Processing of nonstructural proteins NS4A and NS4B of dengue 2 virus in vitro and in vivo.

The production, from polyprotein precursors, of two hydrophobic nonstructural proteins of dengue 2 (DEN2) virus, NS4A and NS4B, was analyzed both in cell-free systems and in infected cells. In DEN2-infected cells, NS4B is first produced as a peptide of apparent size 30 kDa; NS4B is then post-translationally modified, in an unknown way, to produce a polypeptide of apparent size 28 kDa. The rate and extent of NS4B modification was found to be cell-dependent; in BHK cells the half-time for the conversion of the 30-kDa form to the 28-kDa form was 90 min. N-terminal sequence analysis of NS4B suggests that the N-terminus is produced by an enzyme with a specificity similar to that of signalase. Low levels of a putative polyprotein, NS4AB, were also found in mammalian cells, but not mosquito cells, infected with DEN2, suggesting that a small proportion of DEN2 4A/4B cleavage can occur post-translationally or that some nonstructural polyproteins escape normal processing. Cleavage of the 4A/4B bond in infected cells required expression of DEN2 sequences in addition to those in NS4A and NS4B, as NS4AB produced in cells by a vaccinia expression system was not cleaved. NS4AB produced in cells by a vaccinia expression system was modified post-translationally, presumably in the same way as NS4B. We show that upon translation of DEN2 polyproteins in a cell-free system, the N-terminus of NS4A is generated by cleavage by the viral nonstructural proteinase NS3 and that processing of DEN2 polyproteins occurs with a preferred, but nonobligatory order.     

Production of yellow fever virus proteins in infected cells: identification of discrete polyprotein species and analysis of cleavage kinetics using region-specific polyclonal antisera

Flavivirus proteins are produced by translation of a single long open reading frame and a complex series of cotranslational and post-translational proteolytic cleavages. To study these processing events in yellow fever virus (YF)-infected cells, polyclonal antisera recognizing C, prM, E, NS1, NS2B, NS3, NS4B, and NS5 were generated using peptide and fusion protein immunogens. Evidence suggests that production of the structural protein precursors involves rapid cotranslational processing consistent with signalase cleavages. The synthesis of the NS1 glycoprotein involves cleavage of polyprotein precursors (t1/2 approximately 10 minutes) which probably contain portions of the NS2A gene product. Endoglycosidase F treatment or labeling in the presence of tunicamycin suggests that YF prM and NS1 each have two N-linked oligosaccharides. NS2B is produced without any identifiable precursors or associated polyprotein species. Processing of the NS3-4-5 region is complex and occurs rapidly. A series of polyproteins can be detected whose molecular weights correlate with the cleavage sites defined by available N-terminal amino acid sequence data. However, convincing precursor-product relationships between these polyproteins and the mature NS3 and NS5 proteins could not be demonstrated. In contrast, NS4B appears to be produced by cleavage of a discrete precursor believed to be NS4AB. N-terminal sequence data for the putative NS4AB product has tentatively defined the NS3-4A cleavage site. A scheme for in vivo processing of the YF polyprotein is presented and discussed.     

Activation of dengue protease autocleavage at the NS2B-NS3 junction by recombinant NS3 and GST-NS2B fusion proteins

Dengue virus possesses a protease complex made up of the non-structural proteins NS2B and NS3. This protease complex catalyzes autocleavage (cis) at the junction between NS2A and NS2B as well as between NS2B and NS3. It also catalyzes trans cleavage at the junctions between NS3 and NS4A as well as NS4B and NS5. The cis cleavage at the NS2B-NS3 junction has been demonstrated in Escherichia coli by linking a 40-residue hydrophilic segment of NS2B to a NS3 N-terminal protease domain carrying the NS2B-NS3 cleavage site. To explore whether the hydrophilic segment could be further shortened, residues from both N- and C-termini of the NS2B hydrophilic segment were deleted. The results indicate that the four C-terminal's consecutive Glu residues could be deleted, each one leading to a further loss of activity, whereas the N-terminal boundary needed to be absolutely preserved. To examine whether an NS2B peptide could be expressed independently and added to activate the NS3 protease domain, the hydrophilic region of NS2B was fused to the C-terminus of glutathione-S-transferase (GST). This recombinant protein was soluble in bacteria and easily purified by affinity chromatography. Without removing the GST, the fusion protein activated the NS3 protease domain allowing it to function at the adjacent NS2B-NS3 junction. Thus, the findings reported below have produced a feasible alternative for the assay of dengue viral protease and this should facilitate the development of a screening method for inhibitors of dengue protease.     

