Virus-induced Protein Research Articles

Non-cytopathic infection of rhabdomyosarcoma cells by coxsackie B5 virus.

Infection of rhabdomyosarcoma (RD) cells by coxsackie B5 virus (CBV5) was non-cytopathic, although low titres of infectious virus were produced after 24 h post-infection. The extent of CBV5 replication in RD cells increased after sequential passage of the virus in these cells. The RD cells from the first cycle of CBV5 infection were recovered and maintained in culture for 3 months (equivalent to 21 passages) releasing infectious virus throughout this period; these cells were considered to be persistently infected with CBV5 and were designated piRD cells. Coxsackie virus antigen was demonstrated in a small proportion of piRD cells by immunofluorescence staining. High resolution two-dimensional polyacrylamide gel electrophoresis was used to analyse the intracellular proteins prepared from piRD cells, three proteins were detected which were absent in uninfected RD cells. These new proteins were similar in charge to virus proteins induced during CBV5 lytic infection of HEp-2 cells. Quantitative densitometry of 2-dimensional protein profiles of piRD and uninfected cells showed no significant disruption of RD cell protein synthesis by the persistent virus infection. Three cloned cell lines were recovered from piRD cells, none of which showed evidence of infectious virus or virus-induced protein synthesis suggesting that the parental cell line was a carrier culture for CBV5.

The UL13 virion protein of herpes simplex virus type 1 is phosphorylated by a novel virus-induced protein kinase.

Herpes simplex virus type 1 (HSV-1) induces a protein kinase (PK) activity in infected cell nuclei. In vitro, the enzyme is able to phosphorylate exogenous casein (albeit inefficiently) but not protamine, can use ATP or GTP as a phosphate donor, is stimulated by high salt concentrations and is insensitive to inhibition by heparin. On the basis of these properties, the PK appears to be distinct from previously described cellular enzymes and from the cytoplasmic PK encoded by the viral US3 gene. A major substrate of the enzyme in vitro is a virus-induced protein with an Mr of 57000 (Vmw57). The gene encoding Vmw57 was mapped using recombinants between HSV-1 and HSV-2 to a region of the virus genome containing genes UL9 to UL15. Use of a monospecific rabbit antiserum showed that Vmw57 is a virion structural protein encoded by gene UL13. These results, in conjunction with previous reports that the UL13 protein contains PK sequence motifs, support the notions that the nuclear PK and Vmw57 are identical, and that the observed reactivity is due to autophosphorylation.

DNA-binding proteins of baculovirus-infected cells during permissive and semipermissive replication

Certain insect cell lines have been shown to be permissive (TN-368) or semipermissive (IPLB-LD-652Y) for Autographa califomica nuclear polyhedrosis virus (AcMNPV) replication (McClintock et al., 1986b). In this report DNA-binding proteins were identified in such cell:virus systems by hybridizing Western blots containing uninfected and infected cell proteins with AcMNPV or host DNA probes. In the AcMNPV-infected TN-368 permissive cell system, 8 virus-induced DNA-binding proteins with molecular weights ranging from 67.5K to 18.75K were observed under the highest conditions of stringency. When these DNA-binding proteins were compared to structural proteins of AcMNPV, several appeared to be similar to those observed in SDS-PAGE protein profiles of nonoccluded virus (NOV) and occlusion body (PIBs) preparations. Using an AcMNPV occlusion negative mutant (L1GP- gal3) and an anti-AcMNPV-polyhedrin monoclonal antibody, a major DNA-binding protein (33.0K), observed in the permissive system, corresponded to polyhedrin and to a comigrating virus-induced DNA-binding protein. In the AcMNPV-infected IPLB-LD-652Y semipermissive cell system, no virus-induced DNA-binding proteins were detected. However, several host DNA-binding proteins were present but their ability to bind DNA decreased significantly following infection.

