Temperature-sensitive mouse cell factors for strand-specific initiation of poliovirus RNA synthesis.

Two cell lines, TgSVA and TgSVB, were established from the kidneys of transgenic mice carrying the human gene encoding poliovirus receptor. The cells were highly susceptible to poliovirus infection, and a large amount of infectious particles was produced in the infected cells at 37 degrees C. However, the virus yield was greatly reduced at 40 degrees C. This phenomenon was common to all mouse cells tested. To identify the temperature-sensitive step(s) of the virus infection cycle, different steps of the infection cycle were examined for temperature sensitivity. The results strongly suggested that the growth restriction observed at 40 degrees C was due to reduced efficiency of the initiation process of virus-specific RNA synthesis. Furthermore, this restriction appeared to occur only on the synthesis of positive-strand RNA. Virus-specific RNA synthesis in crude replication complexes was not affected by the nonpermissive temperature of 40 degrees C. In vitro uridylylation of VPg seemed to be temperature sensitive only after prolonged incubation at 40 degrees C. These results indicate that a specific host factor(s) is involved in the efficient initiation process of positive-strand RNA synthesis of poliovirus and that the host factor(s) is temperature sensitive in TgSVA and TgSVB cells.     

Simian virus 40 gene A regulation of cellular DNA synthesis. II. In nonpermissive cells.

The stimulation of host macromolecular synthesis and induction into the cell cycle of serum-deprived G0-G1-arrested mouse embryo fibroblasts were examined after infection of resting cells with wild-type simian virus 40 or with viral mutants affecting T antigen (tsA58) or small t antigen (dl884). At various times after virus infection, cell cultures were analyzed for DNA synthesis by autoradiography and flow microfluorimetry. Whereas mock-infected cultured remained quiescent and displayed either a 2N DNA content (80%) or a 4N DNA content (15%), mouse cells infected with wild-type simian virus 40, tsA58 at 33 degrees C, or dl884 were induced into active cell cycling at approximately 18 h postinfection. Although dl884-infected mouse cells were induced to cycle initially at the same rate as wild type-infected cells, they became arrested earlier after infection and also failed to reach the saturation densities of wild-type simian virus 40-infected cells. Infection with dl884 also failed to induce loss of cytoplasmic actin cables in the majority of the infected cell population. Mouse cells infected with tsA58 and maintained at 39.5 degrees C showed a transient burst of DNA synthesis as reflected by changes in cell DNA content and an increase in the number of labeled nuclei during the first 24 h postinfection; however, after the abortive stimulation of DNA synthesis at 39.5 degrees C shift experiments demonstrated that host DNA replication was regulated by a functional A gene product. It is concluded that both products of the early region of simian virus 40 DNA play a complementary role in recruiting and maintaining simian virus 40-infected cells in the cell cycle.     

Specific Sindbis virus-coded function for minus-strand RNA synthesis.

The synthesis of minus-strand RNA was studied in cell cultures infected with the heat-resistant strain of Sindbis virus and with temperature-sensitive (ts) belonging to complementation groups A, B, F, and G, all of which exhibited an RNA-negative (RNA-) phenotype when infection was initiated and maintained at 39 degrees C, the nonpermissive temperature. When infected cultures were shifted from 28 degrees C (the permissive temperature) to 39 degrees C at 3 h postinfection, the synthesis of viral minus-strand RNA ceased in cultures infected with ts mutants of complementation groups B and F, but continued in cultures infected with the parental virus and mutans of complementation groups A and G. In cultures infected with ts11 of complementation group B, the synthesis of viral minus-strand RNA ceased, whereas the synthesis of 42S and 26S plus-strand RNAs continued for at least 5 h after the shift to 39 degrees C. However, when ts11-infected cultures were returned to 28 degrees C 1 h after the shift to 39 degrees C, the synthesis of viral minus-strand RNA resumed, and the rate of viral RNA synthesis increased. The recovery of minus-strand synthesis translation of new proteins. We conclude that at least one viral function is required for alphavirus minus-strand synthesis that is not required for plus-strand synthesis. In cultures infected with ts6 of complementation group F, the syntheses of both viral plus-strand and minus-strand RNAs were drastically reduced after the shift to 39 degrees C. Since ts6 failed to synthesize both plus-strand and minus-strand RNAs after the shift to 39 degrees C, at least one common viral component appears to be required for the synthesis of both minus-strand and plus-strand RNAs.     

