Virus-specific Polypeptides Research Articles

Expression in Escherichia coli of the large genomic segment of infectious pancreatic necrosis virus

A complete cDNA clone of the larger A segment of the genome of infectious pancreatic necrosis virus (IPNV) was expressed in Escherichia coli in an effort to develop a vaccine for IPNV in fish. When the cDNA insert was positioned in the correct orientation to the pUC19 lacZ promoter, the viral proteins VP2, NS, and VP3 were synthesized and processed as observed in infected cells. When the insert was placed in the opposite orientation, VP3 and a 38-kDa virus-specific polypeptide were also synthesized. In addition, specific deletions made from the 3' end into the NS gene of the cloned A segment led to inactivation of the NS proteolytic activity and subsequently, the synthesis of an unprocessed VP2-NS polyprotein precursor. Antiserum to this polyprotein distinguished NS (28.5 kDa) from VP3 (31 kDa) and led to the identification of a previously undescribed 38-kDa virus-specific polypeptide in infected cells. Thus, both internal translational initiation and proteolytic cleavage could lead to the synthesis of VP2, NS, and VP3 from a single mRNA with a single open reading frame. A trpE expression vector, pATH2, was used to synthesize large quantities of the A-segment-encoded proteins in bacteria. The resulting bacterial lysate was very effective in inducing protective immunity in rainbow trout fry.

Cell-free translational analysis of the processing of infectious pancreatic necrosis virus polyprotein

A cDNA clone of the large genomic segment of infectious pancreatic necrosis virus (IPNV) was inserted into transcription vectors and used for production of RNA transcripts of various lengths. These RNA transcripts were used to prime the synthesis of virus-specific polypeptides in a rabbit reticulocyte translation system. Full-length transcripts resulted in the synthesis of processed viral proteins pVP2, NS, and VP3. Transcripts which were deleted in the VP2 coding region did not affect the processing of NS or VP3 and similarly, deletions in VP3 did not affect the formation of NS or VP2. However, deletions which extended into the NS coding region resulted in a loss of protease activity and the production of truncated precursor polypeptides. The virus-specific proteolytic activity could not be inhibited by specific antisera and a trans activity for the viral protease could not be demonstrated.

Analysis of a glycoprotein of human herpesvirus 6 (HHV-6) using monoclonal antibodies

The virus-specific polypeptides of human herpesvirus 6 (HHV-6) were studied. Six hybridoma clones secreting monoclonal antibodies were established. Localized cytoplasmic antigen and surface antigens were found in and on the infected cells, respectively, by using the immunofluorescent test with those antibodies. In the neutralization test, two clones (OHV3 and OHV9) neutralized HHV-6, but the other five monoclonal antibodies did not. When infected cells were labeled with [ 3S]methionine and the antigens were immunoprecipitated by OHV3 and OHV9 followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, two polypeptides with molecular weights of 98,000 (98K) and 92K were detected. Furthermore, it was found that these polypeptides were glycosylated, because they were labeled with [ 14C]mannose. Pulse-chase studies revealed that precursor molecules were cotranslationaly glycosylated to produce 98K glycoprotein and they were replaced by 92K glycoprotein. Endo-β- N-acetylglucosaminidases H and F reduced those glycoproteins to putative precursor molecules of 75 kDa. This indicates that N-linked high-mannose sugars associate to 98K and 92K glycoproteins.

Frog virus 3-mediated translational shut-off: frog virus 3 messages are translationally more efficient than host and heterologous viral messages under conditions of increased translational stress

Frog virus 3 rapidly and selectively blocks host cell translation while synthesizing more than 60 virus-specific polypeptides. Previous work indicated that virus infection led to activation of a kinase that phosphorylated and, as a consequence, inactivated eIF-2. Although phosphorylation of eIF-2 could explain the rapid decline in host cell translation, it could not explain how viral protein synthesis persisted in the face of host shut-off. To explain this phenomenon, we speculated that viral messages, either as a consequence of their higher translational efficiency or their greater abundance, were able to outcompete host messages for the remaining translational initiation complexes. To test this hypothesis, the relative translational efficiency of three characteristic FV3 messages was measured against that of several model messages. Translational efficiency was determined by monitoring the resistance (and hence the competitiveness) of a given transcript to increasing concentrations of salt in vitro and in vivo. In both rabbit reticulocyte lysates and wheat germ extracts, FV3 messages were more resistant to supra-optimal concentrations of potassium acetate than globin message and three BMV transcripts. In vivo, FV3 polypeptides were synthesized in the presence of salt concentrations that blocked host cell protein synthesis. These results suggest that the selective translation of FV3 messages in virus-infected cells may partly be due to the higher translational efficiency of viral messages. Structural features that contribute to translational efficiency are discussed.