Yellow fever virus NS2B-NS3 protease: charged-to-alanine mutagenesis and deletion analysis define regions important for protease complex formation and function

Charged-to-alanine substitutions and deletions within the yellow fever virus NS2B-NS3(181) protease were analyzed for effects on protease function. During cell-free translation of NS2B-3(181) polyproteins, mutations at three charge clusters markedly impaired cis cleavage activity: a single N-terminal cluster in the conserved domain of NS2B (residues ELKK(52-55)) and two in NS3 (ED(21-22), and residue H(47)). These mutations inhibited other protease-dependent cleavages of a transiently expressed nonstructural polyprotein, although differential effects occurred. NS2B and NS3(181) proteins harboring these mutations were impaired in their ability to associate for trans cleavage activity. N-terminal deletions in NS3 also implicated residues ED(21-22) in the association with NS2B. Deletions within NS2B revealed that the conserved domain alone provided minimal cofactor activity, with optimal function requiring both flanking hydrophobic regions. NS2B-3(181)- and NS3(181)-green fluorescent protein fusion proteins were used to determine the intracellular distribution of the protease complex. The former localized in membrane-based vesicular structures, whereas the latter localized poorly. The data suggest that NS2B-NS3 complex formation requires charge interactions involving the N-terminus of the conserved domain of NS2B and 22 N-terminal residues of NS3. A role for the putative transmembrane regions of NS2B in targeting of NS3 to intracellular membranes is also suggested.     

NS2B/3 proteolysis at the C-prM junction of the tick-borne encephalitis virus polyprotein is highly membrane dependent

The replication of tick-borne encephalitis virus (TBEV), like that of all flaviviruses, is absolutely dependent on proteolytic processing. Production of the mature proteins C and prM from their common precursor requires the activity of the viral NS2B/3 protease (NS2B/3(pro)) at the C-terminus of protein C and the host signal peptidase I (SPaseI) at the N-terminus of protein prM. Recently, we have shown in cell culture that the cleavage of protein C and the subsequent production of TBEV particles can be made dependent on the activity of the foot-and-mouth disease virus 3C protease, but not on the activity of the HIV-1 protease (HIV1(pro)) (Schrauf et al., 2012). To investigate this failure, we developed an in vitro cleavage assay to assess the two cleavage reactions performed on the C-prM precursor. Accordingly, a recombinant modular NS2B/3(pro), consisting of the protease domain of NS3 linked to the core-domain of cofactor NS2B, was expressed in E. coli and purified to homogeneity. This enzyme could cleave a C-prM protein synthesised in rabbit reticulocyte lysates. However, cleavage was only specific when protein synthesis was performed in the presence of canine pancreatic microsomal membranes and required the prevention of signal peptidase I (SPaseI) activity by lengthening the h-region of the signal peptide. The presence of membranes allowed the concentration of NS2B/3(pro) used to be reduced by 10-20 fold. Substitution of the NS2B/3(pro) cleavage motif in C-prM by a HIV-1(pro) motif inhibited NS2B/3(pro) processing in the presence of microsomal membranes but allowed cleavage by HIV-1(pro) at the C-prM junction. This system shows that processing at the C-terminus of protein C by the TBEV NS2B/3(pro) is highly membrane dependent and will allow the examination of how the membrane topology of protein C affects both SPaseI and NS2B/3(pro) processing.     

Identification and analysis of truncated and elongated species of the flavivirus NS1 protein

The flavivirus non-structural glycoprotein NS1 is often detected in Western blots as a heterogeneous cluster of bands due to glycosylation variations, precursor-product relationships and/or alternative cleavage sites in the viral polyprotein. In this study, we determined the basis of structural heterogeneity of the NS1 protein of Murray Valley encephalitis virus (MVE) by glycosylation analysis, pulse-chase experiments and terminal amino acid sequencing. Inhibition of N-linked glycosylation by tunicamycin revealed that NS1 synthesised in MVE-infected C6/36 cells was derived from two polypeptide backbones of 39 kDa (NS1(o)) and 47 kDa (NS1'). Pulse-chase experiments established that no precursor-product relationship existed between NS1(o) and NS1' and that both were stable end products. Terminal sequencing revealed that the N- and C-termini of NS1(o) were located at amino acid positions 714 and 1145 in the polyprotein respectively, consistent with the predicted sites based upon sequence homology with other flaviviruses. Expression of the NS1 gene alone or in conjunction with NS2A by recombinant baculoviruses demonstrated that the production of NS1' was dependent on the presence of NS2A, indicating that the C-terminus of the larger protein was generated within NS2A. A smaller form (31 kDa) of NS1 (deltaNS1) was also identified in MVE-infected Vero cultures, and amino acid sequencing revealed a 120-residue truncation at the N-terminus of this protein. This corresponds closely with the in-frame 121-codon deletion at the 5' end of the NS1 gene of defective MVE viral RNA (described by Lancaster et al. in 1998), suggesting that deltaNS1 may be a translation product of defective viral RNA.     

