Nucleotide sequence of the 24S subgenomic messenger RNA of a vaccine strain (HPV77) of rubella virus: comparison with a wild-type strain (M33)

A full-length cDNA clone for the 24S subgenomic mRNA of the vaccine strain (HPV77) of rubella virus has been isolated from a cDNA library made from the RNAs of infected cells. Starting from the first Met start codon, the 24S mRNA codes for a precursor protein of 1063 amino acids (aa). This precursor encodes a capsid protein of 300 aa, and two envelope proteins, E1 (481 aa) and E2 (282 aa). Both the E1 and E2 proteins are preceded by a stretch of 21 hydrophobic aa, characteristic of a signal peptide, and each has three putative glycosylation sites in the polypeptide chains. Comparison between the structural proteins of the vaccine and the wild-type (wt; M33) strains of rubella virus, revealed that the E2 protein of the vaccine strain differs, in its apparent Mr, by approx. 3 kDa, from the wt strain. The difference could be due to decreased glycosylation of the vaccine strain E2 protein, as revealed by [3H]mannose incorporation studies. Five single-aa changes in the structural proteins occurred during the attenuation process, one each in the capsid and the E1 protein and three in the E2 protein. The change of Thr-412----Ile in the E2 protein results in the loss of a putative glycosylation site at Asn-410, which offers a plausible explanation for decreased glycosylation of the E2 protein from the vaccine strain of rubella virus.     

Topology of the non-structural rotavirus receptor glycoprotein NS28 in the rough endoplasmic reticulum.

The rotavirus non-structural glycoprotein (NS28), the receptor for the virus core during budding into the lumen of the rough endoplasmic reticulum (RER), is 175 amino acids long and possesses an uncleaved signal sequence and two amino-terminal glycosylation sites. Utilizing one of three potential hydrophobic domains, the protein spans the membrane only once, with the glycosylated amino-terminal region oriented to the luminal side of the ER and the carboxy-terminal region to the cytoplasmic side. To localize sequences involved in translocation of NS28, we constructed a series of mutations in the coding regions for the hydrophobic domains of the protein. Mutant protein products were studied by in vitro translation and by transfection in vivo. In transfected cells, all mutant forms localize to the ER, and none are secreted. In vitro, each of the three hydrophobic domains is able to associate with microsomes. However, glycosylation and proteolysis of wild-type and mutant forms of NS28 indicates that the wild-type protein is anchored in the membrane only by the second hydrophobic domain, leaving approximately 131 residues exposed on the cytoplasmic side for receptor - ligand interaction.     

Multiple glycoproteins synthesized by the smallest RNA segment (S10) of bluetongue virus.

The genome of bluetongue virus, an orbivirus, consists of 10 double-stranded RNAs, each encoding at least one polypeptide. The smallest RNA segment (S10) encodes two minor nonstructural proteins, NS3 and NS3A, the structures and functions of which are not understood. We have expressed these two proteins in mammalian cells by using the T7 cytoplasmic transient expression system. Using a deletion mutant (lacking the first AUG initiation codon), we have demonstrated that the second initiation codon is used to initiate the synthesis of NS3A protein and that the two initiation codons are responsible for the synthesis not only of NS3 and NS3A but also of high-molecular-weight forms of both proteins. These higher-molecular-weight forms (GNS3 and GNS3A) are glycosylated. We have also demonstrated that the carbohydrate chains of GNS3 and GNS3A could be further modified by heterogeneous extension to polylactosaminoglycan forms. The glycosylated and nonglycosylated forms are found in similar intracellular locations in the Golgi complex. In the presence of cycloheximide, NS3 and NS3A immunofluorescence staining was pronounced in the Golgi complex, confirming that NS3 and NS3A are competent for transport to the Golgi apparatus after synthesis. We conclude that S10 gene products are integral membrane glycoproteins.     

Sequence analysis of the 4.7-kb BamHI-EcoRI fragment of the equine herpesvirus type-1 short unique region.