Myristoylation of the poliovirus polyprotein is required for proteolytic processing of the capsid and for viral infectivity

The poliovirus polyprotein is cotranslationally linked to myristic acid at its amino-terminal glycine residue. We investigated the role of myristoylation in the viral replication cycle by site-directed mutagenesis of this glycine codon. Synthetic full-length RNA transcripts carrying a Gly-to-Ala mutation (G4002A) gave no infectious virus on transfection into permissive cells (HeLa). However, mutant viral RNA was replicated in the transfected cells, albeit at a reduced level. The virus-specific polypeptide P1, the precursor for the capsid proteins, was found in HeLa cells transfected with wild-type or mutant RNA, but only the wild-type P1 was myristoylated; the G4002A mutant P1 was not myristoylated. We also introduced the G4002A mutation into an in vitro transcription-translation vector encoding poliovirus P1 precursor. Processing of the mutant precursor by poliovirus-infected cell lysate (providing 3Cpro and 3CDpro activities) was severely inhibited, whereas the normally inefficient cleavage by purified 3Cpro was not affected. These results suggest that the myristic acid moiety of the P1 precursor may be required for efficient processing by 3CDpro.

Intracellular virus-induced polypeptides of pestivirus border disease virus

Intracellular virus specific polypeptides of pestivirus, border disease virus (BDV) in bovine turbinate cells were analysed by radio-immunoprecipitation with specific antisera. Eleven viral polypeptides with molecular weights of 220, 165, 118, 84, 66, 58, 55, 53, 45, 37 and 31 kDa, respectively, were detected in infected cells. Of these, the 165, 118, 84, 66, 58, 55, 53, 45 and 31 kDa proteins were found to be glycosylated. Comparative studies indicated that the polypeptides induced by BDV share many antigenic epitopes with those of the polypeptides induced by bovine viral diarrhea virus (BVDV), a serologically related virus of the same genus, pestivirus. The polypeptide profile of BDV appeared to be more similar to that of the noncytopathic BVDV strain NY1 compared to that of cytopathic BVDV strains NADL and Singer. Peptide mapping analysis of homologous polypeptides from BVDV and BDV confirmed their structural relatedness.

Analysis of polypeptides synthesized in bovine respiratory syncytial virus-infected cells

Ten virus-specific polypeptides ranging in molecular weight from approximately 200k to 11k were identified in bovine respiratory syncytial virus (BRSV-)infected cells. Time course analysis of the induction of the viral polypeptides indicated that they could be detected as early as 30 min post-infection and their synthesis reached a plateau 12 h after infection. Cell free translation of total infected-cell mRNA in a rabbit reticulocyte system yielded 7 proteins corresponding in size to virus-specific proteins synthesized in BRSV-infected cells. The P protein was highly phosphorylated; G and F were identified as glycoproteins by [3H]glucosamine labeling. Glycosylation of G protein was largely resistant to tunicamycin, suggesting that the majority of the carbohydrate residues are attached via O-glycosidic bonds, whereas the F protein was N-linked glycosylated. Tunicamycin caused a drastic reduction in the yield of infectious virus titer indicating that the carbohydrate moieties serve a critical role in the infectious cycle of BRSV.

