Capsid Polypeptide Research Articles

A detection method for infectious pancreatic necrosis virus (IPNV) based on reverse transcription (RT)‐polymerase chain reaction (PCR)

Abstract. A rapid, sensitive and highly specific detection method for infectious pancreatic neerosis virus (IPNV), based on reverse transcription (RT) polymerase chain reaction (PCR) has been developed. The specificity of the assay is provided by the oligonucleotide primers selected from the IPNV major capsid polypeptide VP2 gene. For each primer combination only one major product is obtained when amplifieation is performed using IPNV double‐stranded RNA from two different viral strains, Sp and VR‐299, as the initial template. No products were detected when genomie nueleic acids other than IPNV RNA were used as RT‐PCR templates. The specificity of the amplification products were confirmed by Southern hybridization using a specific cDNA probe. To assess the sensitivity of the method, dilutions of purified IPNV dsRNA total genome were amplified and quantities of as little as 1 pg of purified dsRNA were detected when the amplification product was visualized by silver‐stained polyacrylamidc gel electrophoresis. This technique detected IPNV directly in infected coho salmon, Oncorhynchus kisutch (Walbaum), and rainbow trout, Oncorhynchus mykiss (Walbaum), tissues and fish egg samples, avoiding viral propagation in cell culture. The results show that this RT‐PCR amplification method is useful for the direct tissue detection of IPNV.

Giardiavirus double-stranded RNA genome encodes a capsid polypeptide and a gag-pol-like fusion protein by a translation frameshift.

Giardiavirus is a small, nonenveloped virus comprising a monopartite double-stranded RNA genome, a major protein of 100 kDa, and a less abundant polypeptide of 190 kDa. It can be isolated from the culture supernatant of Giardia lamblia, a parasitic flagellate in human and other mammals, and efficiently infects other virus-free G. lamblia. A single-stranded copy of the viral RNA can be electroporated into uninfected G. lamblia cells to complete the viral replication cycle. Giardiavirus genomic cDNA of 6100 nt was constructed and its sequence revealed the presence of two large open reading frames that are separated by a -1 frameshift and share an overlap of 220 nt. The 3' open reading frame contains all consensus RNA-dependent RNA polymerase sequence motifs. A heptamer-pseudoknot structure similar to those found at ribosomal slippage sites in retroviruses and yeast killer virus was identified within this overlap. Immunostudies using antisera against synthesized peptides from four regions in the two open reading frames indicated that the 100- and 190-kDa viral proteins share a common domain in the amino-terminal region. But the 190-kDa protein makes a -1 switch of its reading frame beyond the presumed slippage heptamer and is therefore a -1 frameshift fusion protein similar to the gag-pol fusion protein found in retroviruses.

Polioviruses with Natural Recombinant Genomes Isolated from Vaccine-Associated Paralytic Poliomyelitis

To determine how oral poliovirus vaccine (Sabin) strains evolve during replication in humans and to confirm the etiology of vaccine-associated paralytic poliomyelitis (VAPP), we examined 70 vaccine-derived strains isolated from VAPP cases. Two distant sequences of the poliovirus genome were targeted for a double restriction fragment length polymorphism assay (RFLP) of reverse-transcribed genomic segments amplified by PCR, an extension of the method that we described previously (Balanant et al., 1991). One (RFLP-1) was a 480-long nucleotide sequence coding for the N-terminal part of the VP1 capsid polypeptide, situated in the 5′ third of the vital genome (nucleotides 2401-2880). The other (RFLP-3D1) was a 291-long nucleotide sequence coding for a part of the viral polymerase, situated near the 3′ end of the genome (nucleotides 6086-6376). Strain-specific restriction profiles could be generated for different field isolates by using three restriction enzymes in each case: HaeIII, DdeI, and HpaII for RFLP-1 and HaeIII, DdeI and RsaI for RFLP-3D1. With few exceptions, the vaccine-specific RFLP profiles were found to be conserved in both regions during replication of these viruses in humans. Thus, RFLP could be used as a marker so as to identify the origin of viral isolates at both ends of their genome. Whether viral isolates were vaccine-derived was determined by using strain-specific monoclonal antibodies and RFLP-1. Among the 70 isolates, 21 of the 43 type 2 strains and 15 of the 22 type 3 strains had a recombinant genome. None of the 5 type 1 Sabin-derived isolates was found to be recombinant. Both intertypic vaccine/vaccine and vaccine/non-vaccine recombinants were detected. Partial nucleotide sequencing confirmed the RFLP results in all cases that were investigated. In one case, it was possible to predict the recombination junction site from the restriction profiles. This site was more precisely localized by sequencing. The C 6203 > U nucleotide substitution, which is suspected to contribute to the reversion toward neurovirulence of the attenuated Sabin 1 strain, was detected in almost all the recombinant genomes containing Sabin 1-specific sequences at the 3′ extremity. This mutation was detected by identification of the modified RsaI profile in the RFLP-3D1. The results presented in this paper suggest that recombination, alone or together with mutation, might be one of the mechanisms of the reversion toward neurovirulence of attenuated vaccine strains and of the natural evolution of poliovirus.