Positive identification of NS4A, the last of the hypothetical nonstructural proteins of flaviviruses

A radiolabeled protein migrating in SDS-polyacrylamide gels near the core protein C of Kunjin virus-infected cells was isolated and subjected to N-terminal amino acid sequencing. Comparisons with the translation sequence deduced from the known nucleotide sequence identified a hydrophobic protein of 149 amino acids located in the polyprotein sequence between NS3 and NS4B, thus establishing its identity as NS4A with a calculated Mr 16,100. The cleavage sites identified at the N- and C-termini are KR decreases S....parallel....VAA decreases, both representing consensus sequences defined previously for Kunjin and other flaviviruses.     

Mosquito densonucleosis virus non-structural protein NS2 is necessary for a productive infection

Mosquito densonucleosis viruses synthesize two non-structural proteins, NS1 and NS2. While NS1 has been studied relatively well, little is known about NS2. Antiserum was raised against a peptide near the N-terminus of NS2, and used to conduct Western blot analysis and immuno-fluorescence assays. Western blots revealed a prominent band near the expected size (41 kDa). Immuno-fluorescence studies of mosquito cells transfected with AeDNV indicate that NS2 has a wider distribution pattern than does NS1, and the distribution pattern appears to be a function of time post-infection. Nuclear localization of NS2 requires intact C-terminus but does not require additional viral proteins. Mutations ranging from complete NS2 knock-out to a single missense amino acid substitution in NS2 can significantly reduce viral replication and production of viable progeny.     

NS3 serine protease of bovine viral diarrhea virus: characterization of active site residues, NS4A cofactor domain, and protease-cofactor interactions

The gene expression of bovine viral diarrhea virus (BVDV), a pestivirus, occurs via translation of a hypothetical polyprotein that is processed cotranslationally and posttranslationally by viral and cellular enzymes. A protease located in the N-terminal region of nonstructural (NS) protein NS3 catalyzes the cleavages, leading to the release of NS4A, NS4B, NS5A, and NS5B. Our study provides experimental evidence that histidine at position 1658 and aspartic acid at position 1686 constitute together with the previously identified serine at position 1752 (S1752) the catalytic triad of the pestiviral NS3 serine protease. Interestingly, a mutant protease encompassing an exchange of the active site S1752 to threonine still showed residual activity. This finding links the NS3 protease of pestiviruses to the capsid protease of Sindbis virus. Furthermore, we observed that the minimal protease domain of NS3 encompasses about 209 amino acids. The NS3 protease was found to be sensitive to N-terminal truncation because a deletion of 6 amino acids significantly reduced the cleavage efficiency at the NS4A/4B site. Larger N-terminal deletions also impaired the activity of the enzyme with respect to the other cleavage sites but to a different degree at each site. The NS3 protease of BVDV has previously been shown to depend on NS4A as cofactor. We demonstrate here that the central region of NS4A represents the cofactor domain. Furthermore, coprecipitation studies strongly suggest an interaction between NS4A and the N-terminal region of NS3. Besides the remarkable similarities observed between the pestiviral NS3 protease and the corresponding enzyme of hepatitis C virus (HCV), our results suggest a common ancestry between these enzymes and the capsid protease of Sindbis virus.     

Complex formation between hepatitis C virus NS2 and NS3 proteins

Hepatitis C virus (HCV) NS2 and NS3 proteins as well as the NS3 protease cofactor NS4A are essential for the replication of the virus. The presence of in vivo heterodimeric complex between HCV NS2 and NS3 has been suggested by biochemical studies. Detailed characterization of the interactions between these viral proteins is of great importance for better understanding their role in viral replication cycle and represents attractive target for antiviral agents. In this study, we demonstrated in vivo interactions between HCV NS2 and NS3 proteins using an epitope tagging technique. For this purpose NS2, NS3 and NS4A were expressed in fusion with two different tags in Cos7 cells. Immunofluorescence analysis and co-immunoprecipitation with tag-specific antibodies revealed the existence of biologically important NS3/NS4A and NS3/NS2 complexes. Similar complexes were detected also in Huh7 cells infected with Semliki Forest virus vectors expressing NS2 and NS3 or NS23 precursor polyprotein. The formation of complex between NS2 and NS3 was found not to depend on whether the proteins were expressed individually or in form of common precursor. This observation suggests the existence of direct interaction between these two proteins that may have importance for the formation of the whole HCV replication complex.     

