Viral Structural Polypeptides Research Articles

Chimeric Hepatitis B core antigen virus-like particles displaying the envelope domain III of dengue virus type 2

BackgroundDengue is a global public health problem for which no drug or vaccine is available. Currently, there is increasing interest in developing non-replicating dengue vaccines based on a discrete antigenic domain of the major structural protein of dengue viruses (DENVs), known as envelope domain III (EDIII). The use of bio-nanoparticles consisting of recombinant viral structural polypeptides, better known as virus-like particles (VLPs), has emerged as a potential platform technology for vaccine development. This work explores the feasibility of developing nanoparticles based on E. coli-expressed recombinant Hepatitis B virus core antigen (HBcAg) designed to display EDIII moiety of DENV on the surface.FindingsWe designed a synthetic gene construct encoding HBcAg containing an EDIII insert in its c/e1 loop. The fusion antigen HBcAg-EDIII-2 was expressed in E. coli, purified to near homogeneity using Ni+2 affinity chromatography and demonstrated to assemble into discrete 35–40 nm VLPs by electron microscopy. Competitive ELISA analyses showed that the EDIII-2 moieties of the VLPs are accessible to anti-EDIII-2-specific monoclonal and polyclonal antibodies, suggesting that they are surface-displayed. The VLPs were highly immunogenic eliciting high titer anti-EDIII-2 antibodies that were able to recognize, bind and neutralize infectious DENV based on ELISA, immunofluorescence and virus-neutralization assays.ConclusionThis work demonstrates that HBcAg-derived nanoparticles can serve as a useful platform for the display of DENV EDIII. The EDIII-displaying nanoparticles may have potential applications in diagnostics/vaccines for dengue.

Bovine lactoferrin inhibits echovirus endocytic pathway by interacting with viral structural polypeptides

Lactoferrin, an 80 kDa bi-globular iron-binding glycoprotein belonging to the transferrin family, is a pleiotropic factor with potent antimicrobial and immunomodulatory activities, present in breast milk, in mucosal secretions, and in the secondary granules of neutrophils. Recently, we have shown that bovine lactoferrin prevents the early phases of echovirus infection and also acts as a survival factor inhibiting viral-induced apoptosis. In the present research we investigated the mechanism of bovine lactoferrin anti-echoviral effect demonstrating that echovirus enters susceptible cells by an endocytic pathway and that lactoferrin treatment is able to prevent viral genome delivery into the cytoplasm. It is likely that lactoferrin interaction with echovirus capsid proteins induces alterations that stabilize the conformation of the virion making it resistant to uncoating. Taken together, the results of our study show that the inhibition of echovirus 6 infectivity by lactoferrin is dependent on its interaction not only with cell surface glycosaminoglycan chains but also with viral structural proteins demonstrating that this glycoprotein targets the virus entry process.

Accumulation of Non-Viral 45.5K and 44K Polypeptides Inversely Correlates with that of Viral Structural Polypeptides in the Midgut of the Silkworm, Bombyx mori, Infected with B. mori Densovirus Type 2

Accumulation of Non-Viral 45.5K and 44K Polypeptides Inversely Correlates with that of Viral Structural Polypeptides in the Midgut of the Silkworm, Bombyx mori, Infected with B. mori Densovirus Type 2

Antiviral activity of lactoferrin towards naked viruses.

It is well known that lactoferrin (Lf) is a potent inhibitor towards several enveloped and naked viruses, such as rotavirus, enterovirus and adenovirus. Lf is resistant to tryptic digestion and breast-fed infants excrete high levels of faecal Lf, so that its effect on viruses replicating in the gastrointestinal tract is of great interest. In this report, we analysed the mechanism of the antiviral action of this protein in three viral models which, despite representing different genoma and replication strategies, share the ability to infect the gut. Concerning the mechanism of action against rotavirus, Lf from bovine milk (BLf) possesses a dual role, preventing virus attachment to intestinal cells by binding to viral particles, and inhibiting a post adsorption step. The BLf effect towards poliovirus is due to the interference with an early infection step but, when the BLf molecule is saturated with Zn+2 ions, it is also capable of inhibiting viral replication after the viral adsorption phase. The anti-adenovirus action of BLf takes place on virus attachment to cell membranes through competition for common glycosaminoglycan receptors and a specific interaction with viral structural polypeptides. Taken together, these findings provide further evidence that Lf is an excellent candidate in the search of natural agents against viral enteric diseases, as it mainly acts by hindering adsorption and internalisation into cells through specific binding to cell receptors and/or viral particles.

