The RNA-unwinding activity of hepatitis C virus non-structural protein 3 (NS3) is positively modulated by its protease domain

The nonstructural protein 3 (NS3) of hepatitis C virus contains a protease domain at its amino terminus and RNA helicase domain at its carboxyl terminus. To identify optimal NS3 protein for developing screening assays, we expressed full-length NS3 protease/helicase and helicase domains from both HCV type 1a (H77 strain) and 1b (Con1 strain), using either E. coli or baculovirus expression systems. Our studies showed that the full-length NS3 proteins, either with or without the presence of the NS4A domain, from either strains were at least 10-fold more efficient than the corresponding helicase domains in unwinding partial duplex RNA substrates. These findings provide a rationale for the use of full-length NS3 in high throughput screening assays to identify potent small molecule inhibitors of this important target of HCV.     

A CCHC metal-binding domain in Nanos is essential for translational regulation.

The Drosophila Nanos protein is a localized repressor of hunchback mRNA translation in the early embryo, and is required for the establishment of the anterior-posterior body axis. Analysis of nanos mutants reveals that a small, evolutionarily conserved, C-terminal region is essential for Nanos function in vivo, while no other single portion of the Nanos protein is absolutely required. Within the C-terminal region are two unusual Cys-Cys-His-Cys (CCHC) motifs that are potential zinc-binding sites. Using absorption spectroscopy and NMR we demonstrate that the CCHC motifs each bind one equivalent of zinc with high affinity. nanos mutations disrupting metal binding at either of these two sites in vitro abolish Nanos translational repression activity in vivo. We show that full-length and C-terminal Nanos proteins bind to RNA in vitro with high affinity, but with little sequence specificity. Mutations affecting the hunchback mRNA target sites for Nanos-dependent translational repression were found to disrupt translational repression in vivo, but had little effect on Nanos RNA binding in vitro. Thus, the Nanos zinc domain does not specifically recognize target hunchback RNA sequences, but might interact with RNA in the context of a larger ribonucleoprotein complex.     

Bluetongue virus non-structural protein 1 is a positive regulator of viral protein synthesis

ABSTRACT: BACKGROUND: Bluetongue virus (BTV) is a double-stranded RNA (dsRNA) virus of the Reoviridae family, which encodes its genes in ten linear dsRNA segments. BTV mRNAs are synthesised by the viral RNA-dependent RNA polymerase (RdRp) as exact plus sense copies of the genome segments. Infection of mammalian cells with BTV rapidly replaces cellular protein synthesis with viral protein synthesis, but the regulation of viral gene expression in the Orbivirus genus has not been investigated. RESULTS: Using an mRNA reporter system based on genome segment 10 of BTV fused with GFP we identify the protein characteristic of this genus, non-structural protein 1 (NS1) as sufficient to upregulate translation. The wider applicability of this phenomenon among the viral genes is demonstrated using the untranslated regions (UTRs) of BTV genome segments flanking the quantifiable Renilla luciferase ORF in chimeric mRNAs. The UTRs of viral mRNAs are shown to be determinants of the amount of protein synthesised, with the pre-expression of NS1 increasing the quantity in each case. The increased expression induced by pre-expression of NS1 is confirmed in virus infected cells by generating a replicating virus which expresses the reporter fused with genome segment 10, using reverse genetics. Moreover, NS1-mediated upregulation of expression is restricted to mRNAs which lack the cellular 3[PRIME] poly(A) sequence identifying the 3[PRIME] end as a necessary determinant in specifically increasing the translation of viral mRNA in the presence of cellular mRNA. CONCLUSIONS: NS1 is identified as a positive regulator of viral protein synthesis. We propose a model of translational regulation where NS1 upregulates the synthesis of viral proteins, including itself, and creates a positive feedback loop of NS1 expression, which rapidly increases the expression of all the viral proteins. The efficient translation of viral reporter mRNAs among cellular mRNAs can account for the observed replacement of cellular protein synthesis with viral protein synthesis during infection.     

