Virus-induced Proteins Research Articles

A Study of the Measles Virus-Induced Proteins Incorporated into the Cell Membrane

The polypeptide composition of a measles virus isolate from a case of subacute sclerosing panencephalitis, the Halle strain, and the distribution of these polypeptides in the cell membrane have been investigated. In purified virus preparations, four major polypeptides were identified with molecular weights corresponding to 80K, 62K, 42K and 37K daltons. A minor band was found at 69K and several others with molecular weights +/- 100K. By specifically labeling the proteins exposed at the surface of BGM cells either persistently or lytically infected with the Halle virus, it was shown that only the 80K and 42K viral polypeptides were present in the cell membrane. Furthermore, it was observed that PAGE profiles of cellular polypeptides differed between infected and noninfected cells.

Mechanisms of infection with Epstein-Barr virus III. The synthesis of proteins in superinfected Raji cells

Superinfection of Raji cells at high multiplicities with EBV leads to the synthesis of at least nine virus-induced polypeptides. Inhibition of viral DNA synthesis with phosphonoacetic acid greatly reduces the synthesis of certain virus-induced proteins while others are made in apparently normal amounts.

A viral-induced protein in the nuclei of cells infected with fowlpox virus

The proteins found in the nuclear and cytoplasmic fractions of fowlpox virus-infected and mock-infected chick embryo cells were examined by SDS-polyacrylamide-gel electrophoresis. At 24 hr PI a viral-induced 37,000-dalton protein was present in infected cytoplasm, and was still being synthesized, although in lesser amounts, at 48 hr. No viral-induced proteins were identified in the nuclear fraction of infected cells at 24 hr PI, but by 36 hr PI a new 36,000-dalton protein had appeared; increased synthesis of this protein was seen at 48 hr PI. There was a general decline in the synthesis of host cell proteins during the period of viral infection.

Cyclic synthesis of human cytomegalovirus-induced proteins in infected cells

Synthesis of most human embryo lung cellular proteins is more sensitive to inhibition by a hypertonic condition than is synthesis of cytomegalovirus (CMV)-induced proteins. Results using the selective suppression of host cell protein synthesis by the hypertonic condition indicate that virus-induced proteins are synthesized in a cyclic manner.

The polypeptides of adenovirus IX. Partial purification of early proteins and observations on late proteins of type 5 adenovirus

The identification and partial purification of adenovirus type 5 (Ad 5)-induced early proteins were achieved. Mock- and virus (high multiplicity)-infected HeLa cells were labeled for 90 min with [ 35S]- or [ 3H]methionine at 5 hr after infection in the presence of cytosine arabinoside. Labeled polypeptides from these cells were sequentially fractionated with a four-step extraction procedure: (1) 10 m M NaCl, (2) 1.7 M NaCl, (3) 2% Triton X-100, and (4) 6 M urea plus 2% Triton X-100. At each step, polypeptides from mock-infected and infected cell fractions were analyzed by DEAE-Sephadex chromatography and sodium dodecyl sulfate-polyacrylamide-gel electrophoresis (SDS-disc-PAGE) followed by autoradiography. Six virus-induced early proteins were discerned: El, 42K (E1A), E2, E2A, E2B, and E3. Polypeptides of Ad 5 virions and top components and polypeptides induced at the late stage of adenovirus type 5 infection were analyzed by SDS-PAGE. They were similar to the polypeptide constituents of adenovirus type 2 (Ad 2), except that the hexon and fiber of type 5 had mobilities in polyacrylamide (PA) different from those of type 2. The results eliminate the possibility that polypeptides taking part in Ad 5 morphogenesis differ greatly from those of Ad 2.

Identification of the herpes simplex virus DNA polymerase gene

THERE is at present much interest in the herpes viruses because they have been associated with human cancer and cause latent infections with recurrent disease. They are also of interest because they form a relatively simple but apt model for the processes of transcription and DNA replication in eukaryotic cells. We have studied the virus-induced proteins involved in DNA replication, and present here results that give definitive evidence that the virus-induced DNA polymerase is encoded in the virus genome, that locate the gene for virus DNA polymerase, and also show that the enzyme is essential for virus replication.

