Restricted replication of adenovirus type 2 in mouse balb/3T3 cells

Summary The nature of the infection of mouse B3T3 cells by adenovirus type 2 (Ad2) has been studiedin vitro. Following infection with an adsorbed MOI of 225, more than 90 percent of the cells synthesized both early and late virus-specific antigens. In contrast, the yield of progeny virus varied from only 2 × 10⁴ to 2 × 10⁶ FFU/2 × 10⁵ cells. The range in yields was related, in part, to the number of cell generations from the time of the initial subcloning, the yield increasing with passage level. Infectious center analysis suggested that fewer than 0.5 percent of infected cells synthesized progeny virus.Analysis of DNA synthesis in infected multiplying B3T3 cells demonstrated that cellular DNA synthesis began to be shut off at 12 hours p.i., a time when viral DNA synthesis was beginning. The maximum rate of viral DNA synthesis was approximately 12 percent of that in infected human cells. In contrast to infected multiplying cells, infection of quiescent B3T3 cell cultures resulted in the induction of cellular, along with viral, DNA synthesis. Analysis of late gene expression detected synthesis of most viral polypeptides, but revealed greater than 90 percent reductions in the rate of synthesis of polypeptides II, III, IV, and IX, as compared with infected human cells.With 8 FiguresThese studies were supported by NIH Research Grant No. CA-08851 and NIH Training Grant No. CA-09069.     

Murine cytomegalovirus gene expression during nonproductive infection in go-phase 3T3 cells

Murine 3T3 cells, maintained in the Go-phase by deprivation of serum, were infected with murine cytomegalovirus (MCMV) and examined for the synthesis of viral DNA, RNA, and proteins. No viral DNA replication could be detected, although the viral genome remained viable since infectious centers persisted. Viral RNA was analyzed by reassociation kinetics with ¹²⁵I-MCMV DNA. At early and late times after infection, the percentage of viral DNA transcribed amounted to 18% and 21%, respectively (comprising one abundance class), in contrast to 26% and 38%, respectively (two abundance classes), at corresponding times during productive infection in growing 3T3 cells. Five viral polypeptides were detected in the Go-phase cells, but these did not include structural polypeptides. When infected Go-phase cells were exposed to serum and fresh medium, viral DNA synthesis occurred, and progeny virions appeared subsequently. Thus this system may prove useful for analyzing a latent herpes infection in vitro.     

Heterozygosis and genetic recombination in herpes simplex type 1 virus

Two- and three-factor crosses with temperature-sensitive (ts) and syncytial plaque morphology (syn) mutants of herpes simplex type 1 virus have been used to study the possible role of syn-syn⁺ mixed plaque-forming virus in genetic recombination. Under the conditions of a standard genetic cross, recombinants first appear about 6 hr after infection, the time of formation of the first infectious progeny virus, and their frequency progressively rises until about 20 hr postinfection. During this period the frequency of mixed plaques remained constant at approximately 5%. The frequency of mixed plaques and recombinants were both increased several-fold when crosses were made in the presence of the DNA synthesis inhibitor 5-fluorodeoxyuridine (FUdR). In view of the genetic instability of the partially heterozygous genomes of mixed plaque forming virus this result is interpreted to mean that mixed plaques identify virus which is an intermediate in recombinant formation and that the molecular structure of their genome is probably that of a partial heteroduplex. Measurements of deoxynucleoside triphosphate pools showed that FUdR inhibited the large increase in the dTTP pool size which normally accompanies HSV-1 infection of BHK cells.     

Viral interference of HSV-1: Properties of the intracellular viral progeny DNA

Intracellular progeny DNA was isolated and characterized from cells infected with standard herpes simplex virus or from cells coinfected with standard virus and with a virus stock obtained by serial passages at high multiplicity of infection (HP virus). The latter was shown to contain an excess of variant virus particles interfering with the production of infectious progeny virus. The ratio of plaque-forming to interfering virus in the HP virus stock used in this study was determined to be $\text{1}\text{10}$. In both infections similar amounts of unit length viral DNA were synthesized. Restriction endonuclease digestion of intracellular viral progeny DNA yielded fragment patterns showing that the majority of the DNA molecules present in standard and HP HSV DNA yield the same restriction fragments. That the typical end fragments could be demonstrated suggests a correct processing of concatemeric precursor DNA into HSV unit length DNA. Despite the obvious similarity of the two DNA species the infectivity in transfection assays of progeny DNA formed in HP virus-infected cells was by three log lower than that of standard DNA. As shown by controls involving cotransfections with standard viral DNA and heterologous DNA, this difference can be attributed to the presence in HP DNA of HSV DNA molecules that interfere with the plaque formation by standard DNA. An alteration in various steps of DNA processing such as DNA methylation and incorporation of ribonucleotides into the DNA was demonstrated not to correlate with interference. Both in HP and standard virus DNA preparations methylation was below the level of detection of 10 5-methylcytosine residues per unit length of viral DNA. Low values were also obtained for the uridine content of the DNA. Almost 100% of the radioactivity incorporated could be recovered as deoxyribonucleosides and less than 0.07% as uridine.     

Inactivation of human cytomegalovirus by sodium periodate oxidation

Summary Human cytomegalovirus (HCMV), oxidized by sodium periodate (NaIO₄), is incapable of giving rise to viral progeny in cell culture. At a NaIO₄ concentration as low as 5 MM, there is a loss of at least 6 logs of viral infectivity which occurs very rapidly (less than 5 min). Further, the inactivation is a first-order reaction depending on the periodate concentration. Adsorption to the cell surface, penetration into cells, and penetration of the viral DNA into cell nuclei were found to occur identically in mock oxidized and oxidized HCMV. