Equine herpesvirus type 1 infected cell polypeptides: evidence for immediate early/early/late regulation of viral gene expression

EHV-1 polypeptide synthesis was examined in productively infected rabbit kidney and hamster embryo cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses of extracts from [35S]methionine- and 3H-amino acid-labeled-infected and mock-infected cultures revealed the presence of 30 infected cell-specific polypeptides (ICPs) which ranged in apparent molecular weights from 16.5K to 213K. Twenty-two of these ICPs comigrated with virion structural proteins. Four ICPs (203K, 176K, 151K, 129K) were detected in extracts of infected cultures labeled in the presence or absence of actinomycin D (Act D) immediately after release from a 4-hr treatment with cycloheximide (CH). These polypeptides, which were designated as EHV-1 immediate early (alpha) ICPs, were not detected in unblocked (non-CH-treated) infected cells. The most abundant ICP was a 31.5K nonstructural protein which, in addition to a 74K protein, was detected in unblocked infected cells at 2-3 hr postinfection. These proteins appeared to be regulated as early (beta) ICPs, since neither protein was observed in Act D-treated cultures released from CH block. Twelve ICPs were classified as late (gamma) polypeptides on the basis of their reduced synthesis in cultures in which viral DNA replication was inhibited by phosphonoacetic acid. All but one (40K) of these late ICPs corresponded to virion structural proteins.     

Characterization of two abundant mRNAs of Autographa californica nuclear polyhedrosis virus present late in infection

Cytoplasmic poly(A)(+) RNA isolated from Spodoptera frugiperda cells late after infection with Autographa californica nuclear polyhedrosis virus (30-40 hr pi) was fractionated according to size on denaturing methyl mercury gels. Two major RNA species (1.4 kb and 0.75 kb) and several minor RNA species were detected by ethidium bromide staining. The predominant RNA species of about 1.4 kb was considered to be polyhedrin mRNA because (1) in vitro translation of the RNA, which was eluted from methyl mercury gels, yielded a polypeptide of MW 33K, which comigrated with polyhedrin. (2) When poly(A)+ RNA was fractionated on a sucrose column and then translated in vitro, the distribution and abundance profiles of a 33K polypeptide product and of 1.4-kb RNA were similar. (3) The 33K polypeptide made in vitro and purified polyhedrin gave rise to similar patterns of peptides when digested with S. aureus V8 protease. The polyhedrin mRNA (1.4 kb) hybridized to BamHI-F and HindIII-V AcNPV DNA fragments and hybridization selection with BamHI-F AcNPV DNA yielded a 33K polypeptide, which comigrated with polyhedrin. The second RNA species (0.75 kb in size) hybridized to overlapping EcoRI-P and HindIII-Q regions of the AcNPV genome and translated into a methionine deficient polypeptide of MW = 8K. It was synthesized in large quantities late in the infection and appeared to be coordinately expressed with polyhedrin in infected cells. The 8K polypeptide was detected as early as 15 hr pi and was still synthesized at 60 hr pi.     

Protein synthesis in pig kidney cells infected with herpes simplex virus type 1

Two polypeptides of apparent mol. mass 87,000 and 35,000 were identified in pig kidney cells infected with herpes simplex virus type 1 (HSV-1) after reversing the cycloheximide block. The synthesis of the polypeptide 87,000 declined from 22 hr post infection (p.i.). Its production was prevented by actinomycin D added to the infected cells after removal of cycloheximide. Evidence is presented that the polypeptide 35,000 may be of the cellular origin.     

Protein analysis of herpes simplex virus latency in vitro established with cycloheximide

Herpes simplex virus (HSV)-specific protein synthesis was examined during establishment of HSV latency and reactivation of virus in human embryonic lung cells treated with cycloheximide and incubated at 40.5 degrees. Eight viral proteins, identified during the first two days of establishment of latency at 40.5 degrees, were undetectable by Day 3. At least two synthesized proteins were present during the maintenance phase of latency. Reactivation of HSV (viral protein 135K) was first detected in latently infected cultures between 2 and 3 hr after superinfection with human cytomegalovirus (HCMV). During this period an 82K protein with the same molecular weight as one of the HCMV immediate-early proteins (82 and 75K) was detected in the immunoprecipitates of latently infected cultures with anti-HSV serum. Thus, this HSV latency system can be used to analyze protein synthesis and clarify reactivation of HSV by HCMV superinfection.     

Structure and expression of adenovirus type 12 E1B 58K protein in infected and transformed cells: studies using antibodies directed against a synthetic peptide

We have studied the kinetics of synthesis of the early adenovirus type 12 (Ad12) E1B 58K tumor antigen during lytic infection and analysed its half-life, intracellular localization and phosphorylation in infected KB and transformed hamster (HA12/7) cells. Our analysis has been based on immunoprecipitations using antibodies directed against a synthetic peptide corresponding to the carboxy-terminal end of the E1B 58K protein. Its synthesis was first detectable approximately 8 h after infection and reached a maximum at about 20 h. There is a slight decrease of synthesis late after infection although its level of production is rather high throughout the infectious cycle. The half-life of the Ad12 E1B 58K polypeptide is 2-3 h in infected cells, but strikingly higher (less than 10 h) in the Ad12-transformed cell line HA12/7. Pulse-chase experiments combined with cell fractionation and immunofluorescence studies suggested that about 50% of the amount of the 58K polypeptide accumulates in the nucleus of infected KB cells at least at late times after infection, but only approximately 10% in Ad12-transformed cells. The 58K polypeptide is phosphorylated in both infected and transformed cells. Analysis of the products of acid hydrolysis indicates phosphorylation to equal amounts of serine and threonine. The implications of all these findings for possible roles of the E1B 58K tumor antigen in lytic infection and transformation are discussed.     

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.     

Machupo virus polypeptides: Identification by immunoprecipitation

Summary The most abundant protein in purified Machupo virions (Corvallo strain) labelled with¹⁴C-Protein hydrolysate is a 64K polypeptide which is associated with virion RNAs. Another structural polypeptide, 37K, solubilized by nonionic detergent seems to be a major surface glycoprotein. In addition to this, a 78K polypeptide and a minor 50K polypeptide have been detected.In Machupo virus infected cells three virus-specific polypeptides similar in size to those described for structural polypeptides were immunoprecipitated with anti-Machupo virus serum. The most abundant virus-specific polypeptide was nonglycosylated (64K, NP), and the others were glycosylated polypeptides (78K and 37K). The synthesis of NP and 78K polypeptides was recognized at the beginning of a log phase of virus replication. Pulse-chase experiments as well as experiments with an arginine analogue, canavanine (to block proteolytic processing) suggest that 78K is a precursor for structural glycoproteins of Machupo virions.With 4 Figures     

Early DNA-binding polypeptides of Epstein-Barr virus

The kinetics of Epstein-Barr virus (EBV)-specific protein synthesis has been studied after induction of P3HR-1 cells with n-butyrate. The majority of the EBV polypeptides became detectable on SDS-polyacrylamide gel electrophoresis between 15 and 18 hr after addition of the drug. Overall viral protein synthesis was at maximum after about 24 hr. Some polypeptides appeared significantly earlier (152K, 55K, 51K) while others were synthesized during a limited period of the cycle only (65K). At least four of the polypeptides detected (152K, 134K, 55K, 51K) bound to DNA cellulose columns. The EBV specificity of the polypeptides was confirmed by immunoprecipitation and by two-dimensional gel analysis. The 152K polypeptide had a pI around 8 whereas the other polypeptides varied between basic and acidic pI. The DNA binding proteins isolated by DNA cellulose chromatography represent a subclass of the virus-specific early proteins that can be immunoprecipitated from the total cell extract.     