The sequences of reovirus serotype 3 genome segments M1 and M3 encoding the minor protein mu 2 and the major nonstructural protein mu NS, respectively

The sequences of the M1 and M3 genome segments of reovirus serotype 3 strain Dearing, which encode protein mu 2, a minor capsid, component, and protein mu NS, one of the two nonstructural proteins, are reported. They are 2304 and 2235 base pairs long, respectively, and proteins mu 2 and mu NS comprise 736 and 719 amino acids, respectively. This completes the sequencing of the reovirus serotype 3 genome: it comprises 23,549 basepairs. Neither protein mu 2 nor protein mu NS possesses any sequence similarity to any protein sequence in gene banks, nor any of the commonly recognized motifs indicative of specialized function. Protein mu 2 has a higher alpha-helix content (36%) than other capsid proteins; for it, the ratio of amino acids in alpha-helix/beta-sheet configuration is 1.2, whereas that of typical reovirus capsid proteins ranges from 0.5 to 0.9. Thus it is not a typical capsid protein. Protein mu NS has a very high alpha-helix content (about 50%; alpha-helix/beta-sheet ratio 2.5), which is very similar to that of the other nonstructural reovirus protein, protein sigma NS. The C-terminal regions of mu NS and various myosins exhibit periodic sequence similarity elements indicative of helical structure. Protein mu NS exists in two forms in infected cells: protein mu NS and a protein, mu NSC, which lacks a region of about 5 kDa at its N-terminus. Pulse-chase analysis in vivo suggests that protein mu NSC is not a cleavage product of protein mu NS; further, protein mu NSC is formed along with protein mu NS in in vitro protein synthesizing systems, whereas protein mu 1C, the cleavage product of protein mu 1, is not. It is likely, therefore, that protein mu NSC is a primary translation product, formed either by ribosomes reading through the first initiation codon of m1 messenger RNA at position 14 and initiating at codon 42, or by de novo internal initiation at this codon.     

Classical swine fever virus NS5A regulates viral RNA replication through binding to NS5B and 3'UTR

In this report, classical swine fever virus (CSFV) NS5A inhibit viral RNA replication when its concentration reached and surpassed the level of NS5B. Three amino acid fragments of CSFV NS5A, 137-172, 224-268 and 390-414 individually were shown to be essential to NS5B binding. The former two fragments were independently necessary for regulation of viral RNA replication and correlated with NS5B and 3′UTR binding activity. We also found that amino acids W143, V145, P227, T246, P257, K399, T401, E406 and L413 of CSFV NS5A were essential to NS5B binding activity. Furthermore, these amino acids were shown to be necessary for viral RNA replication and infection and conserved in NS5A proteins of CSFV, BDV, BVDV and HCV. These results indicated that NS5A may regulate viral RNA replication by binding to NS5B and 3′UTR. NS5A can still regulate viral RNA synthesis through binding to 3′UTR when binding to NS5B is not available.     

A comparative analysis of the substrate permissiveness of HCV and GBV-B NS3/4A proteases reveals genetic evidence for an interaction with NS4B protein during genome replication

The hepatitis C virus (HCV) serine protease (NS3/4A) processes the NS3-NS5B segment of the viral polyprotein and also cleaves host proteins involved in interferon signaling, making it an important target for antiviral drug discovery and suggesting a wide breadth of substrate specificity. We compared substrate specificities of the HCV protease with that of the GB virus B (GBV-B), a distantly related nonhuman primate hepacivirus, by exchanging amino acid sequences at the NS4B/5A and/or NS5A/5B cleavage junctions between these viruses within the backbone of subgenomic replicons. This mutagenesis study demonstrated that the GBV-B protease had a broader substrate tolerance, a feature corroborated by structural homology modeling. However, despite efficient polyprotein processing, GBV-B RNAs containing HCV sequences at the C-terminus of NS4B had a pseudo-lethal replication phenotype. Replication-competent revertants contained second-site substitutions within the NS3 protease or NS4B N-terminus, providing genetic evidence for an essential interaction between NS3 and NS4B during genome replication.     