Influenza virus-induced proteins in nuclei and cytoplasm of infected cells

The protein fraction induced by the WSN strain of influenza virus, which has a molecular weight of about 25,000, was isolated from infected chick embryo fibroblasts by extraction into acidic chloroform-methanol or by column chromatography with 6 M guanidine hydrochloride. The isolated material was analyzed in two dimensions by peptide mapping and the results showed the presence of membrane (M) and the nonstructural (NS) proteins. Both proteins are associated with isolated nuclei, cytoplasm, and polysomes and are distinct proteins. Subfractionation of nuclei indicated that NS was associated with nucleoli or components cosedimenting with nucleoli whereas M remained in the nucleoplasm.

DNA synthesis in mouse embryo fibroblast cells infected with murine cytomegalovirus

The overall rate of DNA synthesis in mouse embryo fibroblast (MEF) cells infected with murine cytomegalovirus (MCMV) decreased immediately after infection, reaching its minimum at 10–12 hr postinfection (PI), and then increased gradually. CsCl equilibrium centrifugation analysis of synthesis of both host and viral DNA in MEF cells infected with MCMV revealed the following: (1) Host cell DNA synthesis was inhibited by more than 95% by 10–12 hr PI. (2) Viral DNA synthesis began at 10–12 hr PI, and by 22–24 hr PI the rate was slightly higher than the rate of host cell DNA synthesis observed at 0–2 hr PI. (3) Viral DNA synthesis occurred without the concurrent synthesis of any significant amount of host cell DNA, indicating that there was preferential viral DNA synthesis. The inhibition of host DNA synthesis occurred when cells were infected with either uv- or heat-inactivated MCMV. This occurred in the absence of both cytopathic effects (CPE) and the synthesis of viral-induced proteins. In these instances, the suppression of host DNA synthesis was observed only during the first 12 hr PI. At 12–14 hr PI, host DNA synthesis began to increase, and finally reached or surpassed the level seen in mock-infected cells. Since the suppression of the host DNA synthesis occurred in the absence of viral-induced protein synthesis, the possibility that the suppression is caused by one or more of the structural proteins of the infecting viral particles is discussed.

Detection of adenovirus type 2-induced early polypeptides using cycloheximide pretreatment to enhance viral protein synthesis

(35S) methionine-labeled polypeptides synthesized by adenovirus type 2-infected cells have been analyzed by polyacrylamide gradient gel electrophoresis and autoradiography. Cycloheximide (CH) was added to infected cultures to accumulate early viral mRNA relative to host cell mRNA. This allowed viral proteins to be synthesized in increased amounts relative to host proteins after removal of CH and pulse-labeling with (35S)methionine. During the labeling period arabinosyl cytosine was added to prevent the synthesis of late viral proteins. This procedure facilitated the detection of six early viral-induced polypeptides, designated EP1 through EP6 (early protein), with apparent molecular weights of 75,000 (75K), 42K, 21K, 18K, 15K, and 11K. Supportive data were obtained by coelectrophoresis of (35S)- and (3H)methionine-labeled polypeptides from infected and uninfected cells, respectively. Three of these early polypeptides have not been previously reported. CH pretreatment enhanced the rates of synthesis of EP4 and EP6 20- to 30-fold and enhanced that of the others approximately twofold. The maximal rates of synthesis of the virus-induced proteins varied, in a different manner, with time postinfection and CH pretreatment. Since CH pretreatment appears to increase the levels of early viral proteins, it may be a useful procedure to assist their isolation and functional characterization.

Protein synthesis following infection of the blue-green alga Plectonema boryanum with the temperate virus SPI and its ts mutants

The structural proteins of the purified phage and proteins synthesized in virus-infected cells were examined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and subsequent autoradiography. A total of 24 viral-induced proteins were identified, 12 of which are present in the mature virus. The kinetics of synthesis of the various viral proteins demonstrates the presence of “early” and “late” genes. Specific suppression of host protein synthesis by the infecting phage takes place late in the infection cycle. Analysis of the proteins synthesized at the nonpermissive temperature by phage temperature-sensitive ( ts) mutants demonstrates that some of the viral genes have complex pleiotropic effects on viral protein synthesis. The results suggest the presence of positive regulatory genes, and of specific genes involved in the shutoff of host protein synthesis.

The absence of one class of virus-induced protein in arginine-deprived cells infected with pig herpesvirus

Abstract Three arginine-rich proteins, in the molecular weight range of 25,000–45,000, have previously been described in nuclear fractions of BHK 21 cells infected with pig herpesvirus (pseudorabies). Under conditions of arginine deprivation, the synthesis of these proteins is preferentially inhibited in comparison with other virus-induced proteins identifiable on 10% SDS gels. In addition, viral DNA synthesis still occurs, although it is reduced to 50% of the control value. These basic proteins are present in purified nucleocapsids of PrV and are probably internal or “core” proteins. As no mature virus appears in the absence of arginine, it is possible that these proteins are involved in the assembly process.

