Wound tumor virus polypeptide synthesis in productive noncytopathic infection of cultured insect vector cells.

Inoculation of the leafhopper cell line AC-20 with wound tumor virus resulted in a productive noncytopathic infection with no detectable alteration of cellular protein synthesis. Virus-specific polypeptide synthesis, detectable by 8 h postinoculation, increased in a linear fashion, reaching a peak (approximately 10 to 15% of total protein synthesis) by 48 h postinoculation. The rate of viral protein synthesis continued at this level for several days but declined, relative to cellular protein synthesis, as infected cells were passaged. By passage 10, the synthesis of viral polypeptides was reduced to a level approximately 5% of that observed at 48 h postinoculation. Viral protein synthesis was not stimulated by superinfection. Viral antigens and infectious virus persisted in the majority (greater than 90%) of cells in an infected culture even after more than 100 passages. The synthesis of wound tumor virus polypeptides in infected insect vector cells appears to be regulated in a coordinated and selective manner.     

Replication of human cytomegalovirus DNA: lack of dependence on cell DNA synthesis.

Human cytomegalovirus (CMV) DNA synthesis was studied in 5-fluorouracil (FU)-treated and untreated human embryonic lung cells, which differ greatly with respect to the number of cells in the culture synthesizing cellular DNA. CMV DNA synthesis proceeded at the same rate in FU-treated and in untreated cells. CMV infection also reversed the inhibitory effects of FU and activated cellular DNA synthesis in some of the cells in the FU-treated culture. Autoradiographic studies showed that more than 20% of the cells in the infected FU-treated culture synthesized viral DNA when less than 1% had synthesized cellular DNA, indicating that the synthesis of viral macromolecules proceeds in cells that do not synthesize cellular DNA from the time of infection, and that viral DNA synthesis proceeds independently of the host cell DNA synthesis. Combined autoradiographic and immunofluorescence studies of both the FU-treated and untreated infected cells showed that, whereas 20% of the cells in the cultures synthesize viral DNA and viral antigens, only about 3 to 6% of those cells that synthesize cellular DNA also synthesize viral antigen. Thus, productive infection was delayed or inhibited in those cells that were stimulated by CMV infection to synthesize cellular DNA.     

Changes in Deoxyribonucleic Acid Synthesis Regulation in Chinese Hamster Cells Infected with Simian Virus 40

Infection of primary or secondary cultures of Chinese hamster embryo cells with simian virus 40 at a multiplicity of 20 to 50 induced synthesis of the virus-specific intranuclear T antigen in 80 to 90% of the cells within 48 to 72 hr. In the infected cultures, 30 to 50% more cells were recruited into deoxyribonucleic acid (DNA) synthesis than in the controls, whether or not the cultures were confluent. The newly synthesized DNA was mostly cellular, since little virus was produced (as shown by various techniques: immunofluorescence for viral antigen, virus growth curves, and isolation of viral DNA from infected cultures). Transformed cells could be detected a few weeks after infection and produced tumors when inoculated into irradiated animals. Chromosomal changes were observed soon after infection (24 hr). Initially, there was a marked increase in the proportion of polyploid cells (8 to 14%), most of which were chromosomally normal. In a few weeks, a large majority of the infected population was polyploid (30 to 50%). Thus, the polyploid cells have the ability to proliferate. Evidence is presented to suggest that polyploid cells arise by stimulation of cells in the G(1), G(2), or S phases to undergo two or more successive periods of DNA synthesis without an intervening mitosis. With a subsequent loss or redistribution of chromosomal material, this may lead eventually to a biologically transformed cell; thus, it is suggested that the initial event(s) relevant to transformation occurs at the level of control of cellular DNA synthesis.     

Kinetics of DNA and histone messenger RNA synthesis in CV-1 monkey kidney cells infected with simian virus 40.

