Nucleolin interacts with the feline calicivirus 3' untranslated region and the protease-polymerase NS6 and NS7 proteins, playing a role in virus replication

Cellular proteins play many important roles during the life cycle of all viruses. Specifically, host cell nucleic acid-binding proteins interact with viral components of positive-stranded RNA viruses and regulate viral translation, as well as RNA replication. Here, we report that nucleolin, a ubiquitous multifunctional nucleolar shuttling phosphoprotein, interacts with the Norwalk virus and feline calicivirus (FCV) genomic 3' untranslated regions (UTRs). Nucleolin can also form a complex in vitro with recombinant Norwalk virus NS6 and -7 (NS6/7) and can be copurified with the analogous protein from feline calicivirus (p76 or NS6/7) from infected feline kidney cells. Nucleolin RNA levels or protein were not modified during FCV infection; however, as a consequence of the infection, nucleolin was seen to relocalize from the nucleoli to the nucleoplasm, as well as to the perinuclear area where it colocalizes with the feline calicivirus NS6/7 protein. In addition, antibodies to nucleolin were able to precipitate viral RNA from feline calicivirus-infected cells, indicating a direct or indirect association of nucleolin with the viral RNA during virus replication. Small interfering RNA (siRNA)-mediated knockdown of nucleolin resulted in a reduction of the cytopathic effect and virus yield in CrFK cells. Taken together, these results demonstrate that nucleolin is a nucleolar component that interacts with viral RNA and NS6/7 and is required for feline calicivirus replication.     

An RNA-binding protein, hnRNP A1, and a scaffold protein, septin 6, facilitate hepatitis C virus replication

Hepatitis C virus (HCV) is a positive-sense single-stranded RNA virus. NS5b is an RNA-dependent RNA polymerase that polymerizes the newly synthesized RNA. HCV likely uses host proteins for its replication, similar to other RNA viruses. To identify the cellular factors involved in HCV replication, we searched for cellular proteins that interact with the NS5b protein. HnRNP A1 and septin 6 proteins were identified by coimmunoprecipitation and yeast two-hybrid screening, respectively. Interestingly, septin 6 protein also interacts with hnRNP A1. Moreover, hnRNP A1 interacts with the 5'-nontranslated region (5' NTR) and the 3' NTR of HCV RNA containing the cis-acting elements required for replication. Knockdown of hnRNP A1 and overexpression of C-terminally truncated hnRNP A1 reduced HCV replication. In addition, knockdown of septin 6 and overexpression of N-terminally truncated septin 6 inhibited HCV replication. These results indicate that the host proteins hnRNP A1 and septin 6 play important roles in the replication of HCV through RNA-protein and protein-protein interactions.     

Nuclear factors are involved in hepatitis C virus RNA replication

Unraveling the molecular basis of the life cycle of hepatitis C virus (HCV), a prevalent agent of human liver disease, entails the identification of cell-encoded factors that participate in the replication of the viral RNA genome. This study provides evidence that the so-called NF/NFAR proteins, namely, NF90/NFAR-1, NF110/NFAR-2, NF45, and RNA helicase A (RHA), which mostly belong to the dsRBM protein family, are involved in the HCV RNA replication process. NF/NFAR proteins were shown to specifically bind to replication signals in the HCV genomic 5' and 3' termini and to promote the formation of a looplike structure of the viral RNA. In cells containing replicating HCV RNA, the generally nuclear NF/NFAR proteins accumulate in the cytoplasmic viral replication complexes, and the prototype NFAR protein, NF90/NFAR-1, stably interacts with a viral protein. HCV replication was inhibited in cells where RNAi depleted RHA from the cytoplasm. Likewise, HCV replication was hindered in cells that contained another NF/NFAR protein recruiting virus. The recruitment of NF/NFAR proteins by HCV is assumed to serve two major purposes: to support 5'-3' interactions of the viral RNA for the coordination of viral protein and RNA synthesis and to weaken host-defense mechanisms.     

Interaction of the host protein NbDnaJ with Potato virus X minus-strand stem-loop 1 RNA and capsid protein affects viral replication and movement

Plant viruses must interact with host cellular components to replicate and move from cell to cell. In the case of Potato virus X (PVX), it carries stem-loop 1 (SL1) RNA essential for viral replication and movement. Using two-dimensional electrophoresis northwestern blot analysis, we previously identified several host proteins that bind to SL1 RNA. Of those, we further characterized a DnaJ-like protein from Nicotiana benthamiana named NbDnaJ. An electrophoretic mobility shift assay confirmed that NbDnaJ binds only to SL1 minus-strand RNA, and bimolecular fluorescence complementation (BiFC) indicated that NbDnaJ interacts with PVX capsid protein (CP). Using a series of deletion mutants, the C-terminal region of NbDnaJ was found to be essential for the interaction with PVX CP. The expression of NbDnaJ significantly changed upon infection with different plant viruses such as PVX, Tobacco mosaic virus, and Cucumber mosaic virus, but varied depending on the viral species. In transient experiments, both PVX replication and movement were inhibited in plants that over-expressed NbDnaJ but accelerated in plants in which NbDnaJ was silenced. In summary, we suggest that the newly identified NbDnaJ plays a role in PVX replication and movement by interacting with SL1(-) RNA and PVX CP.     

