Virus-induced Proteins Research Articles

DNA-binding proteins in frog virus 3-infected cells.

At least 12 virus-induced DNA-binding proteins with mol. wt. ranging from 14 X 10(3) have been isolated from frog virus (FV 3)-infected fathead minnow cells by DNA affinity chromatography. Two enzymic activities, DNA-dependent DNA polymerase and endodeoxyribonuclease, were present in the DNA-binding proteins; these enzymic activities were similar to those induced by FV 3 in infected cells. A single species of DNA-binding proteins with a mol. wt of 36 000 had very high affinity for single-stranded DNA, but a low one for double-stranded DNA. Proteins with such characteristic affinity for single-stranded DNA destabilize the DNA helix and are essential for viral DNA replication. Thus, the 36 000 mol. wt. DNA-binding protein is a candidate for such a role in FV 3 DNA replication.

Cucumber mosaic virus-induced RNA replicase: Solubilization and partial purification of the particulate enzyme

Cucumber seedlings infected with cucumber mosaic virus (CMV) contain a virus-induced RNA-dependent RNA polymerase (RNA replicase) in both soluble and particulate fractions of plant extracts; no such activity is found in uninfected plants. The extensive purification and characterization of the soluble RNA replicase has been described ( R. Kumarasamy and R. H. Symons, 1979, Virology 96, 622–632) while the characterization of the particulate enzyme is reported here. The particulate RNA replicase was solubilized-by incubation in 150 m M MgS04 at 37° for 5 min; both magnesium and sulfate ions were required for maximum release. Only 5–7% of the protein of the particulate fraction was released under these conditions either in the presence or absence of a variety of salts. The solubilized enzyme was purified about 60-fold by a three-step procedure to a specific activity similar to that of the 10,000-fold purified soluble enzyme. CMV RNA and poly(C)-copying activities copurified through the three steps. Purification was followed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate of the in vitro 3H-labeled proteins. The most prominent virus-induced components were two proteins of M r 100,000 and 110,000 which correspond in size to two virus-induced proteins in the extensively purified soluble RNA replicase. The M r 110,000 protein comigrated with the full length in vitro translation product of CMV RNA 2 while the Mr 100,000 was detectably larger than the full length in vitro translation product of CMV RNA 1.

Epstein-Barr virus-induced proteins. IV. Characterization of an EBV-associated phosphopolypeptide.

Cells of the Epstein--Barr virus (EBV) non-producing lines NC37 and Raji were induced by the tumour promoter TPA and were labelled with 32P. The analysis by immunoprecipitation with human VCA+EA+ sera revealed that a major phosphopolypeptide with a mol. wt. of 58 000 was specifically precipitated. In addition, a phosphopolypeptide of the same size was the dominant phosphopolypeptide detected in EBV-producing B95-8 and P3HR-1 cells as well as in P3HR-1-superinfected NC37 cells. This phosphopolypeptide seemed to be EBV-specific, because it was not detectable in TPA-treated, but EBV genome-negative, Ramos and BJAB cells. Synthesis of this phosphopolypeptide was inhibited in TPA-treated NC37 cells by treatment with Ara-C. VCA+EA+ sera were found to be more immunoreactive with this phosphopolypeptide than VCA+EA- or EBNA+ sera. Based on these results this phosphopolypeptide may have some immunological relatedness to the EBV-specific early antigen (EA) complex defined by immunofluorescence.

Molecular biology of rotaviruses. I. Characterization of basic growth parameters and pattern of macromolecular synthesis.