Temporal regulation of equine herpesvirus type 3 transcription

The transcription of equine herpesvirus type 3 (EHV-3; equine coital exanthema virus) has been examined and found to be temporally regulated into three classes: immediate early (IE), early (E), and late (L). Hybridization of in vivo 32PO 4-labeled transcripts revealed that IE transcript(s) are derived exclusively from the inverted repeat segments (IRs) of the viral genome, while E and L transcripts are not restricted to any specific region of the genome. Northern blot analysis of EHV-3 IE RNA revealed a single transcript of approximately 5.7 kb (3.8 MDa). We have previously shown that transcription of equine herpesvirus type 1 (EHV-1) DNA is temporally regulated and produces a single 6 kb IE RNA which is derived from the IRs segments. In this paper, we show that the EHV-1 and EHV-3 IE RNA species are homologous, reflecting the colinearity of the genomes of these two related viruses. While four IE polypeptides are synthesized in EHV-1 infected cells in the presence of actinomycin D following the removal of a cycloheximide block, only one major IE polypeptide (180 kDa) is detectable in EHV-3 infected cells under these conditions. However, immunoprecipitation of EHV-3 infected cell extracts with polyvalent rabbit antisera to IE1 of EHV-1 revealed at least two other viral specific IE polypeptides.

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.

Herpes Simplex Virus Type 1 Infection of Rat Astrocytes in Primary Culture: Effects of Dibutyryl Cyclic AMP

Monolayer cultures of primary rat astrocytes grown with or without dibutyryl cyclic AMP (dBcAMP) for two weeks or longer were infected with round plaque-forming (Rd) or syncytia-forming (Syn) variants of herpes simplex virus type 1 (HSV-1). Infection with HSV-1 did not stimulate synthesis of glial fibrillary acidic protein (GFAP) or alter the general organization of the intermediate (glial) filaments in astrocyte cultures. However, the dBcAMP-treated astrocytes produced 10- to 100-fold lower titers of cell-free progeny HSV-1 than the untreated astrocyte cultures. Radiolabeled amino acid or glucosamine incorporated into acid precipitable cellular or viral glycoproteins was decreased by 10-25% in dBcAMP-treated astrocytes. Distinctive cell-rounding or syncytial cytopathology was produced by HSV-1 strains infecting untreated astrocytes, but the infected dBcAMP-treated astrocytes displayed only cell-rounding cytopathology. The dBcAMP-related effects on HSV-1 infection were specific to primary astrocyte cultures; they were not observed in HSV-1-infected human fibroblast cultures treated with dBcAMP. Comparison of HSV-1 infection of untreated versus dBcAMP-treated astrocytes suggests that the dBcAMP-induced "reactive" or differentiated state of the astrocyte can affect expression of virus-induced cytopathology and virus-specific polypeptide synthesis. The dBcAMP-treated primary astrocyte culture may afford a non-neoplastic, differentiated in vitro system for studying HSV-neural cell interactions.

Identification of Virus-specific Polypeptides by Monoclonal Antibodies against Serotype 2 Marek's Disease Virus

A total of 41 antibody-secreting hybridoma cells against the HPRS24 strain of Marek's disease virus (MDV) type 2 (MDV2) have been isolated. Of these monoclonal antibodies (MAbs), 24 were found by immunofluorescence tests to react specifically with MDV2-infected cells, but not MDV type 1 (MDV1)- or herpesvirus of turkeys (HVT)-infected cells, while eight reacted with MDV1- or MDV2-infected cells and nine with MDV1-, MDV2- or HVT-infected cells. By using these MAbs, seven classes of MDV type-specific or cross-reactive polypeptides were characterized by immunoprecipitation followed by SDS-PAGE. Among them, a 28K/32K glycoprotein differed from the previously identified gA and gB. The 28K/32K glycoprotein was found on the surface of MDV2-infected cells and in the cytoplasm by an immunofluorescence test with MAbs. In addition, a cross-reactive polypeptide of 25K/29K was also detected in MDV1-infected cells with MAbs reactive with the 28K/32K glycoprotein of MDV2.

Virus-specific polypeptides of human parainfluenza virus type 4 and their synthesis in infected cells