Human DDX56 protein interacts with influenza A virus NS1 protein and stimulates the virus replication

Influenza A viruses (IAV) are enveloped viruses carrying a single-stranded negative-sense RNA genome. Detection of host proteins having a relationship with IAV and revealing of the role of these proteins in the viral replication are of great importance in keeping IAV infections under control. Consequently, the importance of human DDX56, which is determined to be associated with a viral NS1 with a yeast two-hybrid assay, was investigated for IAV replication. The viral replication in knocked down cells for the DDX56 gene was evaluated. The NS1 was co-precipitated with the DDX56 protein in lysates of cells transiently expressing DDX56 and NS1 or infected with the viruses, showing that NS1 and DDX56 interact in mammalian cells. Viral NS1 showed a tendency to co-localize with DDX56 in the cells, transiently expressing both of these proteins, which supports the IP and two-hybrid assays results. The data obtained with in silico predictions supported the in vitro protein interaction results. The viral replication was significantly reduced in the DDX56-knockdown cells comparing with that in the control cells. In conclusion, human DDX56 protein interacts with the IAV NS1 protein in both yeast and mammalian cells and has a positive regulatory effect on IAV replication. However, the mechanism of DDX56 on IAV replication requires further elucidation.     

The NS1 Protein of Influenza A Virus Interacts with Cellular Processing Bodies and Stress Granules through RNA-Associated Protein 55 (RAP55) during Virus Infection

The nonstructural protein (NS1) of influenza A virus performs multiple functions in the virus life cycle. Proteomic screening for cellular proteins which interact with NS1 identified the cellular protein RAP55, which is one of the components of cellular processing bodies (P-bodies) and stress granules. To verify whether NS1 interacts with cellular P-bodies, interactions between NS1, RAP55, and other P-body-associated proteins (Ago1, Ago2, and DCP1a) were confirmed using coimmunoprecipitation and cellular colocalization assays. Overexpression of RAP55 induced RAP55-associated stress granule formation and suppressed virus replication. Knockdown of RAP55 with small interfering RNA (siRNA) or expression of a dominant-negative mutant RAP55 protein with defective interaction with P-bodies blocked NS1 colocalization to P-bodies in cells. Expression of NS1 inhibited RAP55 expression and formation of RAP55-associated P-bodies/stress granules. The viral nucleoprotein (NP) was found to be targeted to stress granules in the absence of NS1 but localized to P-bodies when NS1 was coexpressed. Restriction of virus replication via P-bodies occurred in the early phases of infection, as the number of RAP55-associated P-bodies in cells diminished over the course of virus infection. NS1 interaction with RAP55-associated P-bodies/stress granules was associated with RNA binding and mediated via a protein kinase R (PKR)-interacting viral element. Mutations introduced into either RNA binding sites (R38 and K41) or PKR interaction sites (I123, M124, K126, and N127) caused NS1 proteins to lose the ability to interact with RAP55 and to inhibit stress granules. These results reveal an interplay between virus and host during virus replication in which NP is targeted to P-bodies/stress granules while NS1 counteracts this host restriction mechanism.     

Physical and functional interaction between hepatitis C virus NS5A protein and ovarian tumor protein deubiquitinase 7B

Hepatitis C virus (HCV) NS5A protein plays crucial roles in viral RNA replication, virus assembly, and viral pathogenesis. Although NS5A has no known enzymatic activity, it modulates various cellular pathways through interaction with cellular proteins. HCV NS5A (and other HCV proteins) are reportedly degraded through the ubiquitin–proteasome pathway; however, the physiological roles of ubiquitylation and deubiquitylation in HCV infection are largely unknown. To elucidate the role of deubiquitylation in HCV infection, an attempt was made to identify a deubiquitinase (DUB) that can interact with NS5A protein. An ovarian tumor protein (OTU), deubiquitinase 7B (OTUD7B), was identified as a novel NS5A‐binding protein. Co‐immunoprecipitation analyses showed that NS5A interacts with OTUD7B in both Huh‐7 and HCV RNA replicon cells. Immunofluorescence staining revealed that HCV NS5A protein colocalizes with OTUD7B in the cytoplasm. Moreover, HCV infection was found to enhance the nuclear localization of OTUD7B. The OTUD7B‐binding domain on NS5A was mapped using a series of NS5A deletion mutants. The present findings suggest that the domain I of NS5A is important and the region from amino acid 121 to 126 of NS5A essential for the interaction. Either V121A or V124A mutation in NS5A disrupts the NS5A‐OTUD7B interaction. The results of this in vivo ubiquitylation assay suggest that HCV NS5A enhances OTUD7B DUB activity. Taken together, these results suggest that HCV NS5A protein interacts with OTUD7B, thereby modulating its DUB activity.     