Aichi virus 2A protein is involved in viral RNA replication

The Aichi virus 2A protein is not a protease, unlike many other picornavirus 2A proteins, and it is related to a cellular protein, H-rev107. Here, we examined the replication properties of two 2A mutants in Vero cells and a cell-free translation/replication system. In one mutant, amino acids 36 to 126 were replaced with an unrelated amino acid sequence. In the other mutant, the NC motif conserved in the H-rev107 family of proteins was changed to alanine residues. The two mutations abolished virus replication in cells. The mutations affected both negative- and positive-strand synthesis, the defect in positive-strand synthesis being more severe than that in negative-strand synthesis.     

The varicella-zoster virus ORF66 protein induces kinase activity and is dispensable for viral replication.

Varicella-zoster virus (VZV) open reading frames (ORFs) 47 and 66 encode proteins that are homologous to a family of eukaryotic serine-threonine kinases. Prior studies showed that the VZV ORF47 protein has kinase activity in vitro and is dispensable for replication in cultured cells. To examine the role of the ORF66 protein during infection, we constructed VZV recombinants that are unable to express either the ORF66 protein (ROka 66S) or both the ORF47 and ORF66 proteins (ROka 47S/66S). VZV unable to express ORF66 grew to titers similar to those of the parental VZV (ROka) in vitro; however, VZV lacking both ORF66 and ORF47 grew to titers lower than those of ROka. Nuclear extracts from ROka 66S- or ROka 47S-infected cells showed a 48-kDa phosphoprotein(s); a phosphoprotein with a similar size was not present in nuclear extracts from ROka 47S/66S-infected cells. To determine the role of the ORF66 protein in the phosphorylation of specific VZV-encoded proteins, we immunoprecipitated known VZV phosphoproteins (ORF4, ORF62, ORF63, and ORF68 proteins) from nuclear extracts of phosphate-labeled cells infected with ROka, ROka 66S, or ROka 47S/66S. Each of the VZV phosphoproteins was phosphorylated to a similar extent in the presence or absence of either the ORF66 protein or both the ORF66 and ORF47 proteins. From these studies we conclude (i) neither ORF66 alone nor ORF66 and ORF47 in combination are essential for VZV growth in cultured cells, (ii) ORF66 either is a protein kinase or induces protein kinase activity during infection, and (iii) the VZV phosphoproteins encoded by ORF4, ORF62, ORF63, and ORF68 do not require either ORF66 alone or ORF66 and ORF47 for phosphorylation in vitro.     

The Epstein-Barr virus glycoprotein 110 carboxy-terminal tail domain is essential for lytic virus replication.

To investigate the importance of the Epstein-Barr virus (EBV) glycoprotein 110 (gp110) tail domain in the intracellular localization of gp110 and virus lytic replication, three carboxy-terminal truncation mutants of gp110 were constructed. Deletion of 16 amino acids from the carboxyl-terminal tail resulted in gp110 intracellular localization which was indistinguishable from that of wild-type gp110, whereas deletion of either 41 or 56 amino acids from the carboxyl-terminal tail of gp110 resulted in loss of retention of gp110 in the endoplasmic reticulum and nuclear membrane. None of the gp110 truncation mutants was able to complement EBV(gp110-)+ lymphoblastoid cell lines in transformation assays, indicating the importance of the gp110 tail domain in virus lytic replication. In electron microscopy analysis, no nucleocapsids or enveloped viruses were detected in EBV(gp110-)+ lymphoblastoid cell lines induced for lytic replication.     

A Rab-GAP TBC domain protein binds hepatitis C virus NS5A and mediates viral replication

Hepatitis C virus (HCV) is an important cause of liver disease worldwide. Current therapies are inadequate for most patients. Using a two-hybrid screen, we isolated a novel cellular binding partner interacting with the N terminus of HCV nonstructural protein NS5A. This partner contains a TBC Rab-GAP (GTPase-activating protein) homology domain found in all known Rab-activating proteins. As the first described interaction between such a Rab-GAP and a viral protein, this finding suggests a new mechanism whereby viruses may subvert host cell machinery for mediating the endocytosis, trafficking, and sorting of their own proteins. Moreover, depleting the expression of this partner severely impairs HCV RNA replication with no obvious effect on cell viability. These results suggest that pharmacologic disruption of this NS5A-interacting partner can be contemplated as a potential new antiviral strategy against a pathogen affecting nearly 3% of the world's population.     