The United Kingdom tissue-adapted bovine rotavirus growing in African green monkey kidney (BSC-1) cells was selected as a model system with which to study the detailed molecular virology of rotavirus replication. Study of the kinetics of infectious virus production revealed a fairly rapid replication cycle, with maximum yield of virus after 10 to 12 h at 37 degrees C. Progeny genome synthesis was first detected during the virus latent period at 2 to 3 h postinfection. Study of the kinetics of viral polypeptide synthesis showed that virus rapidly inhibited cellular polypeptide synthesis such that by 4 h postinfection, only virus-induced polypeptides, 15 of which were detected, were being synthesized. No qualitative changes in the pattern of viral polypeptide synthesis were observed during infection, although, based on kinetic synthesis, three quantitative classes of polypeptides were defined. Pulse-chase analysis revealed three post-translational changes in viral proteins, two of which were shown to be due to glycosylation. Tunicamycin inhibition studies were used to identify the putative non-glycosylated precursors of the two glycoproteins. Comparison of the infected-cell polypeptides with those present in purified virions revealed that mot of the virus-induced proteins were incorporated into virions, with only VP9 being a truly nonstructural protein. Some localization of the various polypeptides within the purified virion was achieved by producing viral cores.

The polypeptides induced in Drosophila cells by Drosophila C virus (strain Ouarzazate)

The Ouarzazate strain of Drosophila virus (DCV 0) was grown in Drosophila melanogaster tissue culture cells, and [ 35S]methionine-labeled virions were found to contain a group of major structural proteins with a molecular weight of ≌30,000 as well as several minor proteins of higher molecular weight and a protein of ≌10,000 daltons. Using a range of pulses, chases and gel systems, examination of the intracellular proteins induced by DCV 0 showed the presence of 17 polypeptides not found in uninfected cells. The synthesis of virus-induced polypeptides was extremely asymmetric with a rapid appearance of the major virus structural proteins and a much slower appearance of the lowest molecular weight structural protein (VP4). Processing of virus-induced proteins including the appearance of VP4 was demonstrated using pulse-chase after pulsing with [ 35S]methionine. While the highest molecular weight induced protein found in infected cells was 146,000, pretreatment of cells with iodoacetamide resulted in the appearance of a protein with a molecular weight of ≌200,000. The evidence presented in this paper supports the inclusion of DCV 0 in the Picornaviridae group.

Synthesis of Black Beetle Virus Proteins in Cultured Drosophila Cells: Differential Expression of RNAs 1 and 2

Black beetle virus is an insect virus with a split genome consisting of two single-stranded, messenger-active RNA molecules with molecular weights of 1.0 x 10(6) (RNA 1) and 0.5 x 10(6) (RNA 2), respectively. Virions contained two proteins, beta with a molecular weight of 43,000 (43K) and gamma (5K), and traces of a third protein, alpha (47K). When translated in cell-free extracts of rabbit reticulocytes, RNA 1 directed the synthesis of protein A (104K), whereas RNA 2 synthesized protein alpha. The in vitro translation efficiency of the two RNAs was roughly equal. Infection of cultured Drosophila cells induced the synthesis of five new proteins: A, alpha, beta, gamma, and B (10K), detected by autoradiography of polyacrylamide gels after electrophoresis of extracts from [(35)S]methionine-labeled cultures. All but protein gamma could also be detected by staining with Coomassie brilliant blue, indicating vigorous synthesis of viral proteins. Pulse-chase experiments in infected cells revealed the disappearance of protein alpha and the coordinate appearance of proteins beta and gamma, supporting an earlier proposal that coat protein of mature virions is made by cleavage of precursor alpha. Proteins A and B were stable in such pulse-chase experiments. The three classes of virus-induced proteins, represented by A, B, and alpha, were synthesized in markedly different amounts and with different kinetics. Synthesis of proteins A and B peaked early in infection and then declined, whereas synthesis of coat protein precursor alpha peaked much later. These results suggest that RNA 1 controls early replication functions via protein A (and also possibly protein B), whereas RNA 2 controls synthesis of coat protein required later for virion assembly.