The application of two‐dimensional polyacrylamide gel electrophoresis to medical microbiology: Molecular epidemiology of viruses and bacteria

A variety of molecular methods can be used to identify protein and nucleic acid markers with which to investigate the epidemiology of viruses and bacteria. This paper reviews the application of two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) for studying microbial molecular epidemiology. A small format 2-D PAGE system is described for locating protein markers in group B coxsackie viruses (CVB) and Haemophilus influenzae isolates. Representative isolates of CVB serotypes 2, 4, and 5 were compared by analysing the intracellular proteins present in CVB-infected HEp-2 cells by 2-D PAGE protein gels. Although some of the virus-induced proteins had similar electrophoretic mobilities, the three serotypes could be distinguished from each other on the basis of a major virus-induced protein of molecular weight between 39,000 and 43,000. Protein differences were demonstrated among six serotype 2 CVB (CVB-2) isolates. Four clinical CVB-2 isolates collected over a period of four months had indistinguishable two-dimensional protein profiles. Comparison of the two-dimensional protein profiles of cloned virus stocks prepared from a single clinical CVB isolate demonstrated that it was a heterogeneous virus population. The proteins of nontypable and type-b H. influenzae isolates were compared. Up to 160 proteins, detected by staining with Coomassie Brilliant Blue R, were resolved by 2-D PAGE. Although protein differences between individual bacterial isolates were detected, comparable two-dimensional protein profiles were found for the two groups of H. influenzae isolates. There was no similarity in the two-dimensional protein profiles of H. influenzae and Aeromonas. Potential protein markers were identified that may be useful in long-term studies of H. influenzae epidemiology.

Vaccinia virus induces cell fusion at acid ph and this activity is mediated by the N-terminus of the 14-kDa virus envelope protein

The mechanism by which the large-size poxviruses enter animal cells is not known. In this investigation we show that acid pH treatment of wild-type vaccinia virus-infected cells triggers strong fusion of cells in culture, with an optimum at pH 4.8. We have identified the virus-induced fusion protein as a 14-kDa envelope protein, based on the ability of a 14-kDa specific monoclonal antibody (mAbC3) to block vaccinia virus-induced fusion-from-within and fusion-from-without. We provide genetic evidence for a role of the 14-kDa protein in cell fusion, since insertion of the 14-kDa encoding gene into the genome of nonfusogenic mutant viruses generates heterozygous viruses that now acquire acid pH-dependent fusion activity. DNA sequence analyses of the 14-kDa encoding gene of the mutant viruses, 65-16 and 101-14, reveal N-terminal deletions of 46 and 10 amino acids, respectively. These deletions remove a small hydrophobic region at the N-terminus of the 14-kDa protein and prevent fusion. Our findings demonstrate that vaccinia virus can induce strong fusion of cells in culture at acid pH implying some entry of the virus by endocytosis, that the 14-kDa virus envelope protein is the fusogenic protein, and that the N-terminal proximal region is involved in fusion.

Molecular Cloning of Osmotin and Regulation of Its Expression by ABA and Adaptation to Low Water Potential

In response to adaptation to NaCl, cultured tobacco cells (Nicotiana tabacum L. cv Wisconsin 38) synthesize a major 26 kilodalton protein which has been named osmotin due to its induction by low water potentials. To help characterize the expression of osmotin in adapted cells, a cDNA clone for osmotin has been isolated. Abscisic acid induces messenger RNA encoding osmotin. Levels of this mRNA in adapted cells are approximately 15-fold higher than in unadapted cells. Message for osmotin is present at constant levels through the growth cycle of adapted cells, while in unadapted cells, the level decreases during exponential phase of growth and increases again when the cells approach stationary phase. While abscisic acid induces the message for osmotin, a low water potential environment appears to be required for accumulation of the protein. An osmotic shock to unadapted cells does not increase the amount of message or protein present most likely because this treatment does not induce immediately the accumulation of abscisic acid. The increased expression of osmotin in adapted cells is not correlated with an increase in osmotin gene copy number. Osmotin is homologous to a 24 kilodalton NaCl-induced protein in tomato, as well as thaumatin, maize alpha-amylase/trypsin inhibitor and a tobacco mosaic virus-induced pathogenesis-related protein.