Human cytomegalovirus induced cyclooxygenase-2 in human retinal pigment epithelial cells augments viral replication through a prostaglandin pathway

Cytomegalovirus (CMV) retinitis is characterized by alterations in retinal cell function and host responses to virus replication. The goal of this study was to evaluate the induction of cyclooxygenase-2 (COX-2) and prostaglandin (PGE) in CMV infected human retinal pigment epithelial (RPE) cells and to determine their effect on virus replication. CMV immediate early (IE) protein and COX-2 proteins were identified in RPE cells in retinal tissue sections from patients with CMV retinitis. COX-2 mRNA and protein were induced after CMV infection of human RPE cell cultures. CMV infection of RPE cells induced translocation of NF-kappaB from the cytoplasm to the nucleus. PGE1 and PGE2 were significantly (p<0.001) increased in human RPE cell cultures infected with CMV. Inhibition of CMV IE gene by antisense oligonucleotides abrogated induction of mRNA for COX-2 and protein synthesis of COX-2 and PGE2. PGE enhanced CMV plaque formation and real time PCR analysis revealed that PGE treatment significantly increased CMV DNA copy numbers. These studies demonstrate that when CMV replicates within human RPE cells, COX-2 induction augments virus replication via the PGE pathway. The induction of COX-2 and PGE during retinal CMV infection may augment virus replication and alter a variety of retinal physiological responses.     

Decline in mutation frequency in temperature-sensitive SV40 viruses before viral DNA synthesis.

Lesions that promote reversion from a temperature-sensitive to a wild-type phenotype were induced in temperature-sensitive late mutants of SV40 virus by UV irradiation. When cultures infected with UV-irradiated temperature-sensitive mutants were grown for various times at permissive temperature (35 degrees C) and then at restrictive temperature (39 degrees C), the reversion frequency declined just before the onset of semiconservative DNA synthesis when DNA synthesis began at 32 degrees C. This can be explained by competition between reactions that lead to the onset of viral DNA synthesis and reactions that repair the lesions before the onset of viral DNA synthesis.     

Simian virus 40 gene A regulation of cellular DNA synthesis. I. In permissive cells.

The kinetics of host cellular DNA stimulation by simian virus 40 (SV40) tsA58 infection was studied by flow microfluorometry and autoradiography in two types of productively infected monkey kidney cells (AGMK, secondary passage, and the TC-7 cell line). Prior to infection, the cell populations were maintained predominantly in G0-G1 hase of the cell cycle by low (0.25%) serum concentration. Infection of TC-7 or AGMK cells by wild-type SV40, viable deletion mutant dl890, or by SV40 tsA58 at 33 degrees C induced cells through S phase after which they were blocked with a 4N DNA content in the G2 phase. The infection of TC-7 cells by tsA58 at 41 degrees C, which was a nonpermissive temperature for viral DNA replication, induced a round of cell DNA synthesis in approximately 30% of the cell population. These cells proceeded through S phase but then re-entered the G1 resting state. In contrast, infection of AGMK cells by tsA58 at 41 degrees C induced DNA synthesis in approximately 50% of the cells, but this population remained blocked in the G2 phase. These results indicate that the mitogenic effect of the A gene product upon cellular DNA is more heat resistant than its regulating activity on viral DNA synthesis and that the extent of induction of cell DNA synthesis by the A gene product may be influenced by the host cell.     

High temperature inactivation of cucumber and alfalfa mosaic viruses in Nicotiana rustica cultures

Cucumber mosaic (CMV) and alfalfa mosaic (AlfMV) viruses could not be detected in Nicotiana rustica tissues cultured at 32 °C for 16–18 days or at 40 °C for 5 days, but infectivity remained high in comparable tissue cultured at 22 °C. Incubation of infected cultures at 28–30 °C resulted in an initial reduction followed by a partial recovery in the infectivity of both viruses. The infectivity of CMV in tissues grown between 12 and 32 °C was highest in cultures grown at 12 °C. Although CMV infectivity was not detected in cultures after 16–18 days at 32 °C, virus was eliminated only after a further 30 days at 32 °C. When cultures were transferred from 32 to 22 °C after shorter treatment periods, infectivity rapidly increased to levels higher than those of infected tissues grown continuously at 22 °C. At 40 °C, CMV was eliminated from infected tissues after 9 days and AlfMV after 7 days. Cultures grown continuously at 40 °C deteriorated rapidly but, when grown under diurnal alternating periods of 8 h at 40 °C and 16 h at 22 °C, they remained viable and CMV was also inactivated.     