Histone messenger RNA has been identified in CV-1 monkey kidney cells and its synthesis during the simian virus 40 (SV40) productive cycle has been correlated with the synthesis of cellular DNA and viral DNA. In cultures of CV-1 cells that have reached confluence, infection with SV40/5 (a high-yield clone of SV40) promotes an increase in the rate of cellular DNA synthesis followed by a decline. During this decline the rate of viral DNA synthesis continues to rise and eventually surpasses that of cellular DNA.The synthesis of histone mRNA rises concomitantly with the increase in the synthesis of cellular DNA. This occurs in a fashion similar to that observed when confluent CV-1 cultures are stimulated by the addition of fresh serum to the growth medium. However, whereas in cells stimulated with serum the synthesis of histone mRNA closely parallels that of cellular DNA, in cells infected with SV40, histone mRNA synthesis continues at a high rate even after the decline of cellular DNA synthesis. The rate of histone mRNA synthesis thus appears to he coupled to the total (cellular plus viral) DNA synthesis and not to the synthesis of the host DNA alone. The high rate of synthesis of the F1 histone at late times after infection suggests that histone genes are transcribed co-ordinately.     

Cytomegalovirus early and late membrane antigens detected by antibodies in human convalescent sera.

Using indirect immunofluorescence and a panel of human convalescent-phase sera, we identified cytomegalovirus (CMV) early and late membrane antigens (CMV-EMA and CMV-LMA, respectively) as separate entities on the surfaces of viable CMV-infected fibroblasts starting at 6 to 12 and 36 to 48 h postinoculation, respectively. For expression of CMV-EMA and CMV-LMA, infectious virus and active protein synthesis were required, whereas the expression of CMV-LMA, in addition, required viral DNA synthesis. Our data suggest that CMV-EMA and CMV-LMA form an individual set of CMV antigens that are different from intracellular CMV antigens and possibly (partly) different from the viral envelope.     

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.     

Cell-Dependent Differences in the Production of Infectious Herpes Simplex Virus at a Supraoptimal Temperature

The replication of herpes simplex virus (HSV) was compared in rabbit and hamster cells at optimal and supraoptimal temperatures. Replication occurred in cells of either species at 33 C, but the total infectious virus yield was routinely about 10-fold greater in rabbit cells than in hamster cells. At 39 C, this difference was exaggerated to greater than 100,000-fold. Whereas infectious virus was produced and plaques formed in rabbit kidney cell monolayers at the higher temperature, neither developed in those derived from hamster embryos. Elevating the temperature from 33 C to 39 C at various time intervals after exposure of the cultures to virus revealed that production of infectious virus in hamster cells was completely heat-sensitive up to 6 hr after infection. Specific viral antigens and viral deoxyribonucleic acid (DNA) were synthesized in both rabbit and hamster cell cultures. In addition, cellular DNA synthesis was depressed and cytopathic effects occurred in both cell systems. These cytopathic effects were not observed in cell cultures treated with HSV previously inactivated with ultraviolet light. Compared with parallel cultures at 33 C, the amount of viral DNA synthesized at 39 C was greatly reduced in both systems. In hamster cells, the reduction was twofold greater than in rabbit cells. This cell-dependent thermal inhibition of HSV replication in hamster cells did not occur with vaccinia virus.     

Cell-cell contact promotes DNA synthesis in retinal glia but not in fibroblasts

Contact among retinal glial cells in monolayer cultures of intermediate density stimulated DNA synthesis. The stimulation occurred in passaged cultures lacking neuronal elements and is, therefore, the first demonstration of growth promotion by contact among cells of the same apparent type. In contrast, low levels of association among dermal fibroblasts in cultures of similar density had no stimulatory effect. However, as few as three cellular contacts depressed fibroblast DNA synthesis. This latter observation suggests that the density dependent suppression of fibroblast proliferation observed in confluent cultures begins to be expressed at subconfluent cell densities.     