To localize gene that may encode immunogens potentially important for recombinant vaccine design, we have analysed a region of the equine herpesvirus type-1 (EHV-1) genome where a glycoprotein-encoding gene had previously been mapped. The 4707-bp BamHI-EcoRI fragment from the short unique region of the EHV-1 genome was sequenced. This sequence contains three entire open reading frames (ORFs), and portions of two more. ORF1 codes for 161 amino acids (aa), and represents the C terminus of a possible membrane-bound protein. ORF2 (424 aa) and ORF3 (550 aa) are potential glycoprotein-encoding genes; the predicted aa sequences contain possible signal sequences, N-linked glycosylation sites and transmembrane domains; they also show homology to the glycoproteins gI and gE of herpes simplex virus type-1 (HSV-1), and the related proteins of pseudorabies virus and varicella-zoster virus. The predicted aa sequence of ORF4 shares no homology with other known herpesvirus proteins, but the nucleotide sequence shows a high level of homology with the corresponding region of the EHV-4 genome. ORF5 may be related to US9 of HSV-1.     

Expression of two related nonstructural proteins of bluetongue virus (BTV) type 10 in insect cells by a recombinant baculovirus: production of polyclonal ascitic fluid and characterization of the gene product in BTV-infected BHK cells.

In vitro translation of bluetongue virus (BTV) double-stranded RNA in the rabbit reticulocyte lysate system has shown segment 10 (S10) to code for two related proteins, NS3 and NS3A. The presence of both products in vivo, however, has remained unconfirmed owing to the very low level of synthesis of the S10 gene product(s) in BTV-infected BHK cells. In the present work, a cDNA copy of BTV type 10 (BTV-10) S10 RNA was inserted into Autographa californica nuclear polyhedrosis baculovirus (AcNPV) in lieu of the 5' coding region of the AcNPV polyhedrin gene. Spodoptera frugiperda cells infected with the recombinant baculovirus synthesized two polypeptides, which were shown to represent NS3 and NS3A by Western blot (immunoblot) and peptide map analysis. Antibodies raised to the expressed NS3 by immunization of mice detected both NS3 and NS3A in BTV-10-infected BHK cells but not in purified BTV-10 virus particles. In contrast to in vitro translation of BTV S10 RNA in which NS3 and NS3A are synthesized in equimolar amounts, NS3 was the principle product both in the baculovirus expression system and in vivo in BTV-infected cells. The results indicate the caution which should be exercised when using the rabbit reticulocyte lysate system to predict the pattern of protein synthesis from a gene with alternative start codons. The expressed NS3 and NS3A proteins reacted strongly with sera from sheep infected with homologous and heterologous BTV serotypes, suggesting that the S10 gene products are highly conserved group-specific antigens.     

Hepatitis C virus NS4B targets lipid droplets through hydrophobic residues in the amphipathic helices

Lipid droplets (LD) are dynamic storage organelles that are involved in lipid homeostasis. Hepatitis C virus (HCV) is closely associated with LDs. HCV Core and nonstructural (NS) proteins colocalize with LDs and presumably are involved in virion formation at that site. We demonstrated that HCV NS4B, an integral membrane protein in endoplasmic reticulum (ER), strongly targeted LDs. Confocal imaging studies showed that NS4B localized at the margins of LDs. Biochemical fractionation of HCV-replicating cells suggested that NS4B existed in membranes associated with LDs rather than on the LD surface membrane itself. The N- and C-terminal cytosolic domains of NS4B showed targeting of LDs, with the former being much stronger. In both domains, activity was present in the region containing an amphipathic α-helix, in which 10 hydrophobic residues were identified as putative determinants for targeting LDs. JFH1 mutants with alanine substitutions for the hydrophobic residues were defective for virus replication. W43A mutant with a single alanine substitution showed loss of association of NS4B with LDs and severely reduced release of infectious virions compared with wild-type JFH1. NS4B plays a crucial role in virus replication at the site of virion formation, namely, the microenvironment associated with LDs.     

trans-Complementation of an NS2 Defect in a Late Step in Hepatitis C Virus (HCV) Particle Assembly and Maturation