Study of liver differentiation in vitro

A clonal rat fetal liver cell line that expresses the functions of differentiated liver cells under controllable conditions has been established. Normal fetal liver cells were transformed by a temperature-sensitive A (tsA) mutant (tsA209) of simian virus 40. At the permissive temperature (33 degrees C), the tsA209-transformed liver cell line (RLA209-15) can be cultured indefinitely and cloned readily. The RLA209-15 cells were temperature sensitive for maintenance of the transformed phenotype. These transformed liver cells selectively lost four characteristics of the transformed phenotype at the restrictive temperature (40 degrees C): generation time of the cells increased, the saturation density decreased, the efficiency of growth on nontransformed cell layers decreased, and the ability to clone in soft agar was lost. The transformation can be reversed simply by a shift in temperature. RLA209-15 fetal liver cells synthesized alpha-fetoprotein albumin, and transferrin. At 33 degrees C, the levels of these liver proteins were relatively low. At 40 degrees C the transformed phenotype was lost and the levels of alpha-fetoprotein, albumin, and transferrin were greatly increased. At the restrictive temperature, maximal induction of the synthesis of alpha-fetoprotein, albumin, and transferrin was achieved 3-4 d after the upward shift in temperature. The synthesis of alpha-fetoprotein then decreased; the synthesis of albumin and transferrin, however, was maintained. A second phase of albumin and transferrin synthesis was observed in all cultures after 6 d or more at 40 degrees C. Alpha-Fetoprotein, albumin, and transferrin secreted by RLA209-15 cells were immunologically indistinguishable from authentic alpha-fetoprotein, albumin, and transferrin, respectively. RLA209-15 cells, like primary cultures of hepatocytes and a simian virus 40 tsA255-transformed fetal liver cell line (RLA255-4) reported earlier from this laboratory, responded to glucagon with markedly elevated levels of cyclic AMP. Thus, it appears that glucagon receptors characteristic of hepatocytes are retained in the simian virus 40 tsA-transformed fetal liver cells.     

Inactivation of cucumber mosaic virus in cultured tissues of Nicotiana rustica by diurnal alternating periods of high and low temperature

Cucumber mosaic virus (CMV) was inactivated in infected tissue cultures of Nicotiana rustica after periods of alternating high (32–40 ^oC) and low (22 ^CC) temperature. An increase in the incubation period at high temperatures resulted in greater virus inactivation but more rapid deterioration of the tissue cultures. Optimal programmes for virus inactivation and culture survival were 40 ^oC (8 h) +22 ^oC (16 h) per day for 12 or 18 days, 40 ^oC (16 h) + 22 ^oC (8 h) per day for 12 days or 36 ^oC (20 h) + 22 ^oC (4 h) per day for 12 days. Diurnal treatment periods of variable length and different high temperatures were related by using a concept of hour-degrees above a base temperature. Taking this base temperature as 25 ^oC a linear relation was found between the logarithm of virus infectivity and the number of hour degrees.     

Effect of lipid alkyl chain perturbations on the assembly of bacteriophage PM2.

The lipid-containing bacteriophage PM2 can produce infectious virus in cultures infected at temperatures up to 31.5 degrees C, but not at 34 degrees C. Its host, Pseudomonas BAL-31, grows at 34 degrees C and cultures infected at that temperature undergo lysis. Sucrose-gradient analysis shows that 34 degrees C lysates contain no PM2-like particles. Temperature-shift experiments establish that the thermally sensitive process is late in infection when virus assembly is taking place. Adamantanone, a small hydrophobic molecule that perturbs membrane hydrocarbon zones, prevents the production of infective virus. Concentrations which prevent virus production have no effect on host-cell growth or stability of mature virions. Adamantanone exerts its effects late in the infectious cycle, and lysates amde in its presence contain no PM2-like particles. These experiments, carried out at 25 degrees C, indicate that adamantanone prevents the assembly of stable PM2 virus. Spin-label studies suggest that the lipid alkyl chains of the host-cell membrane are in an "ordered" state at temperatures below about 33 degrees C and undergo a transition to a "disordered" state above that temperature. Furthermore, the addition of adamantanone perturbs the hydrocarbon zones, producing a greater degree of disorder even below 25 degrees C. Our findings suggest that the cell membrane can function and grow with the lipid alkyl chains in either the "ordered" or "disordered" state, but that the "ordered" state must be maintanined for PM2 assembly to occur.     