Heat shock cognate protein 70 controls Borna disease virus replication via interaction with the viral non-structural protein X

Borna disease virus (BDV) is a non-segmented, negative-sense RNA virus and has the property of persistently infecting the cell nucleus. BDV encodes a 10-kDa non-structural protein, X, which is a negative regulator of viral polymerase activity but is essential for virus propagation. Recently, we have demonstrated that interaction of X with the viral polymerase cofactor, phosphoprotein (P), facilitates translocation of P from the nucleus to the cytoplasm. However, the mechanism by which the intracellular localization of X is controlled remains unclear. In this report, we demonstrate that BDV X interacts with the 71 kDa molecular chaperon protein, Hsc70. Immunoprecipitation assays revealed that Hsc70 associates with the same region of X as P and, interestingly, that expression of P interferes competitively with the interaction between X and Hsc70. A heat shock experiment revealed that BDV X translocates into the nucleus, dependent upon the nuclear accumulation of Hsc70. Furthermore, we show that knockdown of Hsc70 by short interfering RNA decreases the nuclear localization of both X and P and markedly reduces the expression of viral genomic RNA in persistently infected cells. These data indicate that Hsc70 may be involved in viral replication by regulating the intracellular distribution of X.     

A histidine-rich and cysteine-rich metal-binding domain at the C terminus of heat shock protein A from Helicobacter pylori: implication for nickel homeostasis and bismuth susceptibility

HspA, a member of the GroES chaperonin family, is a small protein found in Helicobacter pylori with a unique histidine- and cysteine-rich domain at the C terminus. In this work, we overexpressed, purified, and characterized this protein both in vitro and in vivo. The apo form of the protein binds 2.10 +/- 0.07 Ni(2+) or 1.98 +/- 0.08 Bi(3+) ions/monomer with a dissociation constant (K(d)) of 1.1 or 5.9 x 10(-19) microm, respectively. Importantly, Ni(2+) can reversibly bind to the protein, as the bound nickel can be released either in the presence of a chelating ligand, e.g. EDTA, or at an acidic pH (pH((1/2)) 3.8 +/- 0.2). In contrast, Bi(3+) binds almost irreversibly to the protein. Both gel filtration chromatography and native electrophoresis demonstrated that apo-HspA exists as a heptamer in solution. Unexpectedly, binding of Bi(3+) to the protein altered its quaternary structure from a heptamer to a dimer, indicating that bismuth may interfere with the biological functions of HspA. When cultured in Ni(2+)-supplemented M9 minimal medium, Escherichia coli BL21(DE3) cells expressing wild-type HspA or the C-terminal deletion mutant clearly indicated that the C terminus might protect cells from high concentrations of external Ni(2+). However, an opposite phenomenon was observed when the same E. coli hosts were grown in Bi(3+)-supplemented medium. HspA may therefore play a dual role: to facilitate nickel acquisition by donating Ni(2+) to appropriate proteins in a nickel-deficient environment and to carry out detoxification via sequestration of excess nickel. Meanwhile, HspA can be a potential target of the bismuth antiulcer drug against H. pylori.     

MicroRNA regulation of human protease genes essential for influenza virus replication

Influenza A virus causes seasonal epidemics and periodic pandemics threatening the health of millions of people each year. Vaccination is an effective strategy for reducing morbidity and mortality, and in the absence of drug resistance, the efficacy of chemoprophylaxis is comparable to that of vaccines. However, the rapid emergence of drug resistance has emphasized the need for new drug targets. Knowledge of the host cell components required for influenza replication has been an area targeted for disease intervention. In this study, the human protease genes required for influenza virus replication were determined and validated using RNA interference approaches. The genes validated as critical for influenza virus replication were ADAMTS7, CPE, DPP3, MST1, and PRSS12, and pathway analysis showed these genes were in global host cell pathways governing inflammation (NF-κB), cAMP/calcium signaling (CRE/CREB), and apoptosis. Analyses of host microRNAs predicted to govern expression of these genes showed that eight miRNAs regulated gene expression during virus replication. These findings identify unique host genes and microRNAs important for influenza replication providing potential new targets for disease intervention strategies.     