Expression of Bottom Component RNA of Cowpea Mosaic Virus in Cowpea Protoplasts

Upon inoculation of cowpea protoplasts with the bottom component of cowpea mosaic virus, at least six virus-induced proteins (with sizes of 170, 110, 87, 84, 60, and 32 kilodaltons) are synthesized, but not the capsid proteins (37 and 23 kilodaltons). These bottom-component-induced proteins were studied with respect to their genetic origin and mode of synthesis. The analyses were based on their electrophoretic peptide patterns resulting from partial digestion with Staphylococcus aureus protease V8. Comparison of the peptide patterns of the virus-induced proteins with those of the cowpea mosaic virus RNA-coded polypeptides produced in rabbit reticulocyte lysate showed that the 170- and 32-kilodalton polypeptides, which are the first viral products in cowpea mosaic virus-infected cells, were actually coded by the bottom component RNA of the virus. The 110-, 87-, and 84-kilodalton polypeptides, and possibly the 60-kilodalton polypeptide, appeared to have amino acid sequences in common with the 170-kilodalton polypeptide, demonstrating that they were virus coded as well. The results indicated that cowpea mosaic virus bottom component RNA was translated in vivo into a single 200-kilodalton polyprotein from which probably all bottom-component-specific proteins arose by three successive cleavages.

Isolation and replication of an occlusion body-deficient mutant of the Autographa californica nuclear polyhedrosis virus

A spontaneous mutant of the Autographa californica nuclear polyhedrosis virus which was deficient in viral occlusion body formation was studied in Trichoplusia ni tissue culture cells. Virus multiplication could be divided into four phases based on growth curves and the sequential appearance of 25 viral-induced proteins identified by pulse labeling and polyacrylamide gel electrophoresis. During the latent phase, five viral-induced proteins with molecular weights of 68, 47, 35, and 30 thousand (K) were detected. The synthesis of 17 additional viral-induced proteins with molecular weights ranging from 64 to 14K was detected during the nonoccluded virus (NOV) phase, 10 to 16 hr postinoculation (pi). During the transition from NOV to occluded virus formation, 16 to 34 hr pi, the synthesis of occlusion body matrix protein (33K) and two additional proteins (15.6 and 13K) was detected. During the occlusion phase of replication, after 34 hr pi, NOV synthesis ceased; however, the synthesis of the 25 viral-induced proteins continued. Posttranslational cleavage was not detected in pulse-chase experiments. The occlusion body-deficient mutant induced lower levels of occlusion body matrix protein synthesis than the wild type virus. The decrease in amount of occluded virions was accompanied by a corresponding increase in the number of NOV particles produced.

Polypeptide synthesis and phosphorylation in Epstein-Barr virus-infected cells.

Epstein-Barr virus superinfection of the human lymphoblastoid cell line Raji, a Burkitt lymphoma-derived line that contains Epstein-Barr virus genomes in an episomal form, results in the sequential synthesis of 29 detectable proteins, which range in molecular weight from approximately 155,000 to 21,000, and in the shutoff of the bulk of host protein synthesis within 6 to 9 h after infection. There are three classes of virus-induced proteins; these are an early class, consisting of eight proteins synthesized by 6 h postinfection, an intermediate class, containing two proteins synthesized 9 h postinfection, and a late class, consisting of five proteins synthesized 12 h postinfection. In addition, there is a fourth class of polypeptides, called persistent, that are found both before and after superinfection. The rates of synthesis of the proteins fall into three patterns; these are pattern A, in which the rate of synthesis decreases, pattern B, in which the rate of synthesis remains steady, and pattern C, in which the rate of synthesis increases after the initial appearance of the polypeptide. Both 9-(2-hydroxy-ethoxymethyl)guanine (acyclovir) and phosphonoacetic acid inhibit the appearance of one intermediate protein and at least three late proteins. Seven polypeptides are phosphorylated at different times after infection.