Characterization of an Epstein-Barr virus nuclear antigen 2 variant (EBNA 2B) by specific sera

The Jijoye Epstein-Barr virus (EBV) strain is characterized by a substitution of 1.8 kb in the C-terminal part of EBNA 2 gene compared to B95-8 or M-ABA virus. Protein immunoblot analysis using human sera against EBNA 2 indicated that an immunological variant to the EBNA 2 of B95-8 (type A) is encoded by the Jijoye virus (type B). In order to generate a specific EBNA 2B antiserum the NaeI/NsiI DNA fragment of the Jijoye virus containing 237 bp of the C-terminus from the EBNA 2B gene was cloned in an E. coli expression vector (pME3). The resulting fusion protein contained 79 C-terminal amino acids of the viral protein and a 37,000 Da part of the bacterial anthranilate synthase. Rabbit antisera generated against this fusion protein reacted specifically with two proteins of 73,000 and 77,000 Da from Jijoye cells and three other cell lines carrying type B virus, while no proteins could be identified in the type B cell line BL 29. In addition, using these sera directed against the pME3 fusion protein, no reaction could be observed with the EBNA 2A protein from the B95-8 and several other cell lines containing type A virus.     

Mutational analysis of the West Nile virus NS4B protein

West Nile virus NS4B is a small hydrophobic nonstructural protein approximately 27 kDa in size whose function is poorly understood. Amino acid substitutions were introduced into the NS4B protein primarily targeting two distinct regions; the N-terminal domain (residues 35 through 60) and the central hydrophobic domain (residues 95 through 120). Only the NS4B P38G substitution was associated with both temperature-sensitive and small-plaque phenotypes. Importantly, this mutation was found to attenuate neuroinvasiveness greater than 10,000,000-fold and lower viremia titers compared to the wild-type NY99 virus in a mouse model. Full genome sequencing of the NS4B P38G mutant virus revealed two unexpected mutations at NS4B T116I and NS3 N480H (P38G/T116I/N480H), however, neither mutation alone was temperature sensitive or attenuated in mice. Following incubation of P38G/T116I/N480H at 41 °C, five mutants encoding compensatory substitutions in the NS4B protein exhibited a reduction in the temperature-sensitive phenotype and reversion to a virulent phenotype in the mouse model.     

Hepatitis C virus (HCV) NS2 protein up-regulates HCV IRES-dependent translation and down-regulates NS5B RdRp activity

Chronic hepatitis C virus (HCV) infection often leads to liver cancer. The HCV NS2 protein is a hydrophobic transmembrane protein that associates with several cellular proteins in mammalian cells. In this report, we investigated the function of NS2 protein on HCV replication and translation by using a transient cell-based expression system. Cells co-transfected with pcDNA3.1 (−)-NS2 and the dual-luciferase reporter construct containing the HCV IRES were used to detect the effect of NS2 protein on HCV translation. Cells co-transfected with pcDNA3.1(−)-NS2, pcDNA-NS5B and a reporter plasmid were used to detect the effect of NS2 protein on HCV replication. The results showed that HCV NS2 protein up-regulated HCV IRES-dependent translation in a specific and dose-dependent manner in Huh7 cells but not in HeLa and HepG2 cells, and NS2 protein inhibited NS5B RdRp activity in a dose-independent manner in all three cell lines. These findings may suggest a novel mechanism by which HCV modulates its NS5B replication and IRES-dependent translation and facilitates virus persistence. Y. She and Q. Liao contributed equally to this work.     

Primary structure of the glycoprotein E2 of coronavirus MHV-A59 and identification of the trypsin cleavage site

The nucleotide sequence of the peplomer (E2) gene of MHV-A59 was determined from a set of overlapping cDNA clones. The E2 gene encodes a protein of 1324 amino acids including a hydrophobic signal peptide. A second large hydrophobic domain is found near the COOH terminus and probably represents the membrane anchor. Twenty glycosylation sites are predicted. Cleavage of the E2 protein results in two different 90K species, 90A and 90B (L.S. Sturman, C. S. Ricard, and K. V. Holmes (1985) J. Virol. 56, 904-911), and activates cell fusion. Protein sequencing of the trypsin-generated N-terminus revealed the position of the cleavage site. 90A and 90B could be identified as the C-terminal and the N-terminal parts, respectively. Amino acid sequence comparison of the A59 and JHM E2 proteins showed extensive homology and revealed a stretch of 89 amino acids in the 90B region of the A59 E2 protein that is absent in JHM.