Inhibition of Cellular Protein Synthesis by Simultaneous Pretreatment of Host Cells with Fowl Plague Virus and Actinomycin D: a Method for Studying Early Protein Synthesis of Several RNA Viruses

A method is described for analysis of viral protein synthesis early after infection when minute amounts of viral proteins are effectively concealed by large amounts of produced host-specific proteins. The method is superior to a radioimmune assay, since all virus-induced proteins can be measured independent of their immunological reactivity. Host-specific protein synthesis can be suppressed by infection with fowl plague virus. Addition of actinomycin C 1.25 h postinfection does not prevent this suppression, but it does block effectively the formation of fowl plague virus-specific proteins. Such cells synthesize only small amounts of cellular proteins, as revealed by polyacrylamide electrophoresis. They can be superinfected with several different enveloped viruses, however, without significant diminution of virus yeilds. In pretreated cells the eclipse is shortened for Semliki Forest virus, Sindbis virus, and vesicular stomatitis virus, but prolonged for Newcastle disease virus. The onset of protein synthesis, specific for the superinfecting virus, could be clearly demonstrated within 1 h after superinfection. At this time, in cells superinfected with Semliki Forest virus, great amounts of NSP 75 (nonstructural protein; molecular weight, 75 X 10(3)) and reduced amounts of the core protein C could be deomonstrated. The precursor glycoprotein NSP 68 is followed by a new polypeptide, NSP 65: three proteins with molecular weights exceeding 100 X 10(3) were observed which are missing later in the infectious cycle. Similar results were obtained after superinfection with Sindbis virus. The formation of a new polypeptide with a molecular weight of about 80 X 10(3) was detected. After superinfection with vesicular stomatis virus or Newcastle disease virus the formation of new proteins, characteristic for the early stage of infeciton, was not observed.

Bacteriophage T4 baseplate components. III. Location and properties of the bacteriophage structural thymidylate synthetase.

Two T4D thymidylate synthetase (td) temperature-sensitive mutants have been isolated and characterized. Both mutants produce heat-labile phage particles. This observation supports the view that this viral-induced protein is a phage structural component. Further, antiserum to td has been shown to block a specific step in tail plate morphogenesis. The results indicated that the td protein is largely covered by the T4D tail plate gene 11 protein. Since the phageinduced dihydrofolate reductase (dfr) also is partially covered by the gene 11 protein, it appears that td was adjacent to the tail plate dfr. This location has been confirmed by constructing a T4D mutant which is dfrtstdts and showing that these two tail plate constituents interact and give altered physical properties to the phage particles produced. A structural relationship for the tail plate folate, dfr, and td has been reported.

Herpes simplex virus proteins: DNA-binding proteins in infected cells and in the virus structure

DNA-binding proteins have been isolated from BHK21 C13 cells infected with herpes simplex virus type 1 strain 17 syn + by native and denatured DNA affinity chromatography. Sixteen proteins have been identified as specific to the infected cell on SDS polyacrylamide gradient gel electrophoresis and their molecular weights range from 145 × 10 3 to 10.5 × 10 3. After infection with herpes simplex virus many of the mock infected cell specific DNA binding proteins are no longer produced and this “switch off” of host cell protein synthesis is rapid. The virus-induced DNA-binding proteins fall into two quantitative classes and there is evidence for temporal control of their synthesis. One of the virus-induced DNA-binding proteins has a molecular weight identical to a polypeptide found in full but not empty particles and another binds preferentially to denatured DNA.

Virus-induced proteins in pseudorabies-infected cells. II. Proteins of the virion and nucleocapsid

Analysis of purified naked and enveloped nucleocapsids of pseudorabies virus with high-resolution techniques has allowed a reassessment of their protein composition. Enveloped particles are shown to contain at least 20 proteins whose molecular weights are in the range 20,000 to 230,000. Naked nucleocapsids contain one major and seven minor proteins in the molecular weight range 20,000 to 155,000. Phosphorylation of at least one virion protein is shown to take place in vivo. These results demonstrate that pseudorabies virus is similar in its protein complement to other herpesviruses which have recently been examined.