Nonstructural protein 5B of hepatitis C virus

Since its identification in 1989, hepatitis C virus has been the subject of extensive research. The biology of the virus and the development of antiviral drugs are closely related. The RNA polymerase activity of nonstructural protein 5B was first demonstrated in 1996. NS5B is believed to localize to the perinuclear region, forming a replicase complex with other viral proteins. It has a typical polymerase structure with thumb, palm, and finger domains encircling the active site. A de novo replication initiation mechanism has been suggested. To date, many small molecule inhibitors are known including nucleoside analogues, non-nucleoside analogues, and pyrophosphate mimics. NS5B interacts with other viral proteins such as core, NS3, 4A, 4B, and 5A. The helicase activity of NS3 seems necessary for RNA strand unwinding during replication, with other nonstructural proteins performing modulatory roles. Cellular proteins interacting with NS5B include VAMP-associated proteins, heIF4AII, hPLIC1, nucleolin, PRK2, a-actinin, and p68 helicase. The interactions of NS5B with these proteins might play roles in cellular trafficking, signal transduction, and RNA polymerization, as well as the regulation of replication/translation processes.     

Persistence of bovine viral diarrhea virus is determined by a cellular cofactor of a viral autoprotease

Polyprotein processing control is a crucial step in the life cycle of positive-strand RNA viruses. Recently, a vital autoprotease generating an essential viral replication factor was identified in such a virus, namely, the pestivirus bovine viral diarrhea virus. Surprisingly, the activity of this protease, which resides in nonstructural protein 2 (NS2), diminishes early after infection, resulting in the limitation of viral RNA replication. Here, we describe that a cellular chaperone termed Jiv (J-domain protein interacting with viral protein) acts as a cofactor of the NS2 protease. Consumption of the intracellular Jiv pool is responsible for temporal regulation of protease activity: overexpression of Jiv interfered with regulation and correlated with increased accumulation of viral RNA; downregulation of the cellular Jiv level accelerated the decline of protease activity and reduced intracellular viral RNA levels and virion production. Accordingly, the amount of a cellular protein controls pestiviral replication by limiting the generation of active viral protease molecules and replication complexes. Importantly, this unique mechanism of replication control is essential for maintenance of the noncytopathogenic phenotype of the virus and thereby for its ability to establish persistent infections. These results add an entirely novel aspect to the understanding of the molecular basis of viral persistence.     

Cellular Vimentin Regulates Construction of Dengue Virus Replication Complexes through Interaction with NS4A Protein

Dengue virus (DENV) interacts with host cellular factors to construct a more favorable environment for replication, and the interplay between DENV and the host cellular cytoskeleton may represent one of the potential antiviral targeting sites. However, the involvement of cellular vimentin intermediate filaments in DENV replication has been explored less. Here, we revealed the direct interaction between host cellular vimentin and DENV nonstructural protein 4A (NS4A), a known component of the viral replication complex (RC), during DENV infection using tandem affinity purification, coimmunoprecipitation, and scanning electron microscopy. Furthermore, the dynamics of vimentin-NS4A interaction were demonstrated by using confocal three-dimensional (3D) reconstruction and proximity ligation assay. Most importantly, we report for the first time the discovery of the specific region of NS4A that interacts with vimentin lies within the first 50 amino acid residues at the cytosolic N-terminal domain of NS4A (N50 region). Besides identifying vimentin-NS4A interaction, vimentin reorganization and phosphorylation by calcium calmodulin-dependent protein kinase II occurs during DENV infection, signifying that vimentin reorganization is important in maintaining and supporting the DENV RCs. Interestingly, we found that gene silencing of vimentin by small interfering RNA induced a significant alteration in the distribution of RCs in DENV-infected cells. This finding further supports the crucial role of intact vimentin scaffold in localizing and concentrating DENV RCs at the perinuclear site, thus facilitating efficient viral RNA replication. Collectively, our findings implicate the biological and functional significance of vimentin during DENV replication, as we propose that the association of DENV RCs with vimentin is mediated by DENV NS4A.