Recent studies using cell culture infection systems that recapitulate the entire life cycle of hepatitis C virus (HCV) indicate that several nonstructural viral proteins, including NS2, NS3, and NS5A, are involved in the process of viral assembly and release. Other recent work suggests that Ser-168 of NS2 is a target of CK2 kinase–mediated phosphorylation, and that this controls the stability of the genotype 1a NS2 protein. Here, we show that Ser-168 is a critical determinant in the production of infectious virus particles. Substitution of Ser-168 with Ala (or Gly) ablated production of infectious virus by cells transfected with a chimeric viral RNA (HJ3-5) containing core-NS2 sequences from the genotype 1a H77 virus within the background of genotype 2a JFH1 virus. An S168A substitution also impaired production of virus by cells transfected with JFH1 RNA. This mutation did not alter polyprotein processing or genome replication. This defect in virus production could be rescued by expression of wt NS2 in trans from an alphavirus replicon. The trans-complementing activities of NS2 from genotypes 1a and 2a demonstrated strong preferences for rescue of the homologous genotype. Importantly, the S168A mutation did not alter the association of core or NS5A proteins with host cell lipid droplets, nor prevent the assembly of core into particles with sedimentation and buoyant density properties similar to infectious virus, indicating that NS2 acts subsequent to the involvement of core, NS5A, and NS3 in particle assembly. Second-site mutations in NS2 as well as in NS5A can rescue the defect in virus production imposed by the S168G mutation. In aggregate, these results indicate that NS2 functions in trans, in a late-post assembly maturation step, perhaps in concert with NS5A, to confer infectivity to the HCV particle.     

Hepatitis C virus NS5A protein interacts with lysine methyltransferase SET and MYND domain‐containing 3 and induces activator protein 1 activation

Hepatitis C virus (HCV) non‐structural protein 5A (NS5A) is a multifunctional protein that is involved in the HCV life cycle and pathogenesis. In this study, a host protein(s) interacting with NS5A by tandem affinity purification were searched for with the aim of elucidating the role of NS5A. An NS5A‐interacting protein, SET and MYND domain‐containing 3 (SMYD3), a lysine methyltransferase reportedly involved in the development of cancer, was identified. The interaction between NS5A and SMYD3 was confirmed in ectopically expressing, HCV RNA replicon‐harboring and HCV‐infected cells. The other HCV proteins did not bind to SMYD3. SMYD3 bound to NS5A of HCV genotypes 1b and 2a. Deletion mutational analysis revealed that domains II and III of NS5A (amino acids [aa] 250 to 447) and the MYND and N‐SET domains of SMYD3 (aa 1 to 87) are involved in the full extent of NS5A‐SMYD3 interaction. NS5A co‐localized with SMYD3 exclusively in the cytoplasm, thereby inhibiting nuclear localization of SMYD3. Moreover, NS5A formed a complex with SMYD3 and heat shock protein 90 (HSP90), which is a positive regulator of SMYD3. The intensity of binding between SMYD3 and HSP90 was enhanced by NS5A. Luciferase reporter assay demonstrated that NS5A significantly induces activator protein 1 (AP‐1) activity, this being potentiated by co‐expression of SMYD3 with NS5A. Taken together, the present results suggest that NS5A interacts with SMYD3 and induces AP‐1 activation, possibly by facilitating binding between HSP90 and SMYD3. This may be a novel mechanism of AP‐1 activation in HCV‐infected cells.     

我国两个不同地区猪繁殖和呼吸综合征病毒分离株ORF5基因序列比例

猪繁殖和呼吸综合征病毒(porcine reproductive and respiratory syndrome virus,PRRSV)为动脉炎病毒科成员之一,可引起感染母猪流产、死胎及断奶仔猪呼吸困难和死亡.该病毒呈球形,有囊膜,大小45nm～83nm,基因组为单股RNA,大小约15kb,有8个阅读框(ORF),分别编码2种非结构蛋白和6种结构蛋白,其中ORF5编码病毒糖基化膜蛋白(GP5)[1,2].GP5蛋白为该病毒主要结构蛋白之一,含有病毒线性中和抗原表位.该蛋白可诱导感染细胞发生细胞凋亡[3,4].目前,PRRSV有欧洲型和美州型两个血清型,其结构蛋白基因同源性为54%～70%[5,6].不同美洲型PRRSV野毒株基因也有一定差异.由ORF5基因推导的氨基酸序列有4个相对保守区,但其N端和C端氨基酸残基可变性较大.由该基因构建的重组质粒具有良好免疫原性[7,8].尽管一些欧美国家已普遍使用Resp PRRS弱毒疫苗,但该病仍时有发生[9,10].我国于1996年亦已证实存在该病,并有不断蔓延趋势,已造成我国养猪业严重经济损失.     