The vaccinia virus G1L putative metalloproteinase is essential for viral replication in vivo

The function of the putative metalloproteinase encoded by the vaccinia virus G1L gene is unknown. To address this question, we have generated a vaccinia virus strain in which expression of the G1L gene is dependent on the addition of tetracycline (TET) when infection proceeds in a cell line expressing the tetracycline repressor. The vvtetOG1L virus replicated similarly to wild-type Western Reserve (WR) virus in these cells when TET was present but was arrested at a late stage in viral maturation in the absence of TET. This arrest resulted in the accumulation of 98.5% round immature virus particles compared to 6.9% at a similar time point when TET was present. Likewise, the titer of infectious virus progeny decreased by 98.9% +/- 0.97% when the vvtetOG1L virus was propagated in the absence of TET. Mutant virus replication was partially rescued by plasmid-encoded G1L, but not by G1L containing an HXXEH motif mutated to RXXQR. Modeling of G1L revealed a predicted structural similarity to the alpha-subunit of Saccharomyces cerevisiae mitochondrial processing peptidase (alpha-MPP). The HXXEH motif of G1L perfectly overlaps the HXXDR motif of alpha-MPP in this model. These results demonstrate that G1L is essential for virus maturation and suggest that G1L is a metalloproteinase with structural homology to alpha-MPP. However, no obvious effects on the expression and processing of the vaccinia virus major core proteins were observed in the G1L conditional mutant in the absence of TET compared to results for the TET and wild-type WR controls, suggesting that G1L activity is required after this step in viral morphogenesis.     

Aichi virus leader protein is involved in viral RNA replication and encapsidation

Aichi virus, a member of the family Picornaviridae, encodes a leader (L) protein of 170 amino acids (aa). The Aichi virus L protein exhibits no significant sequence homology to those of other picornaviruses. In this study, we investigated the function of the Aichi virus L protein in virus growth. In vitro translation and cleavage assays indicated that the L protein has no autocatalytic activity and is not involved in polyprotein cleavage. The L-VP0 junction was cleaved by 3C proteinase. Immunoblot analysis showed that the L protein is stably present in infected cells. Characterization of various L mutants derived from an infectious cDNA clone revealed that deletion of 93 aa of the center part (aa 43 to 135), 50 aa of the N-terminal part (aa 4 to 53), or 90 aa of the C-terminal part (aa 74 to 163) abolished viral RNA replication. A mutant (Delta114-163) in which 50 aa of the C-terminal part (aa 114 to 163) were deleted exhibited efficient RNA replication and translation abilities, but the virus yield was 4 log orders lower than that of the wild type. Sedimentation analysis of viral particles generated in mutant Delta114-163 RNA-transfected cells showed that the mutant has a severe defect in the formation of mature virions, but not in that of empty capsids. Thus, the data obtained in this study indicate that the Aichi virus L protein is involved in both viral RNA replication and encapsidation.     

A splice hepadnavirus RNA that is essential for virus replication.

According to the current model of hepadnavirus gene expression, the viral envelope proteins are produced from unspliced subgenomic RNAs, in contrast to the retroviral mechanism, where the subgenomic env RNA is generated by RNA splicing. We now describe and characterize a novel duck hepatitis B virus RNA species which is derived from the RNA pregenome by loss of a 1.15 kb intron. This RNA (termed spliced L RNA) codes for the large surface protein (L protein), as does the previously described unspliced mRNA (the preS RNA); however, it differs in 5' leader sequence and promoter control. Mutational analysis indicates that the spliced L RNA is functionally important for virus replication in infected hepatocytes and ducks, but not for virus formation from transfected DNA genomes. This suggests that the newly discovered second pathway for L protein synthesis plays a distinct role in an early step in the viral life cycle.     

猪流行性腹泻病毒非结构蛋白nsp9互作宿主蛋白对病毒复制的影响

猪流行性腹泻病毒(porcineepidemicdiarrheavirus,PEDV)导致仔猪和育肥猪发生急性肠道传染病,是危害养猪业最重要的病原体之一。目前发现PEDV能够编码至少16个非结构蛋白,其中nsp9能够结合至单链RNA中,但是其功能机制还不清楚。本研究通过免疫沉淀联合蛋白质谱分析,筛选出潜在的与PEDV nsp9宿主互作蛋白。通过进一步免疫共沉淀(co-immunoprecipitation, Co-IP)和激光共聚焦技术确认了nsp9与热休克蛋白HSPA8、Toll相互作用蛋白Tollip、热休克蛋白HSPA9、线粒体外膜蛋白TOMM70互作。其中,过表达HSPA8使nsp9的表达量先上调而后下调,并促进PEDV的增殖;过表达Tollip使nsp9的表达量显著上调,并抑制PEDV的增殖;过表达TOMM70使nsp9的表达量显著下调,但对PEDV的增殖无明显影响;过表达HSPA9对nsp9的表达以及PEDV的增殖均无明显影响。该研究为探索nsp9互作蛋白在PEDV感染过程中的功能提供了重要信息。     