Bovine lactoferrin inhibits adenovirus infection by interacting with viral structural polypeptides.

We recently demonstrated that lactoferrin, an antimicrobial glycoprotein, can inhibit adenovirus infection by competing for common glycosaminoglycan receptors. This study further characterizes the antiadenovirus activity of the protein, thus demonstrating that lactoferrin neutralizes infection by binding to adenovirus particles and that its targets are viral III and IIIa structural polypeptides.

Efficient formation of influenza virus-like particles: dependence on the expression levels of viral proteins.

It has previously been demonstrated in this laboratory that an influenza virus-like chloramphenicol acetyltransferase (CAT) RNA could be expressed in COS-1 cells that synthesized all ten influenza A virus-encoded proteins from recombinant plasmids. It was also shown that supernatant fluids harvested from these cultures contained virus-like particles (VLPs) that could deliver an enclosed CAT RNA to MDCK cells. Here, it is shown that the levels of expression of the reporter gene in the COS-1 and/or MDCK cells can be altered drastically by modifying the concentrations of the recombinant plasmids transfected in the COS-1 cells. Thus, it was observed that overexpression of NS2 reduced CAT expression in COS-1 cells, whereas overexpression of M2 and NS1 proteins dramatically decreased transmission of the CAT RNA to the MDCK cultures. These results are discussed with reference to the roles of these proteins during virus replication. From these experiments, a ratio of transfected plasmids was found that increased the efficiency of the previously described system by 50-100-fold. Under these optimized conditions, it was demonstrated that VLPs can be formed in the absence of neuraminidase expression and that these VLPs remained aggregated to each other and to cell membranes. Moreover, it is shown that CAT RNA transmission was dependent on specific interactions of the ribonucleoprotein complex with other viral structural polypeptides. These data demonstrate the usefulness of this encapsidation-packaging system for the study of different aspects of the influenza virus life-cycle.

Rice ragged stunt oryzavirus genome segments S7 and S10 encode non-structural proteins of M(r) 68,025 (Pns7) and M(r) 32,364 (Pns10).

The nucleotide sequences of genome segments S7 and S10 of a Thai-isolate of rice ragged stunt virus (RRSV) were determined. The 1938 bp S7 sequence contains a single large open reading frame (ORF) spanning nucleotides 20 to 1843 that is predicted to encode a protein of M(r) 68,025. The 1,162 bp S10 sequence has a major ORF spanning nucleotides 142 to 1,032 that is predicted to encode a protein of M(r) 32,364. This S10 ORF is preceded by a small ORF (nt 20-55) which is probably a minicistron. Coupled in vitro transcription-translation from the two major ORFs gave protein products of the expected sizes. However, no protein was visualised from S10 when the small ORF sequence was included. Proteins were expressed in Escherichia coli from the full length ORF of S7 (P7) and from a segment of the S10 ORF (P10) fused to the ORF of glutathione S-transferase (GST). Neither fusion protein was recognised by polyclonal antibodies raised against RRSV particles. Furthermore, polyclonal antibodies raised against GST-P7 fusion protein did not recognise any virion structural polypeptides. These data strongly suggest that the proteins P7 and P10 do not form part of RRSV particle. This is further supported by observed sequence homology (though very weak) of predicted RRSV P7 and P10 with those of rice dwarf virus (RDV) non-structural proteins Pns6 and Pns9, respectively.

A novel Western blot test containing both viral and recombinant proteins for anticytomegalovirus immunoglobulin M detection.

We devised a novel Western blot (WB) test for anti-human cytomegalovirus (HCMV) immunoglobulin M (IgM) detection which contains viral structural polypeptides, significant portions of recombinant p150 (ppUL32), and a significant portion of the most immunogenic nonstructural protein p52 (ppUL44). This new test was evaluated in latently infected blood donors, pregnant women, and transplant recipients with ongoing HCMV infection and shown to be more sensitive and specific than traditional WB and conventional enzyme immunoassay for the detection of HCMV-specific IgM.

Recombinant vaccinia viruses expressing hepatitis A virus structural polypeptides: detection of an anti-VP0 response in convalescent-phase sera

We have generated a number of recombinant vaccinia viruses which expressed the hepatitis A virus (HAV) structural polypeptides VP1, VP2, VP3, and VP4, either alone or in combination. The relevant sequences encoding these polypeptides were amplified from cloned cDNA by PCR and then cloned into the insertion vector pGS62. The presence of the HAV structural polypeptide-encoding sequences in the recombinant viruses was confirmed by Southern blot analysis, whilst their transcription and translation were demonstrated by Northern (RNA) blot analysis and immunodetection, respectively. Immunoprecipitation studies using these constructs have detected the presence of an anti-VP0 response in human convalescent sera following HAV infection. The significance of this finding and the usefulness of these constructs in studying cell-mediated immunity during recovery from HAV infection are discussed.