A Parvo-like virus persistently infecting a C6/36 clone of Aedes albopictus mosquito cell line and pathogenic for Aedes aegypti larvae

We have isolated and partially characterized from an apparently healthy C6/36 subclone of Aedes albopictus cell line a small icosahedral non-enveloped DNA virus, designated AaPV. This virus proved to be highly pathogenic for Aedes aegypti neonate larvae. Viral infection persisted for over 4 years in the cell culture without any cytopathic effect. Attempts to infect suckling mice, Drosophila melanogaster adults and Spodoptera littoralis larvae with AaPV were unsuccessful. Similarly, the AaPV failed to replicate in vertebrate and Drosophila cell lines. Virions, about 22 nm in diameter, had a buoyant density of 1.43 g/cm 3 and contained three capsid polypeptides with molecular weights of 53, 41 and 40 kDa. A preliminary study of the viral genome indicated the presence of single-stranded DNA. By its biophysical and biochemical properties, this virus appears to be related to the genus Densovirus within the family Parvoviridae, but Jacks serological relationships with the other members of this genus.

Patterns and Prevalence of Hepatitis C Virus Infection in Posttransfusion Non-A, Non-B Hepatitis

Improved serologic and polymerase chain reaction (PCR)-based tests for hepatitis C virus (HCV) infection provided an opportunity to reexamine a posttransfusion follow-up study done from 1969 to 1972. A total of 213 cardiac surgery patients was prospectively followed after receiving an average of 18 units of blood, 24% of which was from paid donors. Serial sera were tested for antibody to recombinant DNA-derived C100-3 and capsid polypeptides; selected cases were also tested against synthetic peptides derived from different regions of the HCV sequence. PCR and RIBA II immunoblot assays were done on selected sera. Each of 55 probable and 5 of 11 possible hepatitis cases who were seronegative before transfusion seroconverted. Anti-HCV seroconversion also occurred in 6 (4%) of 148 subjects without hepatitis. Among subjects followed > 1 year, PCR positivity persisted in 14 (82%) of 17. If the results of this study can be generalized, all bloodborne non-A, non-B hepatitis may be due to HCV.

Rapid generation of monoclonal antibody-secreting hybridomas against African horse sickness virus by in vitro immunization and the fusion/cloning technique

Splenocytes from non-immune mice were stimulated in vitro using an equimolar mixture of factors from mixed lymphocyte reaction (MLR) and from phorbol-12-myristate 13-acetate (PMA) stimulated EL-4 cells, and concomitantly immunized with inactivated African horse sickness virus (AHSV) antigen serotype 4 or viral proteins 2 and 5 from AHSV serotype 9. Fusion with NSO myeloma cells was performed five days after primary or secondary stimulation/immunization. The record of hybridoma growth after a standard method of fusion, expansion of cells and subsequent cloning was compared with a fusion/cloning method in which cells were cloned within 2 to 3 days of the fusion event. Detection of antigen specific antibodies in the hybridoma culture supernatants was successful only with cells derived from primary stimulation/immunizations. Antibodies were detected using an indirect ELISA with the immunizing antigen coated on to the surface of the plates. Monoclonal hybridomas were isolated within 2 to 3 weeks using the fusion/cloning method, compared with the standard method, where it took 4 to 5 weeks. Although the total number of clones isolated from the fusion/cloning method was less than that obtained through the standard method, the yield of specific antibody-producing hybridomas as a percentage of the total picked was often more efficient with the fusion/cloning method. With respect to the immunoglobulin isotype produced, not all of the antibodies could be classified by the ELISA system used; 14% of anti-AHSV positive clones were identified as IgG-secreting cells, 25% as IgM-secreting, 18% were cross-reacting with IgG and IgM, and 43% could not be classified. Similar results in all aspects of the work were obtained whether a crude infected cell extract or purified outer capsid polypeptides VP2/5, from serotype 4 and serotype 9 respectively, were used.