We have studied the structural components of human parainfluenza virus type 4A (PIV-4A) and identified some virus-specific polypeptides by immunoprecipitation with polyclonal and monoclonal antibodies followed by one- or two-dimensional SDS-PAGE. HN polypeptides existed as monomer, disulfide-linked dimer, and disulfide-linked larger oligomer in cells infected with PIV-4A. Interestingly, the nonreduced NP, the nonreduced fusion, and the reduced F1 proteins migrated as doublets. Two F1 polypeptides were derived from different F 1+2 proteins which migrated separately under nonreducing condition. In Vero cells infected with two strains of PIV-4A, two lower-molecular-weight proteins related to NP were detected. Oligopeptide patterns of the lower-molecular-weight protein were similar to those of NP protein synthesized in primary monkey kidney cells. The NP-related low-molecular-weight protein(s) was immunoprecipitated by 1 of 11 monoclonal antibodies against mumps virus NP protein. The MAb also reacted with NP proteins of PIV-2 and SV 5. Thus, the epitope recognized by the MAb was common among PIV-2, PIV-4, mumps virus, and SV 5, suggesting that the epitope might have an important biological function. However, the MAb did not react with the intact NP protein from cells infected with PIV-4, indicating that the epitope of PIV-4A was presented only when NP was cleaved. Phosphorylation was demonstrated for NP and P proteins.

Polypeptides of pneumonia virus of mice. I. Immunological cross-reactions and post-translational modifications.

A murine polyclonal antiserum and monoclonal antibodies have been employed to identify pneumonia virus of mice (PVM) polypeptides in infected cells and to study post-translational modifications. Immunoprecipitation experiments using a murine polyclonal antiserum and a monoclonal antibody directed against a 39K protein have established an antigenic relationship between two PVM proteins and the N and P proteins of human respiratory syncytial virus. Although 20 virus-specific polypeptides have been identified in lysates of infected cells, evidence is presented that some of these are related and that the number of unique polypeptides probably does not exceed 11 or 12. The phosphoproteins of PVM have a pattern of mobilities more like that of the recently described pneumovirus causing rhinotracheitis in turkeys than that of human or bovine respiratory syncytial virus.

Incomplete replication of human parainfluenza virus type 2 in mouse L929 cells

Human parainfluenza virus type 2 (HPIV-2) was tested for its ability to replicate in murine L929 cells. L929 cells were non-permissive for replication of HPIV-2. Interferon produced endogenously played no role in its incomplete replication. The mechanism by which growth of HPIV-2 was suppressed in L929 cells was studied. Synthesis of virus-specific polypeptides, particularly glycoprotein(s), was suppressed in HPIV-2-infected L929 cells. The HN mRNA could scarcely be detected in virus-infected L929 cells.

Assignment of wound tumor virus nonstructural polypeptides to cognate dsRNA genome segments by in vitro expression of tailored full-length cDNA clones

Presumptive full-length cDNA clones of 9 of the 12 wound tumor virus double-stranded RNA genome segments were tailored for efficient in vitro expression by a recently described strategy [ Z. Xu, J. V. Anzola, and D. L. Nuss (1987) DNA6, 505–513]. In vitro synthesized polypeptides specified by synthetic transcripts corresponding to the tailored cDNAs comigrated in polyacrylamide gels with in vivo synthesized viral-specific polypeptides. This analysis confirmed the functional integrity of the tailored cDNA clones and identified cognate genome segments which encode all five viral nonstructural polypeptides as well as four structural polypeptides; two which comprise the capsid, one located in the viral core and one associated with the outer protein coat.

Changes in infectious flacherie virus-specific polypeptides and translatable mRNA in the midgut of the silkworm, Bombyx mori, during larval molt

Mise en evidence du changement significatif, a la fois, des polypeptides specifiques du virus et du RNAm dans l'intestin moyen infecte par l'IFV du ver a soie au moment de la mue larvaire

Pneumovirus-like characteristics of the mRNA and proteins of turkey rhinotracheitis virus

Electronmicroscopy has indicated that turkey rhinotracheitis virus (TRTV), the causative agent of an acute repiratory disease in turkeys, is a member of the Paramyxoviridae family. To determine if TRTV belongs to one of the three defined genera of this family ( Paramyxovirus, Morbillivirus and Pneumovirus) we have analysed the RNA and proteins induced during replication of TRTV in Vero cells. Following replication in the presence of actinomycin D 10 polyadenylated RNA bands, ranging in M r from 0.22 to 2.0 × 10 6, were detected in infected cells; some bands probably contained 2 or more RNA species. Viral proteins were studied after radiolabelling in the presence of [ 35S]methionine and [ 3H]glucosamine. Comparison of the polypeptides in mock-infected and infected cells, virions and nucleocapsids and after lentil-lectin chromatography and immunoprecipitation revealed seven virus-specific polypeptides (p), some of which were glycosylated (gp): gp82 ( M r 82K), gp68, gp53, gp15, p43, p40 and p35. These are considered to be analogous to the large glycopolypeptide (HN, H and G), fusion protein precursor F0, the F protein cleavage products F1 and F2, nucleocapsid (N), phosphorylated (P) and matrix (M) polypeptides, respectively, of the Paramyxoviridae. Two other polypeptides ( M r 200K and 22K) were also detected, as was a glycopolypeptide of M r 97K, probably related to gp82. Tunicamycin inhibited glycosylation of gp53 and gp15 but gp82 was little affected, most glycans still being present on a glycopolypeptide of approximately 79K. This finding, indicating that gp82 has mostly O-linked glycans, considered with the mRNA profile and the molecular weight of the N protein shows that of the three genera in this family, TRTV most closely resembles the Pneumovirus genus.

