K2 protein-induced membranous structures from Potato virus A (PVA)-infected Nicotiana benthamiana plants. For this purpose, the 6K2 protein, which is the main inducer of potyviral membrane rearrangements, was expressed in fusion with an N-terminal Twin-Strep-tag and Cerulean fluorescent protein (SC6K) from the infectious PVA cDNA. A non-tagged Cerulean-6K2 (C6K) virus and the SC6K protein alone in the absence of infection were used as controls. A purification scheme exploiting discontinuous sucrose gradient centrifugation followed by Strep-tag-based affinity chromatography was developed. Both (+)- and (-)-strand PVA RNA and viral protein VPg were co-purified specifically with the affinity tagged PVA-SC6K. The purified samples, which contained individual vesicles and membrane clusters, were subjected to mass spectrometry analysis. Data analysis revealed that many of the detected viral and host proteins were either significantly enriched or fully specifically present in PVA-SC6K samples when compared with the controls. Eight of eleven potyviral proteins were identified with high confidence from the purified membrane structures formed during PVA infection. Ribosomal proteins were identified from the 6K2-induced membranes only in the presence of a replicating virus, reinforcing the tight coupling between replication and translation. A substantial number of proteins associating with chloroplasts and several host proteins previously linked with potyvirus replication complexes were co-purified with PVA-derived SC6K, supporting the conclusion that the host proteins identified in this study may have relevance in PVA replication. [ABSTRACT FROM AUTHOR]

Flavivirus nonstructural protein 5 (NS5) consists of methyltransferase (MTase) and RNA-dependent RNA polymerase (RdRp) domains, which catalyze 5’-RNA capping/methylation and RNA synthesis, respectively, during viral genome replication. Although the crystal structure of flavivirus NS5 is known, no data about the quaternary organization of the functional enzyme are available. We report the crystal structure of dengue virus full-length NS5, where eight molecules of NS5 are arranged as four independent dimers in the crystallographic asymmetric unit. The relative orientation of each monomer within the dimer, as well as the orientations of the MTase and RdRp domains within each monomer, is conserved, suggesting that these structural arrangements represent the biologically relevant conformation and assembly of this multi-functional enzyme. Essential interactions between MTase and RdRp domains are maintained in the NS5 dimer via inter-molecular interactions, providing evidence that flavivirus NS5 can adopt multiple conformations while preserving necessary interactions between the MTase and RdRp domains. Furthermore, many NS5 residues that reduce viral replication are located at either the inter-domain interface within a monomer or at the inter-molecular interface within the dimer. Hence the X-ray structure of NS5 presented here suggests that MTase and RdRp activities could be coordinated as a dimer during viral genome replication. [ABSTRACT FROM AUTHOR]

Hepatitis C virus (HCV) is a positive-strand RNA virus of the Flaviviridae family and a major cause of liver disease worldwide. HCV replicates in the cytoplasm, and the synthesis of viral proteins induces extensive rearrangements of host cell membranes producing structures, collectively termed the membranous web (MW). The MW contains the sites of viral replication and assembly, and we have identified distinct membrane fractions derived from HCV-infected cells that contain replication and assembly complexes enriched for viral RNA and infectious virus, respectively. The complex membrane structure of the MW is thought to protect the viral genome limiting its interactions with cytoplasmic pattern recognition receptors (PRRs) and thereby preventing activation of cellular innate immune responses. Here we show that PRRs, including RIG-I and MDA5, and ribosomes are excluded from viral replication and assembly centers within the MW. Furthermore, we present evidence that components of the nuclear transport machinery regulate access of proteins to MW compartments. We show that the restricted assess of RIG-I to the MW can be overcome by the addition of a nuclear localization signal sequence, and that expression of a NLS-RIG-I construct leads to increased immune activation and the inhibition of viral replication. [ABSTRACT FROM AUTHOR]

Plant viruses are responsible for worldwide production losses of numerous economically important crops. The most common plant RNA viruses are positivesense single-stranded RNA viruses [(+)ss RNA viruses]. These viruses have small genomes that encode a limited number of proteins. The viruses depend on their host's machinery for the replication of their RNA genome, assembly, movement, and attraction to the vectors for dispersal. Recently researchers have reported that chloroplast proteins are crucial for replicating (+)ss plant RNA viruses. Some chloroplast proteins, including translation initiation factor [eIF(iso)4E] and 75 DEAD-box RNA helicase RH8, help viruses fulfill their infection cycle in plants. In contrast, other chloroplast proteins such as PAP2.1, PSaC, and ATPsyn-α play active roles in plant defense against viruses. This is also consistent with the idea that reactive oxygen species, salicylic acid, jasmonic acid, and abscisic acid are produced in chloroplast. However, knowledge of molecular mechanisms and functions underlying these chloroplast host factors during the virus infection is still scarce and remains largely unknown. Our review briefly summarizes the latest knowledge regarding the possible role of chloroplast in plant virus replication, emphasizing chloroplast-related proteins. We have highlighted current advances regarding chloroplast-related proteins' role in replicating plant (+)ss RNA viruses.

Double-stranded RNA (dsRNA) is an important type of RNA that plays essential roles in diverse cellular processes in eukaryotic organisms and a hallmark in infections by positive-sense RNA viruses. Currently, no in vivo technology has been developed for visualizing dsRNA in living cells. Here, we report a dsRNA binding-dependent fluorescence complementation (dRBFC) assay that can be used to efficiently monitor dsRNA distribution and dynamics in vivo . The system consists of two dsRNA-binding proteins, which are fused to the N- and C-terminal halves of the yellow fluorescent protein (YFP). Binding of the two fusion proteins to a common dsRNA brings the split YFP halves in close proximity, leading to the reconstitution of the fluorescence-competent structure and restoration of fluorescence. Using this technique, we were able to visualize the distribution and trafficking of the replicative RNA intermediates of positive-sense RNA viruses in living cells. [ABSTRACT FROM AUTHOR]