Taxol inhibits stimulation of cell DNA synthesis by human cytomegalovirus

The microtubule (MT)-stabilizing drug, taxol, inhibited human cytomegalovirus (CMV)-initiated cell DNA synthesis by up to 100% in serum-arrested mouse embryo (ME) fibroblasts that were abortively infected by CMV. Taxol concentrations known to increase MT polymerization and to stabilize existing MTs (10 to 20 micrograms/ml) blocked CMV-stimulated cell DNA synthesis, while taxol concentrations of 2.5 micrograms/ml, or less, did not. Taxol maximally inhibited CMV initiation of cell DNA synthesis when added 3 h after virus infection and inhibited this initiation by greater than 50% when added up to 12 h after CMV infection. Control experiments suggest that taxol specifically inhibited CMV-stimulated cell DNA synthesis. Pretreatment of CMV stock with taxol did not reduce the stimulatory effect of CMV on cell DNA synthesis and taxol had no detectable effect on CMV-specific early protein synthesis. Moreover, taxol did not appear to alter thymidine pool sizes, affect cell viability, or compromise the DNA synthetic machinery in CMV-infected cells. Since taxol increases tubulin polymerization and inhibits MT disassembly, these results suggest that dynamic changes in MTs or in the pool of free tubulin subunits are necessary for CMV to stimulate cell entry into a proliferative cycle.     

Inhibition of herpes simplex virus type 1 replication in temperature-sensitive cell cycle mutants.

Herpes simplex virus type 1 DNA synthesis and infections progeny production were studied in five different conditional hamster (BHK-21) cell cycle mutants. At the nonpermissive temperature (39.5 degrees C), both events were strongly inhibited in four of these cell lines. The degree of inhibition was a reproducible characteristic of each cell mutant and in two cases was dependent upon the multiplicity of infection. Experiments involving shifts to the nonpermissive temperature at least 3 h postinfection at 33.5 degrees C suggested that the defects in viral replication were not due to faulty adsorption, penetration, or uncoating, whereas experiments involving shifts of infected cells from the nonpermissive temperature to 33.5 degrees C revealed the reversible nature of the inhibition.     

Partial characterization of a soluble antigen preparation from cells infected with human cytomegalovirus: properties of antisera prepared to the antigen.

Soluble antigen (SA) preparations were obtained from cell cultures infected with either the Davis or AD169 strains of cytomegalovirus (CMV). Fractionation of SA preparations through Sephadex G-200 resulted in a molecular weight value ranging from 67,000 to 85,000. Rate-zonal centrifugation produced an approximate value of 5.5S for the CMV antigenic material. Antisera to SA prepared from either AD169- or Davis-infected cells lacked neutralizing activity but produced specific fluorescence confined to CMV intranuclear inclusion material when used in the indirect fluorescent antibody test (IFA). The specific fluorescing inclusion reaction was seen when either AD169 or Davis antisera were used with cells infected with the Davis, AD169, Kerr, or C-87 strains of CMV. Fluorescence was not observed in cells infected with a strain of Herpes simplex type 1, varicella-zoster virus, an EBV transformed lymphocyte line, the Cx-90-3B human CMV transformed hamster embryo cell line or CMV-infected cell cultures treated with cytosine arabinoside (Ara-C) and showing only antigens expressed in the absence of viral DNA synthesis. Antisera prepared to SA preparations obtained from CMV-infected cells apparently react with specific CMV antigens that are dependent on viral DNA synthesis and are common to several strains.     

Conditions necessary for inhibition of protein synthesis and production of cytopathic effect in Aedes albopictus cells infected with vesicular stomatitis virus.

The relationship between the development of cytopathic effect (CPE) and the inhibition of host macromolecular synthesis was examined in a CPE-susceptible cloned line of Aedes albopictus cells after infection with vesicular stomatitis virus. To induce rapid and maximal CPE, two conditions were required: (i) presence of serum in the medium and (ii) incubation at 34 degrees C rather than at 28 degrees C. In the absence of serum, incubation of infected cultures at 34 degrees C resulted in a significant increase in viral protein and RNA synthesis compared with that observed at 28 degrees C. However, when serum was present in the medium, by 6 h after infection protein synthesis (both host and viral) was markedly inhibited when infected cells were maintained at 34 degrees C. RNA synthesis (host and viral) was also inhibited in vesicular stomatitis virus-infected cells maintained at 34 degrees C with serum, but somewhat more slowly than protein synthesis. Examination of polysome patterns indicated that when infected cultures were maintained under conditions which predispose to CPE, more than half of the ribosomes existed as monosomes, suggesting that protein synthesis was being inhibited at the level of initiation. In addition, the phosphorylation of one (or two) polysome-associated proteins was reduced when protein synthesis was inhibited. Our findings indicate a strong correlation between virus-induced CPE in the LT-C7 clone of A. albopictus cells and the inhibition of protein synthesis. Although the mechanism of the serum effect is not understood, incubation at 34 degrees C probably predisposes to CPE and inhibition of protein synthesis by increasing the amount of viral gene products made.     