Bluetongue Virus Nonstructural Protein NS3/NS3a Is Not Essential for Virus Replication

Orbiviruses form the largest genus of the family Reoviridae consisting of at least 23 different virus species. One of these is the bluetongue virus (BTV) and causes severe hemorrhagic disease in ruminants, and is transmitted by bites of Culicoides midges. BTV is a non-enveloped virus which is released from infected cells by cell lysis and/or a unique budding process induced by nonstructural protein NS3/NS3a encoded by genome segment 10 (Seg-10). Presence of both NS3 and NS3a is highly conserved in Culicoides borne orbiviruses which is suggesting an essential role in virus replication. We used reverse genetics to generate BTV mutants to study the function of NS3/NS3a in virus replication. Initially, BTV with small insertions in Seg-10 showed no CPE but after several passages these BTV mutants reverted to CPE phenotype comparable to wtBTV, and NS3/NS3a expression returned by repair of the ORF. These results show that there is a strong selection for functional NS3/NS3a. To abolish NS3 and/or NS3a expression, Seg-10 with one or two mutated start codons (mutAUG1, mutAUG2 and mutAUG1+2) were used to generate BTV mutants. Surprisingly, all three BTV mutants were generated and the respective AUG^Met→GCC^Ala mutations were maintained. The lack of expression of NS3, NS3a, or both proteins was confirmed by westernblot analysis and immunostaining of infected cells with NS3/NS3a Mabs. Growth of mutAUG1 and mutAUG1+2 virus in BSR cells was retarded in both insect and mammalian cells, and particularly virus release from insect cells was strongly reduced. Our findings now enable research on the role of RNA sequences of Seg-10 independent of known gene products, and on the function of NS3/NS3a proteins in both types of cells as well as in the host and insect vector.     

Hepatitis C NS3 protease: restoration of NS4A cofactor activity by N-biotinylation of mutated NS4A using synthetic peptides

The NS3 serine protase of Hepatitis C virus (HCV) requires NS4A protein as a cofactor for efficient cleavage at four sites in the nonstructural region. The cofactor activity has been mapped to the central hydrophobic region (aa 22-34) of this 54-amino-acid NS4A protein, and site-directed mutagenesis has identified alternating hydrophobic amino acids, particularly Ile25 and Ile29, as critically important. A double mutant of NS4A cofactor peptide, I25A/I29A, completely abolished the cofactor activity. We now report that the cofactor peptide activity in the I25A/I29A double mutant can be restored specifically by introducing a biotin-aminohexanoic acid fusion at the N-terminus. In addition, a similar N-terminal fusion of biotin-aminohexanoic acid with the wild-type 4A peptide significantly enhanced cofactor activity. Our data corroborate the crystal structure-based hypothesis of hydrophobic interaction between the N-terminus of NS4A and the N-terminal alpha(0) helix of NS3 protease.     

The hepatitis C virus NS4A protein: interactions with the NS4B and NS5A proteins.