Identification of Viral Structural Polypeptides in the Midgut and Feces of the Silkworm, Bombyx mori, Infected with Bombyx Densovirus Type 2

Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) analysis showed that Bombyx mori densovirus type 2 (BmDNV-2) contained six structural polypeptides with apparent molecular weights ( M rs) of 120,000 (120K), 118, 53, 51, 49, and 46.5K. Among these structural polypeptides, four polypeptides were detected by immnnoblot analysis with anti-BmDNV-2 antibody raised against purified BmDNV-2 virions. Using such antibody for immunoblot analysis, these four viral structural polypeptides were identified in the midgut epithelium, midgut contents, and feces from BmDNV-2-infected silkworm larvae. The relative proportion of viral structural polypeptides in the midgut epithelium was different from that in the midgut contents or feces, while midgut contents and feces were similar in their viral structural polypeptide camposition. Conversion of the 53K polypeptide in the midgut epithelium to 51, 49, and 46.5K polypeptides was achieved in vitro at acid and alkaline pHs. Since protease inhibitors specific for cysteine and serine proteases reduced this conversion, proteolytic cleavage appeared to be responsible for this conversion. Peptide mapping of structural polypeptides suggested that 53, 51, 49, and 46.5K polypeptides shared a common primary amino acid sequence. Post-translational modification may be involved in the generation of structural polypeptides and the maturation of the BmDNV-2 virion.

The complete sequence of a human astrovirus.

We have determined the complete genomic sequence of human astrovirus serotype 1 isolated in Newcastle upon Tyne. The genome is 6813 nucleotides long and contains three sequential open reading frames (ORFs). The two closest to the 5' end are linked by a ribosomal frameshifting motif and contain sequence motifs indicative of non-structural virus proteins: serine protease and RNA-dependent RNA polymerase. A nuclear addressing sequence is also located here. The 3' ORF encodes the virion structural polypeptides as a polyprotein precursor. This genomic organization resembles that of the plant virus family Luteoviridae.

RNA-Stimulated NTPase Activity Associated with the p80 Protein of the Pestivirus Bovine Viral Diarrhea Virus

The genomic RNA of pestiviruses contains a single large open frame coding for virion structural proteins and viral nonstructural polypeptides. Based on the presence of specific amino acid sequence motifs, pestivirus nonstructural protein p80 was predicted to be both a serine-type proteinase and a nucleoside triphosphatase (NTPase)/RNA helicase. We previously demonstrated p80 possesses the former activity (Wisherchen and Collett, Virology 184, 341-350, 1991). Here, we provide experimental evidence that this protein is also an RNA-stimulated NTPase. Employing immunoaffinity chromatography, we partially purified a p80 protein analog (p87) from recombinant baculovirus-infected insect cells. We show this preparation contained a specific NTPase activity. This activity was not found in material similarly purified from lysates of baculovirus-infected insect cells not expressing the p87 protein. That the NTPase activity was associated with the p87 polypeptide was demonstrated in two ways. First, the NTPase activity was shown to be completely inhibited by monoclonal antibodies specific to the p80 polypeptide, but was unaffected by monoclonal antibodies to unrelated antigens. Second, radiolabeled ATP could be specifically cross-linked to the p87 polypeptide. NTP hydrolysis by the p87 protein was stimulated by the presence of particular single-strand RNA molecules. Initial enzymologic characterization of the pestivirus p80 NTPase is presented, and the presumptive role of this activity in pestivirus replication is discussed.

Identification of viral structural polypeptides of Thogoto virus (a tick-borne orthomyxo-like virus) and functions associated with the glycoprotein.