The detection of single‐stranded RNA in an isometric virus‐like particle from Shiitake mushroom [Lentinus edodes (Berk.) Sing.

SummaryIsometric virus‐like particles (VLPs) with diameter of approximately 34 nm containing ss‐RNA were purified from abnormal mycelium of Shiitake mushroom, Lentinus edodes (Berk.) Sing. SDS‐polyacrylamide gel elec‐trophoresis demonstrated that the virions contain a single capsid polypeptide with molecular weight of about 22 000 daltons. The nucleic acid extract from purified VLPs preparations showed only one band with a size of approximately 7.3 kilobases. The susceptibility to RNase 1 and S1 digestions, resistance to DNase and thermal denaturation behaviour of the viral genome indicated that it is a single‐stranded RNA. To our knowledge, isometric single‐stranded RNA VLPs isolated from Shiitake mushroom mycelia have not been reported before.

Characterization of a genetic variant of human hepatitis A virus.

Human isolates of hepatitis A (HAV) are a single serotype; however, recent genetic surveys using limited nucleotide sequencing have provided evidence that more than one genotype is responsible for HAV infection in different parts of the world (Jansen et al. [1990]: Proc Natl Acad Sci USA 87:2867-2871; Robertson et al. [1991] J Infect Dis 163:286-292). One of these genotypes was originally isolated from Panamanian owl monkeys (strain PA21), but has subsequently been found associated with human cases of HAV from Sweden in 1979 (H-122) and the United States of America in 1976 (GA76). The nucleic acid sequence of the exposed capsid polypeptide region of GA76 differs from other human HAV sequences by approximately 20%, yet differs by only 2.4% when compared with P1 sequence of the PA21 strain. The 20% nucleic acid variability between GA76 and other human HAV results in limited amino acid changes (3%), while a comparison with PA21 revealed only four homologous amino acid substitutions within VP2, VP3, and VP1 polypeptides. HAV infected stool specimens from Nepal and northern India during 1989 and 1990 were found to contain virus whose genetic makeup was related to the PA21 and GA76 isolates. This genotype of HAV appears to be circulating in some parts of the world where HAV is hyperendemic, and is a potential cause of hepatitis A infection within a susceptible population.

The densovirus of Junonia coenia (Jc DNV) as an insect cell expression vector.

An infectious genome of the Junonia coenia densovirus (Jc DNV) has been recently cloned and sequenced. We investigated the ability of this cloned genome to be used as expression vector by inserting the lacZ gene of Escherichia coli as fusion gene in the major open reading frame (ORF 1) of the viral sequence. The resulting recombinant plasmid designated pBRJlac Z was transfected into insect SPC-SL 52 cells and the expression of beta-galactosidase (beta-gal) was detected qualitatively or quantitatively by using Xgal or ONPG as chromogenic substrates. Western blot analysis revealed that beta-gal was expressed as chimeric capsid-beta-gal polypeptides. This provided evidence that ORF1 codes for structural polypeptides which share a common C-terminal sequence. Construction of plasmids with alterations or deletions in ORF2, 3 or 4, allowed us to implicate nonstructural (NS) functions in viral DNA replication. Deletions in inverted terminal repeats or in NS functions did not abolish expression of capsid polypeptides but reduced it dramatically. Encapsidation of Jlac Z recombinant genome was achieved by trans-complementation with plasmids bearing intact structural and nonstructural functions. Detection of a beta-gal activity in SPC-SL 52 cells following several subcultures post-transfection suggests that Jlac Z recombinant genome could be maintained in an integrative or episomal state.