Hepatitis C virus encodes a large polyprotein precursor that is proteolytically processed into at least 10 distinct products, in the order NH2-C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B -COOH. A serine proteinase encoded in the N-terminal 181 residues of the NS3 nonstructural protein is responsible for cleavage at four sites (3/4A, 4A/4B, 4B/5A, and 5A/5B) in the nonstructural region. NS4A, a 54-residue nonstructural protein which forms a stable complex with the NS3 proteinase, is required as a cofactor for cleavage at the 3/4A and 4B/5A sites and enhances processing at the 4A/4B and 5A/5B sites. Recently reported crystal structures demonstrated that NS4A forms an integral part of the NS3 serine proteinase. In this report, we present evidence that NS4A forms a nonionic-detergent-stable complex with the NS4B5A polyprotein substrate, which may explain the requirement of NS4A for the 4B/5A cleavage. Isoleucine-29 of NS4A, which has been previously shown to be essential for its proteinase cofactor activity and formation of the NS3 complex, was found to be important for the interaction between NS4A and the NS4B5A substrate. In addition, two more hydrophobic residues in the NS4A central region (valine-23 and isoleucine-25) were also shown to be essential for the cofactor activity and for the interaction with either the NS3 proteinase or the NS4B5A polyprotein substrate. Finally, the possible mechanisms by which these viral proteins interact with each other are discussed.     

The membrane spanning domains of protein NS4B from hepatitis C virus

Determination of the membrane spanning domains of highly hydrophobic proteins from its primary structure, i.e., sequence, is cumbersome. However, transmembrane topology is better correlated with protein secondary structure than with the primary one. In this work we have determined the number and location of the transmembrane domains of the highly hydrophobic hepatitis C virus NS4B protein by studying the water-to-bilayer and water-to-interface transfer free energies of thirty-one different hepatitis C virus strains assuming that NS4B forms an α-helical wheel. Additionally, we have studied the effect of a peptide library encompassing the full length of the NS4B protein hepatitis C virus strain 1a_H77 on the phase transitions of DEPE through the use of differential scanning calorimetry. Our findings show that NS4B protein has five transmembrane domains and, as previously suggested, three interfacial segments. One of these segments, segment AH2, could behave similarly to viral pre-transmembrane segments, which would partition into and interact with the membrane and be responsible for the fluctuation of the protein between different topologies and therefore possible locations.     

Multiple Enzymatic Activities Associated with Recombinant NS3 Protein of Hepatitis C Virus

The hepatitis C virus (HCV) nonstructural 3 protein (NS3) contains at least two domains associated with multiple enzymatic activities; a serine protease activity resides in the N-terminal one-third of the protein, whereas RNA helicase activity and RNA-stimulated nucleoside triphosphatase activity are associated with the C-terminal portion. To study the possible mutual influence of these enzymatic activities, a full-length NS3 polypeptide of 67 kDa was expressed as a nonfusion protein in Escherichia coli, purified to homogeneity, and shown to retain all three enzymatic activities. The protease activity of the full-length NS3 was strongly dependent on the activation by a synthetic peptide spanning the central hydrophobic core of the NS4A cofactor. Once complexed with the NS4A-derived peptide, the full-length NS3 protein and the isolated N-terminal protease domain cleaved synthetic peptide substrates with comparable efficiency. We show that, as in the case of the isolated protease domain, the protease activity of full-length NS3 undergoes inhibition by the N-terminal cleavage products of substrate peptides corresponding to the NS4A-NS4B and NS5A-NS5B. We have also characterized and quantified the NS3 ATPase, RNA helicase, and RNA-binding activities under optimized reaction conditions. Compared with the isolated N-terminal and C-terminal domains, recombinant full-length NS3 did not show significant differences in the three enzymatic activities analyzed in independent in vitro assays. We have further explored the possible interdependence of the NS3 N-terminal and C-terminal domains by analyzing the effect of polynucleotides on the modulation of all NS3 enzymatic functions. Our results demonstrated that the observed inhibition of the NS3 proteolytic activity by single-stranded RNA is mediated by direct interaction with the protease domain rather than with the helicase RNA-binding domain.     

Nuclear localization of the NS3 protein of hepatitis C virus and factors affecting the localization.