Protein Synthesis in Sendai Virus-infected Cells

The rate of protein synthesis in chicken embryo cells infected with Sendai virus 18 to 20 h previously was about two times greater than in mock-infected controls. At this time of infection six stable virus-induced proteins, four major structural proteins (P, NH, NP and M) and two non-structural proteins (28K and 61K), were identified by electrophoresis in SDS-polyacrylamide gel of total cell extracts. The structural glycopeptide F was not detected in the infected cell extracts. Pulse-chase experiments showed that PN NP, M and 28K proteins either did not undergo any post-translational processing or the processing occurred very rapidly. By contrast, a glycopeptide NH was apparently derived from one of two unstable precursons, 69K or 63K, which were revealed only after a short pulse. The synthesis of virus-specific proteins appeared to be regulated since its rate varied for individual classes of proteins. In nucleocapsid-particles isolated from infected cells two major structural proteins (P and NP) were found. A minor component with a very large mol. wt. was revealed in these particles as well as in the virus particle.

Proteins Induced in BHK Cells by Infection with Foot-and-Mouth Disease Virus

Although the infection of BHK cells with foot-and-mouth disease virus did not cause a marked inhibition of cellular protein synthesis, the proteins synthesized gradually changed from host-specific to virus-specific. The synthesis of at least thirteen virus-induced proteins was demonstrated by polyacrylamide electrophoretic analysis of the infected cells. Only a small proportion of the virus-induced proteins was incorporated into the mature virus particles. The addition of amino acid analogues caused an accumulation of the larger mol. wt. proteins, suggesting that in infected cells, some cleavages occurred, giving rise to the smaller mol. wt. proteins. The operation of the precursor-cleavage mechanism was more clearly shown in pulse-chase experiments, in which it was found that the proteins were degraded from larger to smaller mol. wt.

Replication process of the parvovirus H-1. IV. H-1-specific proteins synthesized in synchronized human NB kidney cells.

The synthesis of two virus-induced proteins, VP1 (mol wt 92,000) and VP2' (mol wt 72,000), was detected in the cell and in purified virions from H-1 parvovirus propagated in synchronous cell cultures.

Alterations of the immunological specificity of plasma membranes from cells infected with Marek's disease and turkey herpes viruses.

SUMMARY Highly purified plasma membranes were isolated from chicken embryo fibroblasts infected with Marek’s disease virus (MDV) or turkey herpes virus (HVT). The purification was monitored by the incorporation of fucose and marker enzymes specific for plasma membranes. Polyacrylamide gel electrophoresis showed that the membrane preparations contained two new virus-induced proteins. When reacted in the double immunodiffusion test solubilized plasma membranes from MDV-infected cells formed two specific precipitation bands with Marek’s disease immunoglobulins. Antisera prepared against plasma membranes from MDV- or HVT-infected cells neutralized extracellular infectious HVT. After incubation of plasma membranes from MDV-infected cells with Marek’s disease antibodies the buoyant density of the membranes increased from 1.05 g/ml to 1.08 g/ml due to the binding of immunoglobulins to the virus-induced membrane proteins. The Marek’s disease mortality of chickens twice vaccinated with a plasma membrane preparation from HVT-infected cells was reduced by 94%. From the results it was concluded that the plasma membranes acquire new immunologic specificities after infection with MDV or HVT.

Murine virus-induced proteins synthesized by hamster tumor cells transformed by, but not producing, murine sarcoma virus.

The 8303 hamster tumor cells transformed by Moloney strain of murine sarcoma virus (M-MSV), but which do not produce virus, do contain murine virus-induced proteins. The virus-induced proteins within the cell were identified either as free proteins or in association with membranous material, including the plasma membrane. In addition, some were excreted by the 8303 hamster tumor cells into the growth medium. Most virus-induced proteins were larger than 68,000 daltons, and they did not dissociate into components of smaller size in the presence of detergent and a reducing agent. A small amount of virus-induced protein with a molecular weight of less than 20,000 was also found in the hamster tumor cells. No virus-specific proteins with the identical antigenic specificity or size of the major internal group specific antigen (molecular weight about 30,000) of the murine leukemia viruses were present in these cells. There is a common cell surface antigen present in three other tumor cell lines, both virus-producing and non-virus-producing, identical in reactivity to that of the murine virus-induced antigen of the 8303 hamster tumor cell. This antigen is not present on the cell surface of normal mouse embryo cells.