Identification of a T-cell epitope adjacent to neutralization antigenic site 1 of poliovirus type 1

Proliferative T-cell responses to poliovirus in various strains of mice have been analyzed by using either killed purified virus or capsid protein VP1 synthetic peptides. Following immunization of mice with inactivated poliovirus type 1 (PV1), a specific proliferative response of their lymph node CD4+ T cells was obtained after in vitro stimulation with purified virus. In mice immunized with PV1, PV2, or PV3, a strong cross-reactivity of the T-cell responses was observed after in vitro stimulation with heterologous viruses. By using various strategies, a dominant T-cell epitope was identified in the amino acid 103 to 115 region of capsid polypeptide VP1, close by the C3 neutralization epitope. The T-cell response to VP1 amino acids 103 to 115 is H-2 restricted: H-2d mice are responders, whereas H-2k and H-2b mice do not respond to this T-cell epitope. Immunization of BALB/c (H-2d) mice with the uncoupled p86-115 peptide, which represents VP1 amino acids 86 to 115 and contains both the T-cell epitope and the C3 neutralization epitope, induced poliovirus-specific B- and T-cell responses. Moreover, these mice developed poliovirus neutralizing antibodies.

Identification of the Coat Protein Gene of Bean Golden Mosaic Geminivirus

An open-reading frame designated 1R1, on DNA component 1 of bean golden mosaic geminivirus (BGMV), has been identified as the coat protein gene. A DraI restriction fragment of BGMV DNA1 that includes 1R1 was inserted into the SmaI pKK223-3 expression vector. The 32 kD protein expressed in Escherichia coli cells reacted with antibodies to the BGMV capsid polypeptide and behaved identically to purified capsid protein in western blots.

Lack of myristoylation of poliovirus capsid polypeptide VP0 prevents the formation of virions or results in the assembly of noninfectious virus particles

We previously described the generation of a set of mutations into a cDNA of poliovirus type 1 in the myristoylation signal of the capsid polypeptide VP4 (D. Marc, G. Drugeon, A.-L. Haenni, M. Girard, and S. van der Werf, EMBO J. 8:2661, 1989). Genomic transcripts synthesized in vitro from the mutated cDNAs were found to be noninfectious upon transfection of permissive cells, and this property correlated with the lack of VP0 myristoylation in vivo. In the study presented here, we analyzed the assembly intermediates that could be recovered from cells transfected with the mutated transcripts. We found that 14S pentamers could still assemble to a certain extent with an unmyristoylated VP0. Furthermore, viral particles sedimenting at 150S and containing capsid polypeptides VP1 to VP4 and virus-specific RNA were detected in the transfected cells. However, these mature virions were less abundant than those recovered after transfection with an infectious transcript, and they were devoid of infectivity. The results suggest that VP0 myristoylation plays a role in the late steps of poliovirus assembly and that the myristate moiety of VP4 may be required in the early steps of poliovirus infection.

Biochemical characterization of the structural and nonstructural polypeptides of a porcine group C rotavirus

Purified virions or radiolabeled lysates of infected MA104 cells were used to characterize the structural and nonstructural polypeptides of a porcine group C rotavirus. At least six structural proteins were identified from purified group C rotavirus by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Of these, two (37,000- and 33,000-molecular-weight polypeptides) were associated with the outer shell, as demonstrated by the ability of EDTA to remove them from the purified virion. The other four polypeptides (molecular weights, 125,000, 93,000, 74,000, and 41,000) were located in the inner shell. The structural or nonstructural nature of a 25,000-molecular-weight protein identified in our studies was unclear. Glycosylation inhibition studies with tunicamycin in infected cells demonstrated that the 37,000- and 25,000-molecular-weight proteins were glycosylated and contained mannose-rich oligosaccharides identified by radiolabeling of the infected cells with [3H]mannose. The 37,000-molecular-weight outer shell glycoprotein was shown by pulse-chase experiments to be posttranslationally processed. The kinetics of viral polypeptide synthesis in infected cells were also studied, and maximal synthesis occurred at 6 to 9 h postinfection. The 41,000-molecular-weight inner capsid polypeptide was the most abundant and was the subunit structure of a 165,000-molecular-weight protein aggregate. Two polypeptides (molecular weights, 39,000 and 35,000) appeared to be nonstructural, as determined by comparison of the protein pattern of radiolabeled infected cell lysates with that of purified virions. Radioimmunoprecipitation was used to examine the serologic cross-reactions between the viral polypeptides of a group C rotavirus with those of a group A rotavirus. No serologic cross-reactivities were detected. The polypeptides of group A and C rotaviruses are compared and discussed.