Specific binding of tombusvirus replication protein p33 to an internal replication element in the viral RNA is essential for replication

The mechanism of template selection for genome replication in plus-strand RNA viruses is poorly understood. Using the prototypical tombusvirus, Tomato bushy stunt virus (TBSV), we show that recombinant p33 replicase protein binds specifically to an internal replication element (IRE) located within the p92 RNA-dependent RNA polymerase coding region of the viral genome. Specific binding of p33 to the IRE in vitro depends on the presence of a C.C mismatch within a conserved RNA helix. Interestingly, the absence of the p33:p33/p92 interaction domain in p33 prevented specific but allowed nonspecific RNA binding, suggesting that a multimeric form of this protein is involved in the IRE-specific interaction. Further support for the selectivity of p33 binding in vitro was provided by the inability of the replicase proteins of the closely related Turnip crinkle virus and distantly related Hepatitis C virus to specifically recognize the TBSV IRE. Importantly, there was also a strong correlation between p33:IRE complex formation in vitro and viral replication in vivo, where mutations in the IRE that disrupted selective p33 binding in vitro also abolished TBSV RNA replication both in plant and in Saccharomyces cerevisiae cells. Based on these findings and the other known properties of p33 and the IRE, it is proposed that the p33:IRE interaction provides a mechanism to selectively recruit viral RNAs into cognate viral replicase complexes. Since all genera in Tombusviridae encode comparable replicase proteins, these results may be relevant to other members of this large virus family.     

"Natively unfolded" VPg is essential for Sesbania mosaic virus serine protease activity

Polyprotein processing is a major strategy used by many plant and animal viruses to maximize the number of protein products obtainable from a single open reading frame. In Sesbania mosaic virus, open reading frame-2 codes for a polyprotein that is cleaved into different functional proteins in cis by the N-terminal serine protease domain. The soluble protease domain lacking 70-amino-acid residues from the N terminus (deltaN70Pro, where Pro is protease) was not active in trans. Interestingly, the protease domain exhibited trans-catalytic activity when VPg (viral protein genome-linked) was present at the C terminus. Bioinformatic analysis of VPg primary structure suggested that it could be a disordered protein. Biophysical studies validated this observation, and VPg resembled "natively unfolded" proteins. CD spectral analysis showed that the deltaN70Pro-VPg fusion protein had a characteristic secondary structure with a 230 nm positive CD peak. Mutation of Trp-43 in the VPg domain to phenylalanine abrogated the positive peak with concomitant loss in cis- and trans-proteolytic activity of the deltaN70Pro domain. Further, deletion of VPg domain from the polyprotein completely abolished proteolytic processing. The results suggested a novel mechanism of activation of the protease, wherein the interaction between the natively unfolded VPg and the protease domains via aromatic amino acid residues alters the conformation of the individual domains and the active site of the protease. Thus, VPg is an activator of protease in Sesbania mosaic virus, and probably by this mechanism, the polyprotein processing could be regulated in planta.     

Domain 3 of non-structural protein 5A from hepatitis C virus is natively unfolded

Hepatitis C virus (HCV) non-structural protein 5A (NS5A) is involved both in the viral replication and particle production. Its third domain (NS5A-D3), although not absolutely required for replication, is a key determinant for the production and assembly of novel HCV particles. As a prerequisite to elucidate the precise functions of this domain, we report here the first molecular characterization of purified recombinant HCV NS5A-D3. Sequence analysis indicates that NS5A-D3 is mostly unstructured but that short structural elements may exist at its N-terminus. Gel filtration chromatography, circular dichroism and finally NMR spectroscopy all point out the natively unfolded nature of purified recombinant NS5A-D3. This lack of stable folding is thought to be essential for primary interactions of NS5A-D3 domain with other viral or host proteins, which could stabilize some specific conformations conferring new functional features.