Thogoto (THO) virus is a tick-borne virus which shares morphological and genetic features with members of the Orthomyxoviridae family although the viral glycoprotein appears to be related to gp64 of baculoviruses. Characterization of THO virus was undertaken to clarify its taxonomic position. Purified virus preparations contained at least six virus-encoded polypeptides with apparent M(r) values ranging from 29K to 92K. A 75K polypeptide was identified as an envelope-associated glycoprotein by Triton X-100 and salt dissociation studies, and by proteolytic degradation of the exposed proteins of the virion. By the same criteria, the nucleoprotein and the matrix protein were identified as the 52K and 29K polypeptides, respectively. Immunofluorescence studies using monoclonal antibodies (MAbs) located the glycoprotein on the external cell membrane and the nucleoprotein in the nucleus of infected cells indicating that virus replication involved a nuclear phase. In addition, the virus displayed haemagglutination and haemolytic activities with an optimum at pH 6. These activities are functions of the viral glycoprotein since they were inhibited by anti-glycoprotein MAbs. The data reported here support the notion that THO virus is a member of the Orthomyxoviridae family but that it should be classified in a group distinct from the other influenza viruses.

A recombinant vaccinia virus expressing hepatitis A virus structural polypeptides: characterization and demonstration of protective immunogenicity.

A recombinant vaccinia virus containing most of the P1 region of hepatitis A virus (HAV) was constructed. Cell lysates of cultures infected with the virus contained HAV proteins detectable by radioimmunoassay. Western blot analysis revealed the presence of a single protein of Mr 60K to 62K, bearing epitopes from structural polypeptides VP4, -3 and -2, and the N terminus of VP1. The size of the protein suggests that at least some of the vaccinia virus thymidine kinase is also expressed. Inoculation of tamarin monkeys with the recombinant virus resulted in the development of a specific anti-HAV immune response which was protective against challenge with a virulent strain of HAV. Recombinant viruses expressing the above region of HAV or the proteins expressed by such viruses may be useful in the development of a vaccine suitable for use in man.

Preparation and characterization of monoclonal antibodies against occluded virions of Bombyx mori nuclear polyhedrosis virus

Six hybridoma cell lines secreting monoclonal antibodies (MAbs) to the structural polypeptides of occluded virions of Bombyx mori nuclear polyhedrosis virus (BmNPV) were established. These MAbs were characterized by immunoblot analysis and classified into three groups on the basis of their specificity to the viral polypeptides of occluded virions. MAbs classified as group 1 are reactive with occluded virion polypeptides exhibiting a smear pattern ranging in molecular weights (MWs) from 16,000 (16K) to 22K, but show no reactivity with the structural polypeptides of nonoccluded virions. In the BmNPV-infected cells, group 1 MAbs react with a single polypeptide with an apparent MW of 17K, suggesting that group 1 MAbs are directed against the 17K polypeptide or its counterpart present in the occluded virions. Group 2 MAbs react with two occluded virion structural polypeptides with MWs of 66 and 82K that are present in both nonoccluded virions and infected cells, and to a lesser extent, with six additional polypeptides of occluded virions. MAbs in group 3 consisting of four different species recognize the 40K polypeptide and one to five additional polypeptides ranging in MWs from 27 to 39K that are present in the infected cells but not detectable in the nonoccluded virions. In vitro translation of cytoplasmic RNA from infected cells and subsequent immunoprecipitation of translation products showed that polypeptides with MWs similar to those of the polypeptides additionally detected by group 3 MAbs were present among the immunoprecipitates. Immunofluorescence microscopy showed that localization of corresponding antigens in the infected cells differed distinctively among MAbs in the different groups.

Biological and biochemical comparison of Mamestra configurata and Mamestra brassicae nuclear polyhedrosis virus isolates pathogenic for the bertha armyworm, Mamestra configurata (Lepidoptera: Noctuidae)

Nuclear polyhedrosis viruses (NPVs) isolated from bertha armyworm, Mamestra configurata, populations and three NPVs from a related host species, Mamestra brassicae, were compared. Bioassays in first-instar M. configurata showed that the origin of a particular NPV was not predictive of its virulence in bertha armyworm. A M. brassicae NPV, MbNPV-NL, was the most virulent isolate tested with an LD 50 of 10.8 occlusion bodies (OBs) per individual at 10 days postinoculation. A second MbNPV, MbNPV-D, was significantly less virulent than the other isolates with an LD 50 of 154.2 OBs per individual. Although not significantly different from MbNPV-NL, the third MbNPV, MbNPV-NL82/1, and the native M. configurata NPV, McNPV-86/1, were less virulent with LD 50s of 41.3 and 20.1 OBs per individual, respectively. Comparisons of virion structural polypeptides by SDS-PAGE analysis indicated that the McNPV isolates had polypeptide profiles which were distinct from those of the MbNPV isolates, which were all similar. Restriction endonuclease analyses of the virus genomes revealed that the two McNPV isolates had almost identical restriction endonuclease fragment patterns which were distinct from those of the MbNPV isolates.