Possible Involvement of Virus-Induced Protein Kinase in the Antiviral State Induced with Interferon-γ against Sindbis Virus

The kinetics of induction of the antiviral state against two RNA viruses, vesicular stomatitis virus (VSV) and Sindbis virus, by human interferons (IFNs)-alpha, -beta, and -gamma was measured and compared with that of 2',5'-oligoadenylate (2-5A) synthetase and protein kinase in cells treated with IFNs. Both enzymes were induced in similar time courses and the induction by IFN-gamma was slower than IFN-alpha or beta. The time course of the induction of antiviral state against VSV almost paralleled with that of the enzyme induction by each IFN species. In contrast, the induction of antiviral state against Sindbis virus with IFN-gamma was as fast as that induced with IFN-alpha or beta, in spite of the slower enzyme induction by IFN-gamma. The addition of actinomycin D at the time of virus challenge did not substantially affect the induction of the antiviral state against VSV, but markedly retarded the establishment of IFN-gamma-induced antiviral state against Sindbis virus. These results suggest that the antiviral machinery against VSV is induced solely by IFN during the pretreatment, but the one against Sindbis virus involves additional cellular component(s) induced shortly after virus infection, especially in the case of IFN-gamma. Sindbis virus, but not VSV, induced a cellular double-stranded (ds) RNA-dependent protein kinase at an early stage of virus replication. The kinase appeared to phosphorylate the same protein as IFN-induced kinase in the IFN-gamma-treated and Sindbis virus-infected cells, leading to an increased phosphorylation level. These results are consistent with the idea that the Sindbis virus-induced protein kinase may be involved in the IFN-gamma-induced antiviral state against Sindbis virus.

Methylation of the promoter for an immediate-early frog virus 3 gene does not inhibit transcription.

Methylation of critical sites within the promoter region of eucaryotic genes has been shown to inhibit transcription by RNA polymerase II. However, although the large DNA virus frog virus 3 (FV3) has a highly methylated genome, it uses host RNA polymerase II for at least the immediate-early stage of transcription. We have previously shown that an FV3-induced trans-acting protein allows transcription from adenovirus promoters inactivated by methylation. Since FV3 immediate-early genes are transcribed in the absence of de novo protein synthesis, it appears that the virus-induced trans-acting protein that allows transcription from methylated templates is not required for transcription of the immediate-early FV3 genes, possibly because they are not methylated in critical regulatory sequences. In this study, we used site-directed mutagenesis to alter the three CpG dinucleotide sequences in the promoter region of an immediate-early FV3 gene and thereby created sites recognized by bacterial methylases. Transient-expression assays demonstrated that neither the mutations nor methylation of the mutated sites inhibited transcription from the FV3 promoter in FV3-infected cells. These findings support the hypothesis that the immediate-early genes of FV3 do not contain methylatable sites in regions critical for transcription. The function of the virus-induced trans-acting protein that can override the inhibitory effect of methylation may therefore be to facilitate the transcription of methylated delayed-early or late FV3 genes.

A protein of molar mass 12 kDa incorporates into the membrane of ASF virus-infected cells

An African swine fever virus-induced protein of molar mass 12 kDa (p12) was studied in virus-infected Vero cells using the monoclonal antibody 18B.B11. Protein p12 is incorporated into the membrane of infected cells about 7 h post-infection and is not present in purified African swine fever virus particles. The synthesis of protein p12 is sensitive to cytosine arabinoside.

Identification of the virus-induced protein of tobacco leaves that resembles the sweet-protein thaumatin

One of the pathogenesis-related proteins, PR-R, induced in tobacco leaves by infection with tobacco mosaic virus, has many of the properties expected of the virus-induced, thaumatin-like protein that has been predicted from mRNA studies. It has a high content of cysteine, a molecular weight near 23 000, and one of its two isoforms has a sequence of 54 amino acids that is identical to that in the predicted protein. Although the circular dichroism spectrum of PR-R resembles that of the sweet-tasting protein thaumatin, the protein does not appear to react with a thaumatin antibody, nor has it a marked sweet taste.