Virus-specific polypeptides of caprine arthritis-encephalitis virus recognized by monoclonal antibodies to virion proteins p24 and p14.

The proteins of an isolate of caprine arthritis-encephalitis virus (CAEV) were analysed by SDS-PAGE and Western blotting. Monoclonal antibodies (MAbs) produced to the main core protein p24 and the small structural protein p14 also recognized two major polypeptides of Mr 41K and 55K in infected cell material, consistent with a precursor role for these gag polypeptides. In addition, the p24 MAbs detected a 33K polypeptide in extracellular virus preparations, while the p14 MAbs reacted strongly with a polypeptide of 18K (corresponding to structural protein p18) and weakly with another of 21K. The use of these MAbs in an indirect fluorescent antibody method revealed an intracytoplasmic location of these viral antigens in both mononucleated and multinucleated (syncytial) infected cells. Cross-reactivity with several other isolates indicated that these MAbs may be useful for diagnosis of CAEV infection.

Turkey rhinotracheitis virus: in vivo and in vitro polypeptide synthesis.

Turkey rhinotracheitis (TRT) virus is a paramyxovirus associated with recent outbreaks of acute respiratory disease in turkeys. On morphological criteria it resembles the pneumoviruses more than the other members of the paramyxovirus family. In this communication we report the identification of five virus-induced polypeptides in TRT virus-infected BS-C-1 cells. The Mr of these polypeptides were estimated as 38K, 35K, 30K, 19K and 15K from their electrophoretic mobility in SDS-polyacrylamide 6 to 15% gradient gels. The virus specificity of four (the 38K, 35K, 30K and 19K Mr polypeptides) of these five was confirmed by their predominance as products of in vitro translation of mRNA from TRT virus-infected BS-C-1 cells. An additional virus-specific polypeptide with an estimated Mr of 22K was revealed by in vitro translation and may be either a non-structural polypeptide or a minor structural protein. All six polypeptides were immunoprecipitated by a murine antiserum. Three other polypeptides (129K, 57K and 45K) were also immunoprecipitated. The 57K, 45K and 15K Mr polypeptides were glycosylated, and the 57K glycopolypeptide may be a disulphide-bonded dimer of the 45K and 15K glycopolypeptides. Another major glycopolypeptide of 83K Mr was observed but its virus-specificity could not be confirmed. Taken together our results tentatively identify eight or nine TRT virus-specified polypeptides and are consistent with the classification of TRT virus as a new member of the genus Pneumovirus.

Analysis of the polypeptides synthesized in rinderpest virus-infected cells.

We have identified, by [35S]methionine labeling, eight major induced proteins and a number of minor proteins in rinderpest virus-infected bovine kidney cells. The polypeptides ranged in molecular weight from 212 to 21.5 kDa. The majority of these polypeptides are virus specific, as demonstrated by immunoprecipitation with rabbit hyperimmune serum against rinderpest. Infected cells radiolabeled with glucosamine contained a 75-kDa polypeptide and a broad band migrating at 80 kDa, both identified as virus specific by immunoprecipitation. Phosphorylated virus-specific proteins of 65 kDa and a complex of polypeptides at 92.5 kDa were also identified. Monospecific and monoclonal antibodies against measles virus and canine distemper virus hemagglutinin, fusion protein, nucleocapsid protein, and phosphoproteins confirmed the identity of the corresponding rinderpest virus-specific polypeptides.