Autographa californica nuclear polyhedrosis virus-induced proteins in tissue culture

The multiplication of Autographa californica nuclear polyhedrosis virus was studied in Trichoplusia ni and Spodoptera frugiperda insect cell lines. Using pulse labeling, 18 viral-induced proteins were identified by polyacrylamide gel electrophoresis. Based on infectivity curves and protein synthesis studies, replication could be divided into four phases. During the early phase of infection, four viral-induced proteins with molecular weights of 45, 35, 34, and 31 thousand daltons could be detected. Maturation of the nonoccluded form of the virus and synthesis of 10 viral-induced proteins with molecular weights of 67, 61, 49, 41, 39, 26, 24, 22, 21, and 16 thousand daltons were first detected during the intermediate phase of replication, 10 to 16 hr postinoculation (p.i.). During the transitional phase from nonoccluded to occluded virus formation (16 to 24 hr p.i.), four additional viral-induced proteins could be detected with molecular weights of 33, 27, 15.7, and 15 thousand daltons. No additional viral-induced proteins were detected during the late phase of occluded virus formation. Post-translational cleavage was not detected in pulse-chase experiments. The addition of varying concentrations of NaCl did not selectively inhibit cellular protein synthesis.

Epstein-Barr virus-induced proteins II. Analysis of surface polypeptides from EBV-producing and -superinfected cells by immunoprecipitation

Cells of the Epstein-Barr virus (EBV)-producing line P3HR-1 induced by the tumor promoter TPA and NC37 cells Superinfected with P3HR-1 EBV were surface labeled with 125I by the lactoperoxidase method and analyzed by immunoprecipitation with human VCA + MA + sera for virus-induced cell surface polypeptides. Two dominant polypeptides with molecular weights of 80,000 and 250,000 were specifically precipitated. In addition, only traces of polypeptides with 130,000 and 140,000 MW were identified on P3HR-1-EBV-producing cells. The surface of superinfected NC37 cells contained two polypeptides of 80,000 and 140,000 molecular weight. Our experiments demonstrated that the synthesis of the 140,000 polypeptide is Ara C sensitive, while the 80,000 polypeptide is Ara-C insensitive. Both polypeptides were found to be identical in size with [ 35S]-methionine- and 125I-labeled 80,000 and 140,000 envelope polypeptides from purified virus particles. These results may indicate that the identified polypeptides carry antigenic determinants of the EBV-induced membrane antigen (MA) complex.

Epstein-Barr virus-induced proteins. III. Analysis of polypeptides from P3HR-I-EBV-superinfected NC37 cells by immunoprecipitation

P3HR-I-EBV induces in superinfected nonproducer NC37 cells at least 13 [ 35S]methionine-labeled polypeptides with molecular weights between 18,000 and 150,000, which were identified by immunoprecipitation using human sera. Application of Ara-C inhibited the synthesis of the two structural polypeptides p150 and p140 while the synthesis of the other polypeptides was not affected.

A type-specific antiserum induced by a major herpesvirus type 1 glycoprotein

A type-specific antiserum was prepared against VP 7/8, the major HSV-1 type-specific glycoprotein. The specificity of this antiserum was demonstrated by both neutralization of HSV-1 infectivity and immunoprecipitation followed by SDS-polyacrylamide gel electrophoresis of the immunoprecipitates. Only minimal cross-reactivity with HSV-2 was observed. This major VP 7/8 glycoprotein was also purified from a host of virus-induced proteins with the specific immunoadsorbent prepared with anti-VP 7/8 serum The one-step purification of this type-specific neutralizing antigen of HSV-1 should allow the development of a sensitive and specific radioimmunoassay.

Biochemical aspects of variation in foot-and-mouth disease virus.

The biochemical basis for variation in foot-and-mouth disease virus (FMDV) has been explored by analysis of the virus RNA and the virus-induced and structural proteins of three isolates of the virus. Two of the isolates were from serotype A and the third was from serotype O. Hybridization studies of the RNAs showed greater than 80% homology between the two type A viruses and about 65% homology between the two type A viruses and the virus of type O. The ribonuclease T1 maps of the three viruses gave distinct patterns typical of FMDV, but did not show that any two of the three viruses were more closely related. The virus-induced primary translation products, P88, P52 and P100 isolated from infected cells, were compared by tryptic peptide analysis. Combinations of 3H- and 14C-leucine-labelled polypeptides were hydrolysed with trypsin and resolved on an ion-exchange column. Much greater differences were found in P88 than in P52 or P100, indicating that the major variation occurs in the region of the genome coding for the structural proteins. Similar analysis of combinations of the structural proteins of the three viruses showed that there were differences in VP1, VP2 and VP3 and these results were supported by those obtained by PAGE analysis of the Staphylococcus aureus V8 protease cleavage products.