An iridescent virus from Simulium vittatum (Diptera: Simuliidae) in Saskatchewan

An iridescent virus was isolated from larvae of the black fly, Simulium vittatum, collected from the South Saskatchewan River. Infected larvae displayed a brilliant blue-green iridescence. The virions occurred in paracrystalline arrays in the cytoplasm of fat body and epithelial cells. The apex-apex diameter of the icosahedral virions measured 127.7 ± 0.9 nm in negatively stained preparations. Characterization of virion structural polypeptides on SDS-polyacrylamide gels resolved 26 polypeptides ranging from 12.7 to 205.8 kDa. Analysis of EcoRI, HindIII, and PstI restriction endonuclease digests of purified viral DNA indicated that the genome had an approximate size of 153 kbp. On the basis of the restriction endonuclease profiles, this iridescent virus is a new isolate from black flies.

Identification of virus-specific polypeptides and translatable mRNAs in the isolated pupal abdomens of the silkworm, Bombyx mori, infected with nuclear polyhedrosis virus

Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that infection of the isolated pupal abdomens of the silkworm, Bombyx mori, with B. mori nuclear polyhedrosis virus (BmNPV) caused generation of a number of polypeptides with a concomitant decrease of cellular polypeptides. These generated polypeptides were not identified as viral structural polypeptides, but were characterized as the degradation products of cellular polypeptides, as evidenced by the reaction with specific antiserum against storage proteins 1 and 2. Immunoblot analysis using anti-BmNPV serum identified at least 14 virus-specific polypeptides in the BmNPV-infected isolated pupal abdomens and showed that 12 of these polypeptides represented authentic structural polypeptides of the virus. In vitro translation and subsequent immunoprecipitation with anti-BmNPV serum showed that translation yielded at least 15 polypeptides at the expense of cellular polypeptides. Time-course experiments showed that the viral structural polypeptides and virus-specific translatable mRNAs were not detectable until 3 days postinoculation. On the basis of the fact that the isolated pupal abdomens are in an arrested state of development, the delayed onset of virus-specific macromolecule production in the infected isolated pupal abdomens, as compared with that in the developing intact pupae, implies that some cellular function associated with the pupal-adult development is an important prerequisite for the efficient commencement of BmNPV replication.

Selective separation of virus proteins and double-stranded RNAs by SDS-KCl precipitation

The total viral structural polypeptides and the double-stranded genomic RNAs of bluetongue virus can be selectively separated by a single SDS-KCl precipitation step. This simple, rapid and highly reproducible method enables greater than 95% recovery and purity of both viral proteins and dsRNAs within 30 min. The serotypic identity of the separated dsRNAs can be analyzed by SDS-PAGE electrophorogram immediately. After a single phenol/chloroform extraction, the dsRNA can also be used as hybridization probes, templates for molecular cloning and direct RNA sequencing. The SDS-KCl-precipitated viral proteins could be used readily for peptide mapping and as immunogens. Polyclonal and monoclonal antibodies raised against SDS-KCl-precipitated viral structural polypeptides were useful in Western immunoblots.

Inhibition of the accumulation of virus-specific translatable mRNA and structural polypeptides by guanidine hydrochloride in the midgut of the silkworm, Bombyx mori, infected with infectious flacherie virus

Effect of guanidine hydrochloride (GH) on the accumulation of translatable mRNA and structural polypeptides of the virus was investigated in the larval midgut of the silkworm, Bombyx mori, infected with infectious flacherie virus (IFV). When GH was ingested continuously by the IFV-infected larvae from the beginning of virus infection, the accumulation of both the viral translatable mRNA and its structural polypeptides was inhibited. The inhibition, however, ended shortly after the cessation of GH ingestion as a result of molting or maturation of IFV-infected larvae. The fact indicates that the inhibitory effect of GH was reversible. Upon reversal of GH treatment, appearance of viral translatable mRNA in the IFV-infected midgut preceded that of viral structural polypeptides by 12 to 24 hr, suggesting the inhibitory effect of GH on accumulation of viral structural polypeptides is secondary to that on translatable mRNA. In an experiment using newly ecdysed fifth instar larvae, it was found that IFV inoculated per os was able to persist in the GH-treated midgut for a significant peroid of time in a state capable of completing its multiplication cycle upon removal of GH treatment. This excludes the possibility that GH acted to inhibit the step of adsorption and/or internalization of the virus inoculated. Thus, our data suggest that the inhibition of IFV multiplication occurs at either the translation of input genomic RNA or the synthesis of virus-specific negative- or positive-stranded RNA.