Kinetics of Nucleic Acid Synthesis in Human Embryonic Kidney Cultures Infected with Adenovirus 2 or 12: Inhibition of Cellular Deoxyribonucleic Acid Synthesis

Infection of human embryonic kidney (HEK) cell cultures with adenovirus types 2 or 12 resulted in an initial drop in the rate of incorporation of (3)H-thymidine into deoxyribonucleic acid (DNA) during the early latent period of virus growth, followed by a marked rise in label uptake. It was shown by cesium chloride isopycnic centrifugation that, after adenovirus 2 infection, there was a decrease in the rate of incorporation of thymidine into cellular DNA. Moreover, DNA-DNA hybridization experiments revealed that, by 28 to 32 hr after infection with either adenovirus 2 or 12, the amount of isolated pulse-labeled DNA capable of hybridizing with HEK cell DNA was reduced by approximately 60 to 70%. Autoradiographic measurements showed that the inhibition of cellular DNA synthesis was due to a decrease in the ability of an infected cell to synthesize DNA. The adenovirus-induced inhibition of host cell DNA synthesis was not due to degradation of cellular DNA. (3)H-thymidine incorporated into cellular DNA at the time of infection remained acid-precipitable, and labeled material was not incorporated into viral DNA. Furthermore, when zone sedimentation through neutral or alkaline sucrose density gradients was employed, no detectable change was observed in the sedimentation rate of this cellular DNA at various times after infection with adenovirus 2 or 12. In addition, there was no increase in deoxyribonuclease activity in cells infected with either virus. Cultures infected for 38 hr with adenovirus 2 or 12 incorporated three to four times as much (3)H-uridine into ribonucleic acid (RNA) as did non-infected cultures. Furthermore, the net RNA synthesized by infected cultures substantially exceeded that of control cultures. The activity of thymidine kinase was induced, but there was no stimulation of uridine kinase.     

Accumulation of human immunodeficiency virus type 1 DNA in T cells: results of multiple infection events.

Human immunodeficiency virus type 1 DNA synthesis was followed in a CD4+ line of T cells (C8166) grown in the presence or absence of a monoclonal antibody to CD4 that blocks infection By 48 h after infection, cultures grown in the presence of the antibody contained approximately 4 copies of human immunodeficiency virus type 1 DNA per cell, whereas those grown in the absence of the antibody contained approximately 80 copies of viral DNA per cell. Most of the viral DNA in cultures grown in the absence of the antibody was present in a broad smear of apparently incomplete viral sequences. In cultures grown in the presence or absence of the antibody, the 9.6-kilobase linear duplex of viral DNA appeared to undergo integration within 24 h of its appearance. These results demonstrate that T cells accumulate unintegrated human immunodeficiency virus type 1 DNA as a result of multiple virions entering cells.     

Establishment of infection by spleen necrosis virus: inhibition in stationary cells and the role of secondary infection

The relationship of two early events in the establishment of infection by avian retroviruses, the inhibition of viral DNA synthesis in stationary avian cells and the secondary infection which occurs after infection of replicating cells, was investigated. When neutralizing antibody to spleen necrosis virus was used to prevent secondary infection, the amount of unintegrated linear spleen necrosis virus DNA detected was much lower in infected stationary cells than in infected replicating cells. The amount of unintegrated linear spleen necrosis virus DNA in stationary cells was less than one copy per cell even at high multiplicities of infection. Viral DNA synthesis resumed after stimulation of the cells to replicate. The time of this viral DNA synthesis was closely correlated with renewed cellular DNA synthesis. In addition, blocking secondary infection of replicating cells prevented the rate of virus production from reaching the high levels usually associated with a normal productive infection by SNV. Virus production increased if secondary infection was allowed. However, this rise in virus production was not proportional to the amounts of viral DNA integrated after secondary infection.     

Synthesis of Proteins and Glycoproteins in Cells Infected with Human Cytomegalovirus