Characterization of a temperature-sensitive mutant of human adenovirus type 7.

The properties of a naturally occurring temperature-sensitive (ts) mutant of human adenovirus type 7 (Ad7) were studied. Mutant Ad7 (19), or E46-, was the nonhybrid adenovirus component derived from the defective simian virus 40 (SV40)-Ad7 hybrid (PARA). Growth of the mutant was restricted at 40.5 degrees C, and the ratios of virus yields in KB cells at 40.5 and 33 degrees C were 10(-2) to 10(-3). Viral DNA synthesis and the synthesis of adenovirus-specific antigens (tumor, capsid, hexon, and penton antigens) appeared normal at the restrictive temperature. The assembly of virus particles was aberrant, as determined by thin-section of infected cells. The infectivity of mutant virions was heat labile at 50 degrees C, suggesting a ts defect in a structural component of the viron. Analysis by polyacrylamide gel electrophoresis of [35S]methionine-labeled polypeptides synthesized in mutant-infected cells suggested that at least the major virion polypeptides were synthesized at the restrictive temperature. A lack of inhibition of host protein synthesis late in mutant infections, as compared with wild-type (WT) infections at both the permissive and nonpermissive temperatures, made quantitation of infected-cell polypeptides difficult. Analysis of the assembly of capsomeres from cytoplasmic extracts of infected cells on sucrose gradients and by non-dissociating polyacrylamide gel electrophoresis suggested that hexon capsomeres were made at 40.5 degrees C. The hexon capsomeres made by the mutant at either 33 or 40.5 degrees C displayed a decreased migration in the non-dissociating gels compared with the WT hexon capsomeres. The molecular weights of the mutant and WT hexon polypeptides were identical. These results suggest that the ts lesion of this group B human Ad7 mutant may be reflected in altered hexons. The mutant Ad7 interfered with the replication of adenovirus types 2 and 21 at the elevated temperature.     

The role of calmodulin in the proliferation of transformed and phenotypically normal tsASV-infected rat cells

NRK cells infected with a temperature-sensitive, transformation-defective mutant of avian sarcoma virus (ASV), tsLA23, behaved as if nontransformed at a nonpermissive 40 degrees C and were rendered quiescent by serum deprivation. These serum-deprived cells were stimulated to start entering S phase about 7 hours after serum addition at 40 degrees C or about 9 hours after shifting the cultures to 36 degrees C, a temperature allowing the production of active viral pp60src and expression of the transformed phenotype. The transit of both serum- and temperature-stimulated tsLA23-NRK cells through later G1 was inhibited by the unrelated calmodulin antagonists W7 and R24571. The former drug was found to block the cells at a point in the cell cycle no more than 2 hours from the G1/S transition. The weaker calmodulin antagonist, W5, was less effective in impairing progression. Thus, calmodulin is likely required for the transit of both transformed and phenotypically normal tsLA23-NRK cells through the later stages of their G1 phases. Cells neoplastically transformed by ASV contain more calmodulin than uninfected, non-neoplastic cells. At the nonpermissive 40 degrees C, the calmodulin content of the tsLA23-NRK cells dropped to the non-neoplastic level. When these phenotypically nontransformed cells were enabled to reenter the cell cycle while still in low-serum medium by a 40 to 36 degrees C shift, they passed through the G1 and S phases and divided without a concomitant rise in the total calmodulin content. Thus, a calmodulin rise does not appear to be required for the expression of one characteristic of transformed cells, i.e., reduced requirement for exogenous growth factors.     

Properties of mammalian cells transformed by temperature-sensitive mutants of avian sarcoma virus.