Specificity of measles and canine distemper virus antibodies

Rabbits, guinea-pigs and a goat were inoculated with purified measles virus strains, and rabbits with canine distemper virus (CDV). Antisera from rabbits did not readily distinguish between measles virus strains, although guinea-pig antiserum was more selective for the homologous virus. No cross-reaction (neutralization, inhibition of haemagglutination or haemolysin) was observed between measles and CDV with these sera. Human sera from healthy adults, subacute sclerosing panencephalitis (SSPE) and multiple sclerosis (MS) patients occasionally contained CDV-neutralizing antibodies although the titre was always lower than that against measles virus. Several human sera neutralized the Edmonston strain better than the Hallé SSPE strain. However, a plaque-purified LEC strain (SSPE) was also neutralized as efficiently by certain human sera. The strain specificity was unmodified by removal of the haemagglutinin (HA) antibody from the sera. Immunoprecipitation and polyacrylamide gel electrophoresis of 35S-measles virus-induced proteins showed that all the polypeptides with the exception of the HA (80K) cross-reacted with CDV dog serum.

Persistence of the cytomegalovirus genome in human cells

A small percentage of human fibroblast cells survived high-multiplicity infection by cytomegalovirus and were isolated as persistently infected cultures. Approximately 30% of the cells were in the productive phase of infection, since virus-specific structural antigens and virions were associated with these cells. The remaining cells contained neither viral structural antigens nor particles. Nuclear DNA from these nonproductive cells contained approximately 120 genome equivalents of viral DNA per cell as determined by reassociation kinetics. In situ hybridization confirmed that nuclei from nonproductive cells contained a significant amount of viral DNA that was distributed in most of these cells. Early virus-induced proteins and antigens were also detected. Nonproductive cells continued to grow, and there was a slow, spontaneous transition of some of these cells to productive viral replication. The majority of the viral DNA in nonproductive cells persisted with restricted gene expression. When infectious virus production was eliminated by growing the persistently infected cultures in the presence of anticytomegalovirus serum, approximately 45 genome equivalents of the viral DNA persisted per cell. The reassociation reaction approached completion. After removal of the antiserum and subculturing, infectious virus production resumed. Therefore, it was assumed that all sequences of the viral genome remained associated with these cells. Restriction of cytomegalovirus gene expression in persistently infected cell cultures is discussed.

Induction of virus-neutralizing antibody by glycoproteins isolated from chicken cells infected with a herpesvirus of turkeys

The production of virus-induced proteins in chicken embryo fibroblast cells infected with a herpesvirus of turkeys was studied. It was found that glycoproteins isolated from membrane-rich fractions of infected cells by affinity chromatography using concanavalin A induced neutralizing and precipitating antibody in rabbits and chickens. After analytical electrophoresis, such isolates were found to contain three polypeptide bands of between 100 x 10(3) and 120 x 10(3) molecular weight not present in glycoprotein extracts of uninfected cells, and these polypeptides were further purified by preparative polyacrylamide gel electrophoresis. Inoculation of chickens with purified material also resulted in the production of precipitating and neutralizing antibody, showing that these high-molecular-weight polypeptides play a role in the humoral immunity to Marek's disease. Challenge of these chickens with virulent Marek's disease virus revealed that a partial protection was afforded by the inoculated glycoproteins.