In cytomegalovirus-infected cells, the rate of protein synthesis was detected as two peaks. One occurred during the early phase of infection, 0 to 36 h postinfection, and the other occurred during the late phase, after the initiation of viral DNA synthesis. Double-isotopic-label difference analysis demonstrated that host and viral proteins were synthesized simultaneously during both phases. In the early phase, approximately 70 to 90% of the total proteins synthesized were host proteins, whereas approximately 10 to 30% were viral, even at a multiplicity of infection of 20 PFU/cell. Virus-related proteins or glycoproteins were referred to as infected-cell specific (ICS). Two ICS glycoproteins (gp145 and 100) were clearly detectable and were synthesized preferentially in the early phase of infection. Their synthesis was concomitant with stimulation of the protein synthesis rate. In the late phase of infection, approximately 50 to 60% of the total protein synthesis was viral and approximately 40 to 50% was host. The ICS proteins and glycoproteins detected during the late phase of infection were viral structural proteins. Infectious virus was not detectable until 48 to 72 h postinfection. An inhibitor of viral DNA synthesis, phosphonoacetic acid, prevented the appearance of the late-phase ICS proteins and glycoproteins, but there was little or no effect on early ICS glycoprotein synthesis. Radiolabeled ICS proteins and glycoproteins were identified by their relative rates of synthesis, by their different electrophoretic mobilities compared with those of host proteins and host glycoproteins, and by their similar electrophoretic mobilities compared to those of proteins and glycoproteins associated with virions and dense bodies of cytomegalovirus. Structural viral antigens in the infected-cell extracts were removed by immunoprecipitation, using F(ab')(2) fragments of cytomegalovirus-specific antibodies, and identified as described above. The last two criteria were used to identify viral structural ICS proteins and glycoproteins. Although approximately 35 structural proteins were found to be associated with purified virions and dense bodies, the continued synthesis of host cell proteins complicated their identification in infected cells. Nevertheless, seven of the nine structural glycoproteins were identified as ICS glycoproteins.     

Herpes Simplex Virus Type 2 Functions Expressed During Stimulation of Human Cell DNA Synthesis

Experiments were designed to identify herpes simplex virus type 2 (HSV-2)-specific functions expressed during stimulation of human embryo fibroblast DNA synthesis. Cultures were partially arrested in DNA synthesis by pretreatment with 5-fluorouracil and maintenance in low-serum (0.2%) medium during virus infection. Results showed that continuous [methyl-(3)H]thymidine uptake into cellular DNA was ninefold greater in HSV-2-infected than in mock-infected cultures measured after 24 h of incubation at 42 degrees C. Shifting mock-infected cultures from low- to high-serum (10%) medium also caused some stimulation, but [methyl-(3)H]thymidine uptake was only twofold greater than in cells maintained with low serum. Plating efficiencies of both HSV-2-infected and mock-infected cells at 42 degrees C were essentially the same and ranged from 37 to 76% between zero time and 72 h of incubation. De novo RNA and protein syntheses were continuously required for HSV-2 stimulation of cellular DNA synthesis. HSV-2 infection markedly enhanced transport, phosphorylation, and rate of incorporation of [methyl-(3)H]thymidine into cellular DNA, starting at 3 h and reaching a maximum by 12 h; after 12 h, these processes gradually declined to low levels. In mock-infected cells these processes remained at low levels throughout the observation period. Pretreatment of cells with interferon or addition of arabinofuranosylthymine at the time of virus infection inhibited stimulation caused by HSV-2. 5-Bromodeoxyuridine density-labeled experiments revealed that HSV-2 stimulates predominantly semiconservative DNA replication and some DNA repair. Stimulation of [methyl-(3)H]thymidine into cellular DNA correlated with detection of virus-specific thymidine kinase activity. In conclusion, HSV-2 stimulation of cellular DNA synthesis appeared to involve at least four virus-specific functions: induction of thymidine transport, HSV-2 thymidine kinase activity, semiconservative replication, and repair of cellular DNA.     

Human cytomegalovirus stimulates host cell RNA synthesis.

Human cytomegalovirus infection of human fibroblast cells (WI-38) induced cellular RNA synthesis. The RNA synthesis in infected cultures preceded the synthesis of viral DNA and progeny virus by approximately 24 h. RNA species synthesized in infected cells included ribosomal 28S and 18S; and 4S transfer RNA; all were markedly increased in comparison to uninfected cells. This induction of host cell RNA synthesis was dependent upon a protein(s) that was synthesized during the early stages of infection.     