Replication of Heliothis zea Nuclear Polyhedrosis Virus in Insect Cell Culture: Virus-induced and Structural Proteins

Summary The replication of Heliothis zea nuclear polyhedrosis virus was studied in the H. zea (IPLB-HZ-1075A) insect cell line. Infectious extracellular virus was first detected at 24 h post-infection (p.i.) and reached a maximum titre of 1 × 107 p.f.u./ml at 90 h p.i. Viral DNA synthesis was detected by 14 h p.i. and increased exponentially until 32 h p.i. Using [35S]methionine pulse-labelling, 38 virus-induced proteins were identified, ranging in mol. wt. from 13K to 128K. Virus-induced protein synthesis was temporally regulated, with new proteins detected between 8 and 28 h p.i. Host cell protein synthesis continued throughout the virus replication cycle (48 h), although at gradually decreasing rates. Fifteen extracellular virus structural proteins were identified, and all but one comigrated with virus-induced proteins. Thirty-two structural proteins of tissue culture-derived, occluded virus were identified using silver staining procedures.

Analysis of <i>Herpesvirus sylvilagu</i><i>s</i>-lnduced Proteins in Infected Rabbit Kidney Cells by Two-Dimensional Gel Electrophoresis

Two-dimensional polyacrylamide gel electrophoresis showed that structural virus proteins (VP) were first synthesized in the following time course: VP9 and VP16 during the pulse-labeling period 2-4 h postinfection; VP5 (the major capsid protein), VP23, VP25, and VP39 during the pulse-labeling period 4-6 h postinfection, VP10 and VP41 during the pulse-labeling period 6-8 h postinfection, and VP12 and VP34 during the pulse-labeling period 8-10 h postinfection. Once synthesis began, all of the virus-induced structural proteins, except VP23, were synthesized during the remainder of the one-step virus growth cycle. Synthesis of a nonstructural 45-kilodalton (Kd) virus-induced protein complex began 4-6 h postinfection and continued for the next 20 h. The complex was resolved into two sets of spots, designated the A and B series. The A series consisted of 7 species of proteins with a molecular weight of 45 Kd ranging in isoelectric points from 6.16 to 5.90. The B series consisted of 4 species having a molecular weight of 43.5 Kd and an isoelectric point identical to the homologous protein in the A series. One immediate-early virus-induced protein (VIP115) was identified. Early events in virus infection include selective inhibition of the host protein hgl, glycosylation, and the synthesis of the 45- and 48-Kd virus-induced glycoprotein complexes. The D/E virus-induced protein doublet is synthesized from the infecting virus genome, but its glycosylation is dependent on viral DNA replication.

Varicella zoster virus glycoprotein gpI is selectively phosphorylated by a virus-induced protein kinase.

Varicella zoster virus glycoprotein I (VZV gpI; Mr 98,000) was phosphorylated in virus-infected human cell monolayers, while two other major VZV glycoproteins (gpII and gpIII) were not similarly modified. Phosphorylation of VZV gpI was not blocked by inhibitors of glycosylation, nor were the phosphoryl groups enzymatically removed by endoglycosidases. Phosphoamino acid analysis revealed the presence of phosphoserine and phosphothreonine residues on the polypeptide backbone. The selective nature of the phosphorylation event was further demonstrated in vitro by a protein kinase (Mr 50,000), which was present in virus-infected cells but absent from uninfected cells or purified virions. The enzyme catalyzed the transfer of 32Pi from [gamma-32P]ATP to gpI but not to gpII and gpIII. Like VZV gpI, this virus-induced protein kinase was also a constituent of the plasma membrane of live VZV-infected cells.