Studies of the effects of 5-iodo-2′-deoxyuridine on the formation of adenovirus type 2 virions and the synthesis of virus-induced polypeptides

The antiviral activity of 5-iodo-2′-deoxyuridine (IdUrd) on human adenovirus type 2 (Ad2) was examined. The loss of infectivity, resulting from the presence of IdUrd in the medium, was greater than either the decrease in the number of viral particles produced or the increase in yield of incomplete particles. A “new” species of viral particle with a lower buoyant density than other known incomplete particles was identified at the higher concentrations of IdUrd tested. In the presence of 10 μg/ml of IdUrd, the specific infectivity of the complete particles was 0.04 per cent that of control. No difference in mobilities or molar ratios of the structural proteins derived from these completed virions was detected by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The buoyant density of the viral DNA derived from virions grown in the presence of IdUrd was higher in CsCl than that of control viral DNA, although both samples of DNA sedimented at the same rate in neutral and alkaline sucrose density gradients. The loss of infectivity after replication in media supplemented with IdUrd is associated not only with a reduced yield in total particles formed and an enhanced formation of incomplete particles, but also with the production of noninfectious substituted complete virions. The biosynthesis of virus-induced polypeptides in the presence or absence of IdUrd was analyzed. No apparent inhibition of early virus-induced proteins was detected even at very high doses of the analog. On the other hand, the synthesis of late viral proteins was inhibited markedly; the degree of inhibition was related directly to the concentration of IdUrd in the medium. Our studies suggest that IdUrd exerts its antiviral effect after incorporation into viral progeny DNA. The loss in the biological integrity of such substituted DNA molecules is not accompanied by fragmentation. The molecular basis for the inhibitory effect of IdUrd on the replication of Ad2 remains to be elucidated.

Murine Cytomegalovirus-induced Protein Synthesis

Murine cytomegalovirus (MCMV)-induced protein synthesis in mouse embryo fibroblast (MEF) cells was studied using polyacrylamide gradient SDS gel electrophoresis and autoradiography. Synthesis of at least 14 virus induced proteins (VIPs) was consistently detected in a lytic cycle. They were designated VIPs 132, 118, 99, 98, 88, 81, 76, 74, 58, 56, 51, 38, 36 and 33 on the basis of their mol. wt. Judging from the pattern of the rate of protein synthesis, VIPs can be classified into three groups: group A VIPs were synthesized actively for a brief period of time and then their synthesis was no longer detectable. This group included two major VIPs, 98 and 88 and three minor VIPs, 58, 56 and 38. Group B VIPs 81, 74, 36 and 33 were similar to group A except that, following a brief period of active synthesis, a low level of synthesis continued during the entire lytic cycle. Group C VIPs 132, 118, 99, 76 and 51 were synthesized at low steady levels at all times after initiation and seemed to accumulate slowly. According to temporal sequences of initiation of VIP synthesis, these proteins can also be divided into three groups: immediate early, early and late VIPs. The synthesis of the immediate early VIPs 132, 98, 88, 81, 76, 74 and 38 was initiated immediately after virus infection. The early VIPs included 58, 56, 51, 36 and 33 and their synthesis was initiated from 1 to 3 h post-infection. VIPs 118, 99 and several minor VIPs were first synthesized during 12 to 13 h post-infection which corresponded to the time of initiation of virus DNA synthesis and they are classified as late VIPs. Cycloheximide reversal experiments indicated that the initiation of synthesis of early VIPs must be preceded by the synthesis of immediate early VIPs. In the presence of actinomycin D, the immediate early VIPs (0 to 1 h post-infection) were not synthesized indicating that immediate early VIPs are translated from virus mRNA synthesized after virus infection.

Nuclease activity associated with the Ustilago maydis virus induced killer proteins.

An in vitro nuclease activity was found to be associated with the purified killer proteins of Ustilago maydis. The proteins are effective against single stranded RNA, single and double stranded DNA. Endonucleolytic activity was confirmed by cleavage of circular molecules of 0x174 and PM2. Double stranded RNA did not appear to serve as a substrate.