Heterogeneity in the replicating population of cultured human epidermal keratinocytes

We studied the replication of keratinocytes in stratified squamous epithelia. Other studies have revealed functional and morphological heterogeneity in the replicating population of such cells. To examine possible kinetic heterogeneity, we determined the cell-cycle lengths of replicating cells in cultures of human epidermal keratinocytes. A double-label assay was developed, which measures the time between two successive cycles of DNA synthesis. The first cycle of DNA synthesis was marked by pulse labeling cultures for a brief period with 14C-thymidine (dThd), and the second cycle was detected by labeling at a later time with bromodeoxyuridine (BrdUrd). The time taken for the 14C-labeled DNA to become doubly labeled with BrdUrd was shown to correspond to the length of the cell cycle. In subconfluent cultures in which the cell number increased at an exponential rate, the average cell-cycle time was 21.5 h. In confluent cultures in which desquamation was balanced by cell renewal, the average cell cycle was 31.5 h. However, in confluent cultures, three populations of replicating cells were evident, these having cycle times of 22, 33, and 40 h. In subconfluent cultures, there was no clear evidence for cell-cycle heterogeneity of the replicating cells, although the most rapidly cycling cells in these cultures had a cycle time (16 h) considerably less than the most rapidly cycling cells in the confluent cultures (21 h). It is possible that the rapidly cycling cells seen in the subconfluent cultures were stem cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fate of Infecting Simian Virus 40-Deoxyribonucleic Acid in Nonpermissive Cells: Integration into Host Deoxyribonucleic Acid

Nonpermissive 3T3 cells were infected with purified superhelical simian virus 40 (SV40) deoxyribonucleic acid I (DNA I). One hour after infection, approximately 60% of the intracellular SV40 DNA was converted to relaxed forms. One day after infection, all intracellular SV40 DNA was present as slow-sedimenting material, and no SV40 DNA I was detectable. At 2 days after infection there appeared viral DNA sequences cosedimenting with cellular DNA during alkaline velocity centrifugation. Furthermore, by both alkaline equilibrium gradient centrifugation and by DNA-ribonucleic acid hybridization analysis, covalent linkage of viral DNA sequences to cellular DNA was demonstrated. Integration of SV40 DNA into cellular DNA did not appear to require DNA synthesis, although DNA synthesis followed by mitotic division of the cells enhanced the amount of viral DNA integrated. Based on data obtained by two different methods, it was calculated that 1,100 to 1,200 SV40 DNA equivalents must be integrated per cell by 48 hr after infection.     

Destruction of L Cells by Mengo Virus: Use of Interferon to Study the Mechanism

Interferon, when added to L cells, inhibited the synthesis of infectious Mengo viral ribonucleic acid, hemagglutinins, and infectious virus by 85 to 95%. Serum-blocking antigens were also reduced by the action of interferon, but threefold excess amounts of these antigens accumulated in interferon-treated cultures above the amounts expected for the quantity of infectious virus that was produced in these cultures. Radioautographic analysis showed that 28 to 36% of the cells of an interferon-treated population synthesized viral ribonucleic acid and 36 to 47% produced viral antigens as determined by an immunofluorescence technique. Despite the reductions in synthesis of viral components, all cells in an interferon-treated culture underwent cytopathic effects at the same time as cells in infected cultures which had not been treated with interferon. The results are compatible with the hypothesis that the cell destruction which results from the infection of L cells with Mengo virus is due to a protein which is coded for by the virus but is not a component of the mature virion.     

Deoxyribonucleic Acid Replication in Simian Virus 40-Infected Cells: III. Comparison of Simian Virus 40 Lytic Infection in Three Different Monkey Kidney Cell Lines

A comparative study of simian virus 40 (SV40) lytic infection in three different monkey cell lines is described. The results demonstrate that viral deoxyribonucleic acid (DNA) synthesis and infectious virus production begin some 10 to 20 hr earlier in CV-1 cells and primary African green monkey kidney (AGMK) cells than in BSC-1 cells. Induction of cellular DNA synthesis by SV40 was observed in CV-1 and AGMK cells but not with BSC-1 cells. Excision of large molecular weight cellular DNA to smaller fragments was easily detectable late in infection of AGMK cells. Little or no excision was observed at comparable times after infection of CV-1 and BSC-1 cells. The different kinds of responses of these three monkey cell lines during SV40 lytic infection suggest the involvement of cellular functions in the virus-directed induction of cellular DNA synthesis and the excision of this DNA from the genome.