Intracellular appearance, morphological features and properties of oyster mushroom virus

Viruses in Agaricus hisporus and Lentinula edodes are well-known but have not previously been reported in Pleurotus spp. Two kinds of virus were found in sporophores of Pleurotus sapidus in this study. One occurred as spherical particles 24 nm diam and the other as flexuous rods. The virus preparation of P. sapidus had a typical nucleoprotein ultraviolet spectrum and three peaks were obtained during ISCO sucrose density gradient centrifugation. The nucleic acid derived from the spherical virus was shown to be double-stranded ribonucleic acid (dsRNA) by a positive orcinol reaction and RNase treatment. Four major bands appeared during Polyacrylamide gel electrophoresis, and the molecular weight was determined at 1·3, 1·10, 0·98, and 0·85 × 10°6 using CMV dsRNA and virus dsRNA of Aspergillus niger as markers. Two major components of the virus capsid polypeptide with molecular weights 44 and 22 kDa, respectively, were obtained using discontinuous SDS-PAGE. Immune diffusion tests showed that the anti-serum to P. sapidus virus reacted with P. ostreahis virus and P. florida virus. Ultrathin sections of the septal pore apparatus of P. ostreatus revealed the regular arrangement of the pore cap perforation and the openings. Virus particles appear to be able to pass through the septal dolipore. These results provide new evidence for the translocation of viruses in basidiomycetes.

The 15 amino acid residues preceding the amino terminus of the envelope protein in the yellow fever virus polyprotein precursor act as a signal peptide

The 15 amino acids which precede the sequence of the envelope (E) protein in the yellow fever virus (YFV) polyprotein precursor have been proposed to function as a signal peptide for the E protein (P. Desprès, A. Cahour, C. Wychowski, M. Girard and M. Bouloy; Ann. Inst. Pasteur/Virol., 139, 59–67, 1988). To confirm this hypothesis, recombinant SV40 genomes were constructed in which the sequence of the E protein, or that of the poliovirus VPO capsid polypeptide were placed immediately downstream of and in frame with the sequence of the putative signal peptide, under the control of the late SV40 promoter. The E protein expressed by the hybrid virus SV-E was recognized by two neutralizing monoclonal antibodies directed against the YFV envelope protein. In this construct, the E protein was deleted of its C-terminal transmembrane zone. Therefore, as expected, the protein appeared to be efficiently transported along the exocytic pathway and excreted into the cell culture medium. In addition, when the putative signal peptide was fused in frame with poliovirus polypeptide VPO, the expressed chimeric polypeptide was targeted to the endoplasmic reticulum where it underwent glycosylation.

Identification of Virus Components in Circulating Immune Complexes Isolated During Hepatitis A Virus Infection

Circulating immune complexes were isolated by conglutinin affinity chromatography during the course of hepatitis A virus infection in a chimpanzee. Characterization of circulating immune complexes showed that most of the hepatitis A virus-specific antibody was IgM, that IgG was present and that C3d and fibronectin were also present. Hepatitis A virus capsid polypeptides were identified in the circulating immune complexes and polypeptides in the molecular weight range of 63 to 67 kDa having immunological determinants common to both C3d and hepatitis A virus. Hepatitis A virus-RNA was detected in these circulating immune complexes using the polymerase chain reaction for in vitro amplification of nucleic acid and suggests the circulating immune complexes contain intact virus.

Steady-state infection by echovirus 6 associated with nonlytic viral RNA and an unprocessed capsid polypeptide.