Subcellular localization of the NS2 and NS3 proteins of hepatitis C virus was analyzed. In stable Ltk transfectants inducibly expressing an NS2-NS3 polyprotein (amino acids [aa] 810 to 1463), processed full-size NS2 (aa 810 to 1026) was detected exclusively in a cytoplasmic membrane fraction. On the other hand, the other processed product, carboxy-truncated NS3 (NS3 deltaC1463; aa 1027 to 1463), was present in both cytoplasmic and nuclear fractions. To further analyze subcellular localization of NS3, NS3 deltaC1459 (aa 1027 to 1459), full-size NS3 (NS3F; aa 1027 to 1657), and both amino- and carboxy-truncated NS3 (NS3 deltaNdeltaC; aa 1201 to 1459) were expressed in HeLa cells by using a vaccinia virus-T7 hybrid expression system. NS3 deltaC1459 and NS3F accumulated in the nucleus as well as in the cytoplasm, exhibiting a dot-like staining pattern. On the other hand, NS3 deltaNdeltaC was localized predominantly in the cytoplasm, suggesting the presence of a nuclear localization signal(s) in the amino-terminal sequence of NS3. NS4A, a viral cofactor for the NS3 protease, inhibited nuclear transport of NS3 deltaC1459 and NS3F, with the latter inhibited to a lesser extent than was the former. Interestingly, wild-type p53 tumor suppressor augmented nuclear localization of NS3 deltaC1459 and NS3F, whereas mutant-type p53 inhibited nuclear localization and augmented cytoplasmic localization of NS3 deltaC1459. However, subcellular localization of NS3 deltaNdeltaC was not affected by either type of p53. Wild-type p53-mediated nuclear accumulation of NS3 deltaC1459 and NS3F was inhibited partially, but not completely, by coexpressed NS4A, with NS3F again affected less prominently than was NS3 deltaC1459.     

Glycan-Dependent Immunogenicity of Recombinant Soluble Trimeric Hemagglutinin

Recombinant soluble trimeric influenza A virus (IAV) hemagglutinin (sHA₃) has proven an effective vaccine antigen against IAV. Here, we investigate to what extent the glycosylation status of the sHA₃ glycoprotein affects its immunogenicity. Different glycosylation forms of subtype H5 trimeric HA protein (sH5₃) were produced by expression in insect cells and different mammalian cells in the absence and presence of inhibitors of N-glycan-modifying enzymes or by enzymatic removal of the oligosaccharides. The following sH5₃ preparations were evaluated: (i) HA proteins carrying complex glycans produced in HEK293T cells; (ii) HA proteins carrying Man₉GlcNAc₂ moieties, expressed in HEK293T cells treated with kifunensine; (iii) HA proteins containing Man₅GlcNAc₂ moieties derived from HEK293S GnTI(−) cells; (iv) insect cell-produced HA proteins carrying paucimannosidic N-glycans; and (v) HEK293S GnTI(−) cell-produced HA proteins treated with endoglycosidase H, thus carrying side chains composed of only a single N-acetylglucosamine each. The different HA glycosylation states were confirmed by comparative electrophoretic analysis and by mass spectrometric analysis of released glycans. The immunogenicity of the HA preparations was studied in chickens and mice. The results demonstrate that HA proteins carrying terminal mannose moieties induce significantly lower hemagglutination inhibition antibody titers than HA proteins carrying complex glycans or single N-acetylglucosamine side chains. However, the glycosylation state of the HA proteins did not affect the breadth of the antibody response as measured by an HA1 antigen microarray. We conclude that the glycosylation state of recombinant antigens is a factor of significant importance when developing glycoprotein-based vaccines, such as recombinant HA proteins.     

Structural characterization of mumps virus fusion protein core

The fusion proteins of enveloped viruses mediating the fusion between the viral and cellular membranes comprise two discontinuous heptad repeat (HR) domains located at the ectodomain of the enveloped glycoproteins. The crystal structure of the fusion protein core of Mumps virus (MuV) was determined at 2.2 A resolution. The complex is a six-helix bundle in which three HR1 peptides form a central highly hydrophobic coiled-coil and three HR2 peptides pack against the hydrophobic grooves on the surface of central coiled-coil in an oblique antiparallel manner. Fusion core of MuV, like those of simian virus 5 and human respiratory syncytium virus, forms typical 3-4-4-4-3 spacing. The similar characterization in HR1 regions, as well as the existence of O-X-O motif in extended regions of HR2 helix, suggests a basic rule for the formation of the fusion core of viral fusion proteins.