The substrate specificity of the protein kinase induced in cells infected with herpesviruses: Studies with synthetic substatres indicate structural requirements distinct from other protein kinases

Synthetic peptides have been used to investigate the site specificity of highly purified virus induced protein kinase, a recently discovered protein kinase isolated from cells infected with α-herpesviruses. The enzyme from cells infected with pseudorabies virus can catalyse the phosphorylation of both seryl and threonyl residues in peptides that contain several arginyl residues on the amino-terminal side of the target residue. At least two arginyl residues are required, and the best substrates examined contain four to six such residues. Virus induced protein kinase differs in site specificity from protein kinase C in being unable to phosphorylate peptides in which multiple arginyl residues are on the carboxyl-terminal side of the target residue, or to phosphorylate peptides in which the arginyl residues are replaced by ornithyl residues. Virus induced protein kinase from cells infected with herpes simples virus type I had similar substrate preferences to virus induced protein kinase from cells infected with pseudorabies virus. Although virus induced protein kinase and the cyclic AMP-dependent protein kinase have several peptide substrates in common, their relative preferences for these (as indicated by K m values) were found to be very different.

Trans-activation of a methylated adenovirus promoter by a frog virus 3 protein.

The high degree of methylation of the frog virus 3 (FV3) genome suggests that FV3-infected cells are capable of transcribing highly methylated DNA. We tested this hypothesis by assaying the transcriptional activity of adenovirus promoters known to be inhibited by methylation. Plasmid constructs containing the E1a and E2aE promoters of adenovirus type 12 linked to the gene for chloramphenicol acetyltransferase [(CAT) EC 2.3.1.28], when methylated and introduced into eukaryotic cells, promoted CAT synthesis only when the cells were subsequently infected with FV3. Mapping of transcriptional initiation sites revealed that the same sites in the E1a promoter were used for the initiation of transcription in uninfected and infected cells. Moreover, Southern blots showed that transfected plasmid DNA from FV3-infected cells was not demethylated. The absence of CAT-specific RNA in transfected cells infected with FV3 in the presence of protein synthesis inhibitors demonstrated that a virus-induced protein was responsible for the trans-activation. Inhibition of transcription from the methylated template by alpha-amanitin indicated that a functional host RNA polymerase II is required for transcription of methylated DNA in FV3-infected cells. The virus-induced trans-acting protein presumably alters either host RNA polymerase II or the methylated DNA template to allow transcription from the methylated adenovirus promoters.

The phosphorylation of ribosomal protein S6 by protein kinases from cells infected with pseudorabies virus.

We examined the ability of protein kinase activities from BHK (baby-hamster kidney) cells infected with pseudorabies virus to catalyse the phosphorylation of ribosomal protein S6 in vitro. When the cytosol from infected cells was fractionated on DEAE-cellulose, 40S ribosomal protein kinase activity was found associated with the two isoforms of the cyclic AMP-dependent protein kinase, protein kinase C and a protein kinase (ViPK, virus-induced protein kinase) only detected in infected cells. The phosphorylation of ribosomal protein by ViPK was of particular interest because the appearance of the protein kinase and the increase in the phosphorylation of protein S6 in infected cells shared a similar time course. At moderate concentrations of KCl the major ribosomal substrate for ViPK was ribosomal protein S7, a protein not found to be phosphorylated in vivo. However, at 600 mM-KCl, or in the presence of 5-10 mM-spermine at 60-150 mM-KCl, the phosphorylation of ribosomal protein S7 was suppressed and ribosomal protein S6 became the major substrate. The maximum stoichiometry of phosphorylation obtained under the latter conditions was 1-2 mol of phosphate/mol of S6, and only mono- and di-phosphorylated forms of S6 were detected on two-dimensional gel electrophoresis. As the infection of BHK cells by pseudorabies virus results in the appearance of phosphorylated species of S6 containing up to 5 mol of phosphate/mol of S6 protein, it appears unlikely that ViPK alone can be responsible for the multiple phosphorylation seen in vivo. Nevertheless, tryptic phosphopeptide analysis did indicate that in vitro ViPK catalysed the phosphorylation of at least one of the sites on ribosomal protein S6 phosphorylated in vivo, so that a contributory role for the enzyme in the phosphorylation in vivo cannot be excluded.

Characteristics of the induction of a new protein kinase in cells infected with herpesviruses.