Fibroblasts from European field vole (Microtus agrestis) and from normal rat kidney (NRK) have been infected by avian sarcoma virus mutants which are temperature-sensitive for the maintenance of transformation. These cells are transformed at 33 degrees C, but show normal cell characteristics in morphology, colony formation in agar, saturation density, sugar uptake and membrane proteins at 39 degrees C and 40 degrees C, the nonpermissive temperatures. Ts mutant virus was rescued from most of the ts transformed cell lines. NRK cells infected by avian sarcoma virus ts mutants and kept at the nonpermissive temperature can be transformed by wild-type avian sarcoma virus. The susceptibility of the temperature-sensitive NRK lines to this transformation is higher than the susceptibility of uninfected NRK at either permissive or nonpermissive temperature.     

Prolongation of herpes simplex virus latency in cultured human cells by temperature elevation.

Treatment of herpes simplex virus type 2 (HSV-2)-infected human fibroblast cells with cytosine arabinoside (ara-C) at 25 microgram/ml resulted in complete inhibition of virus replication. Removal of ara-C after 7 days of treatment ultimately resulted in renewed virus replication, but after a delay of at least 5 days. If however, the temperature was elevated from 37 degrees C to 39.5 to 40 degrees C at the time of ara-C reversal, infectious HSV-2 did not reappear. As long as the cultures were maintained at 39.5 to 40 degrees C (up to at least 128 days), HSV-2 was latent and infectious virus was undetectable. If the temperature was reduced to 37 degrees C at any time during the latent period, infectious virus was always reactivated, but only after a period of incubation at 37 degrees C of a least 11 days. Infectious-center assays performed with latent cultures indicated that only a very small fraction of cells could reactivate virus. The infectious-center titer did not show significant changes during much of the period of latency. This seemed to argue against the possibility that the latent cultures were synthesizing very small amounts of infectious virus. Additional studies were aimed at determining the minimum incubation period at 37 degrees C required to reactivate infectious HSV-2. Latent cultures reduced from 39.5 to 40 degrees C to 37 degrees C for less than 96 h did not yield infectious HSV-2, but those incubated at 37 degrees C for 96 h or more did.     

Modulation by the src oncogene of the effect of inhibitory diffusible factor IDF45

Density-dependent inhibition of growth has been assumed to be under the control of inhibitory molecules diffusing from dense cell cultures. Growth inhibitory factors have been fractionated or purified from medium conditioned by different cell types. In the present work, it was shown that IDF45 (inhibitory factor diffusing from 3T3 cells) decreased DNA synthesis in chick embryo fibroblasts (CEF) and was an inhibitor of CEF growth; this inhibition was reversible. Since similitudes between oncogene products and growth factors have been observed, it was of interest to compare the inhibitory effect of IDF45 upon the stimulation of DNA synthesis induced either by serum or by pp60-src. CEF infected by Ny68 virus (a mutant of Rous sarcoma virus ts for the expression of transformation) were density-inhibited at 41 degrees C, but were stimulated at this temperature by addition of 1% serum. This stimulation was 94% inhibited by IDF45. The same Ny68-infected cells could also be stimulated by transfer to 37 degrees C, the permissive temperature (in the absence of serum). The stimulation of DNA synthesis by src expression was poorly inhibited by IDF45. From our results, it appears that oncogene expression in CEF induces a loss in their sensitivity to IDF45. This would explain why transformed cells escape DDI of growth.     

Consequences of herpes simplex virus type 2 and human cell interaction at supraoptimal temperatures.

The consequences of herpes simplex virus type 2 (HSV-2) and human embryonic fibroblast cell interaction at different temperatures (37, 40, and 42 degrees C) were investigated. Incubation at 37 or 40 degrees C was permissive for HSV-2 inhibition of host DNA synthesis, induction of virus-specific DNA replication, and infectious virus production. The amount of [methyl-3H]thymidine incorporated into viral DNA and the final yield of new infectious virus were significantly reduced at 40 degrees C compared to 37 degrees C. At 42 degrees C, detectable virus-specific DNA synthesis was totally blocked. Maximum stimulation of host cell DNA synthesis at 42 degrees C was measured after a multiplicity of infection of 0.5 to 1.0 PFU/cell. By autoradiography, data indicated that HSV-2 stimulates host cell chromosomal DNA synthesis. Stimulation of thymidine kinase activity with thermostability properties in common with a virus enzyme was detected during the first 24 h of infection at 42 degrees C, after 24 h the enhanced thymidine kinase activity had properties in common with host cell isozymes. The data obtained during this investigation indicated that stimulation of host cell DNA synthesis does not require viral DNA synthesis.