We established a human cell line which was persistently infected (PI) by the normally cytolytic echovirus 6. All of the cultured PI cells contained genome-size viral RNA which was synthesized continuously and incorporated into virus particles. This steady-state infection has been maintained for more than 6 years. In contrast to RNA of wild-type echovirus 6, the viral RNA from PI cells was not lytic when transfected into uninfected, susceptible cells. The capsid polypeptides of the virus particles produced during lytic infections were compared with those of virus particles from PI cells. Wild-type virions contained five polypeptides with molecular masses of 31.5, 27, 25.8, 21.2, and 9.5 kilodaltons. Comparison of polypeptide profiles of virions and empty immature capsids along with peptide analyses by immunoblotting and partial proteolysis of isolated viral proteins identified the cleavage products of the 31.5-kilodalton polypeptide (VP0) as the two smaller polypeptides (VP2 and VP4). The virus particles produced by PI cells as well as cellular extracts of PI cells contained only the three largest proteins (VP0, VP1, and VP3), indicating that VP0 was not processed during persistent infection. The lack of VP2 and VP4 in the defective virus particles coincided with their inability to attach to uninfected, susceptible cells. The maintenance of the steady-state infection of echovirus 6 was not dependent upon the release of virus particles from PI cells.

Use of Ar+ plasma etching to localize structural proteins in the capsid of herpes simplex virus type 1

Partially cored herpes simplex virus type 1 (HSV-1) capsids (B capsids) were eroded in a low-energy (0.5-keV) Ar+ ion plasma under conditions in which the outermost structural proteins were expected to be degraded before more internal ones. After various periods of etching, the proteins remaining intact were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and determined quantitatively by densitometric scanning of the stained gels. The results showed that the major capsid polypeptide (VP5) and two other capsid proteins, VP19 and VP23, were degraded rapidly beginning as soon as capsids were exposed to the ion plasma. In contrast, significant lags were observed for erosion of VP21, VP22a, and VP24, suggesting that these proteins were available to accelerated ions only after other, more external structures had been damaged or eroded away. The results suggest that VP5, VP19, and VP23 are exposed on the surface of the capsid, while VP21, VP22a, and VP24 are found inside the capsid cavity. The experiments are consistent with the view that VP5 constitutes the major structural component of the hexavalent capsomers. It is proposed that VP19 and VP23 may form other surface structures such as the pentavalent capsomers, the capsid floor, or the intercapsomeric fibers.

Overlapping genes in a yeast double-stranded RNA virus.

The Saccharomyces cerevisiae viruses have a large viral double-stranded RNA which encodes the major viral capsid polypeptide. We have previously shown that this RNA (L1) also encodes a putative viral RNA-dependent RNA polymerase (D. F. Pietras, M. E. Diamond, and J. A. Bruenn, Nucleic Acids Res., 16:6226, 1988). The organization and expression of the viral genome is similar to that of the gag-pol region of the retroviruses. The complete sequence of L1 demonstrates two large open reading frames on the plus strand which overlap by 129 bases. The first is the gene for the capsid polypeptide, and the second is the gene for the putative RNA polymerase. One of the products of in vitro translation of the denatured viral double-stranded RNA is a polypeptide of the size expected of a capsid-polymerase fusion protein, resulting from a -1 frameshift within the overlapping region. A polypeptide of the size expected for a capsid-polymerase fusion product was found in virions, and it was recognized in Western blots (immunoblots) by antibodies to a synthetic peptide derived from the predicted polymerase sequence.

Expression in Escherichia coli of the major outer capsid protein of infectious pancreatic necrosis virus

The outer capsid polypeptide, VP2, represents the major neutralizing antigen of infectious pancreatic necrosis virus (IPNV). A 926-bp viral cDNA, encoding an N-terminal truncated VP2, was cloned into the pWR590 expression plasmid family resulting in a C-terminal extension of a truncated Escherichia coli β-galactosidase (βGal) under the control of the lac promoter. When cells transformed by in-phase hybrid plasmids were induced by isopropylthiogalactoside, high levels of the 100-kDa βGal-VP2 fusion protein accumulated within 4 h after induction. The fusion protein reacted in Western blots both with rabbit anti-βGal and with neutralizing mouse anti-VP2 monoclonal antibody. Sera of rabbits immunized with semipurified fusion protein reacted with the VP2 polypeptide in Western blots and with intact purified virus in ELISA and also neutralized IPNV infectivity in a plaque-reduction assay. Out-of-phase hybrid plasmids did not produce the fusion protein but expressed a small amount of structurally discrete VP2-specific sequences probably by internal initiation of translation at an in-phase AUG codon near the 5' end of the VP2 gene.