The appearance of a recently described protein kinase activity (virus-induced protein kinase, ViPK) has been studied during infection of hamster fibroblasts with pseudorabies virus or with herpes simplex virus type 1 (HSV-1). An enzyme activity with comparable catalytic properties was induced in both cases, and had broadly similar kinetics of appearance to that of the viral DNA polymerase. The amount of active ViPK detected depended on the multiplicity of infection, and no ViPK was induced after the viruses had been subjected to irradiation with u.v. light. When cells were infected with the tsK mutant of HSV-1, ViPK was induced at the permissive but not at the restrictive temperature. The ViPK preparations obtained from cells infected with each virus differed in chromatographic properties on anion-exchange and gel-permeation resins. These results indicate that expression of the viral genome is required for induction of ViPK. They suggest that the enzyme may be encoded by the viral genome, but do not provide proof of this.

Studies on Autographa californica nuclear polyhedrosis virus replication in Spodoptera littoralis cells including virus-induced protein synthesis.

The replication of the Autographa californica nuclear polyhedrosis virus in Spodoptera littoralis cells has been investigated. Various cytopathic changes were detected by light and electron microscopy and progeny enveloped virus particles, some occluded within polyhedra, were later seen in the nucleus of infected cells. Infectious virus was released into the growth medium and increased exponentially from ca. 10 to 24 hours post infection and then slowly increased over the next 4 days. In comparison, virus was released ca. 3.5 hours earlier from Spodoptera frugiperda cells. Total rates of DNA and protein synthesis were drastically reduced during the late stages of virus replication when cell death was occurring. By ca. 18 hours post infection, a clear switch from host to virus induced protein synthesis had occurred and a total of 39 virus-induced polypeptides of M.W. 12 to 120 X 10(3) were detected. These included polyhedrin of M.W. 33 X 10(3), which was particularly prominent during the late stages of virus replication, and a major virus structural protein of M.W. 42 X 10(3).

Intracellular Protein Synthesis During Standard and Defective Hz-1 Virus Replication

Summary An isolate of the non-occluded baculovirus Hz-1, containing a high level of defective particles, was recovered after serial passage in Trichoplusia ni (TN-368) tissue culture cells. DNA from defective virions contained deletions of up to 91 kilobase pairs. Defective particles were shown to interfere with the infection or replication of standard virions. Standard virus appeared to be required for replication of defective interfering (DI) particles. Initiation of both standard and DI viral DNA replication occurred at 4 h post-inoculation. Virus-induced protein synthesis was studied by pulse-labelling with [35S]methionine. During defective virus replication in the presence of low amounts of standard virus, there was a significant reduction in the synthesis of 14 proteins (mol. wt. 109, 93, 92, 90, 85, 69, 57, 50, 40, 27.5, 23, 17.5, 17 and 14, all × 10-3) and an increase in the synthesis of five proteins (mol. wt. 104, 75, 41, 37 and 14.2, all × 10-3) as compared to standard virus infections.

Vaccinia virus-infected cells release a novel polypeptide functionally related to transforming and epidermal growth factors.

The recent discovery, that a vaccinia virus (VV) gene encodes a polypeptide with structural homology to transforming growth factor (TGF-alpha) and epidermal growth factor (EGF), led us to look for a virus-induced protein with the predicted biological activity. The supernatants of VV-infected cell cultures were found to contain an acid stable Mr 25,000 polypeptide that competes with EGF for binding to EGF membrane receptors. This VV-induced growth factor (VGF) like EGF and TGF-alpha is mitogenic and stimulates anchorage-independent cell growth in the presence of TGF-beta. However, VGF did not cross-react in a radioimmunoassay specific for small and large forms of TGF-alpha and exhibited minimal cross-reactivity with antisera to EGF. VGF was detectable in the culture medium within 2 hr, and maximal amounts were present 12 hr after infection. The level of VGF was proportional to the multiplicity of VV used. Inhibition of viral DNA synthesis enhanced VGF production, consistent with the hypothesis that VGF is an early gene product encoded by VV. The demonstration of a novel growth factor, released from cells infected with VV, may have important implications regarding the nature of virus-host interactions.