Host cell and virus strain differences in synthesis of immediate early polypeptides in HSV-1 infected cells

The synthesis of the immediate early (IE) polypeptides was analysed in primary rabbit kidney (RK) cells and a stable line of rabbit lung (ZP) cells infected with the syncytial (syn) strain HSZP and the non-syn strain KOS of herpes simplex virus type 1 (HSV-1). Results showed the following: After cycloheximide reversal the infection of RK and ZP cells with HSZP strain led to synthesis of five IE polypeptides (175K, 136K, 87K, 68K, and 63K), while infection of both cell cultures with the KOS strain led to synthesis of significantly reduced amounts of the IE polypeptides. The ability to switch on the expression of non-alpha viral genes was impaired in RK cells infected with the HSZP strain. The IE polypeptides were still detectable without any sign of the non-IE polypeptide synthesis 4 hr after cycloheximide reversal. The observed failure of the IE HSZP polypeptides to undergo posttranslational modification in ZP cells may be the consequence of this phenomenon. In contrast to the KOS IE mRNAs, the HSZP IE mRNAs exhibited a pronounced functional stability in both cell cultures. The IE polypeptides were still synthesized in HSZP-infected cells which had been incubated for 19 hr after cycloheximide reversal in the presence of actinomycin D (Act D). The HSZP strain failed to suppress the host polypeptide synthesis in RK but not in ZP cells. However, the HSZP strain, in contrast to the KOS strain, proved to be defective with respect to the early shutoff of host polypeptide synthesis in both cell cultures.(ABSTRACT TRUNCATED AT 250 WORDS)     

Early functions of the genome of herpesvirus. V. Serological analysis of "immediate-early" proteins.

Immediate-early proteins synthesized by cells treated with cycloheximide during early stages of infection with pseudorabies virus do not react with serum prepared against mature virions. Antiserum against immediate-early proteins was prepared. These sera were used to follow the synthesis of immediate-early proteins during the normal course of infection.Proteins reacting with antiserum against immediate-early proteins are synthesized up to 3 hr postinfection only. Immediate-early proteins are stable and are present in the cells at later stages of infection but do not become part of virions. The polyacrylamide gel electrophoretic (PAGE) profile of the immediate-early proteins synthesized during the normal course of infection is similar to that of proteins synthesized by cells treated with cycloheximide during early stages of infection. Immediate-early proteins are less stable upon storage at 4° than are proteins synthesized at later stages of infection. This treatment alters the PAGE profile of the immediate-early proteins.     

Analysis of immediate-early and early proteins of murine cytomegalovirus in permissive and nonpermissive cells

Summary The immediate-early (IE) and early proteins induced by murine cytomegalovirus (Smith strain) in permissively infected 3T3-L1 murine fibroblasts were identified by SDS polyacrylamide gel electrophoresis. Ten proteins were classified as IE by their time of synthesis and by their synthesis in the presence of actinomycin D following reversal of a cycloheximide mediated protein synthesis block. By exclusion, seven proteins were classified as early class. Eleven of these proteins were precipitated by MCMV antiserum.The role of IE and early proteins in the replication of the virus was studied by infection of a murine macrophage cell line (J 774A.1) and human foreskin fibroblast (HFF) cells. The infections were characterized as nonpermissive by several criteria, including lack of production of infectious virus or viral DNA. However, the major IE and early MCMV proteins were detected in the nonpermissively infected cells. The block to virus replication in the macrophage and human fibroblast cells appeared to occur after the switch from IE to early protein synthesis, but before viral DNA replication.With 7 Figures     

Synthesis of the precursor to avian RNA tumor virus internal structural proteins early after infection.

The synthesis of the 76,000-dalton precursor (Pr 76) of the avian RNA tumor virus internal structural proteins was studied as a function of time after infection of chick embryo fibroblasts (CEF) with avian myeloblastosis virus (AMV). During the course of infection, cells were pulse-labeled with [³⁵S]methionine, lysed, and the labeled viral polypeptide precursor was precipitated with antibody against detergent-lysed AMV. Pr 76 was detected by SDS gel electrophoresis of immune precipitates.The earliest time at which Pr 76 synthesis could be detected was 3 hr after infection. Pr 76 synthesis remained low (about 1% of the level of synthesis several days after infection) and constant from 3 until 7 hr after infection. Between 7 and 9 hr after infection, Pr 76 synthesis increased by fivefold.Cells treated with cycloheximide during the first 8 or 12 hr after infection showed an 85–90% inhibition of Pr 76 synthesis and virus production measured late during infection. This finding does not necessarily imply that early viral protein synthesis is required for a productive infection, because cycloheximide also inhibited chick embryo fibroblast DNA synthesis.If cells were treated prior to and during infection with actinomycin D or cytosine arabinoside, precursor synthesis was still observed early (3 to 7 hr after infection). The amount of precursor synthesized early in the presence of inhibitors was similar to that synthesized in the absence of inhibitors, suggesting that the incoming RNA served as a messenger RNA for Pr 76.     

Rabbit kidney cells abortively infected with human cytomegalovirus are arrested in mitotic phase

Summary In rabbit kidney epithelial cells (RK₁₃) abortively infected with human cytomegalovirus (HCMV), DNA synthesis at 1 or 2 days post-infection was enhanced 4 to 5 fold, compared to mock-infected cells. DNA analysis by isopycnic centrifugation revealed that the DNA newly synthesized in the virus infected RK₁₃ cells was of cellular origin. HCMV infection also caused a marked increase in the mitotic activity of RK₁₃ cells. When semi-confluent RK₁₃ cells were infected more than 20 per cent of cells demonstrated mitosis at 72 hours post-infection although the rate of cell growth was considerably reduced compared to that of uninfected cells. The most frequent chromosomal change observed was fragmentation although other aberrations, gap, break, deletion etc. occurred also.Two immediate-early viral polypeptides with apparent molecular weights 72,000 (72K) and 76,000 (76K) daltons were produced in both RK₁₃ cells and human embryonic lung cells (HEL) by 3 hours post-infection. Synthesis of the 76K polypeptide was greater than that of the 72K polypeptide in non-permissive RK₁₃ cells whereas the reverse occurred in permissive HEL cells. Furthermore, of three early polypeptides which were expressed in productively infected HEL cells two, 88K and 80K, were not detected in abortively infected RK₁₃ cells.These results suggest that the arrest in mitosis of the abortively infected RK₁₃ cells and the subsequent chromosomal changes are associated with the altered expression of immediate-early or early virus functions in these cells.Following HCMV infection permissive HEL cells undergo a characteristic sequence of morphological changes (3) of which cell rounding and contraction are observed in the early stages and could be related to either the action of cellular contractile proteins or changes in the plasma membrane permeability and a concomitant Ca⁺⁺ influx (2). Relaxation and cell enlargement are other changes considered to be initiated by early virus functions although they occur after the commencement of virus DNA synthesis. Thus, the expression of immediate-early and early virus functions has an important role in controlling the sequential morphological changes in infected cells and may also be important in determining whether infection with HCMV will be productive or abortive.With 4 Figures     

The polypeptides of monkeypox virus: I. analysis of the polypeptide synthesis of MPV by SDS-PAGE and by two-dimensional electrophoresis

The time course synthesis of the polypeptides of monkeypox virus (MPV) in cynomolgus monkey kidney cells was studied by one-dimensional SDS-polyacrylamide and by two-dimensional polyacrylamide electrophoresis. Cytosine arabinofuranoside (Ara-C) was used to differentiate early and late proteins. The synthesis of several early polypeptides was shown to continue throughout the time course. The synthesis of late polypeptides was shown to start at the peak of DNA synthesis (5–7 hr postinfection) and to continue throughout the remainder of the time course. Some individual polypeptide bands resolved by one-dimensional SDS-PAGE were shown to consist of multiple polypeptides using two-dimensional electrophoresis. These multiple polypeptide species, i.e., polypeptides of the same molecular weight but different isoelectric points, represented both structural and non-structural viral proteins. About 115 polypeptides are shown to exist in the purified virus by using two-dimensional electrophoresis.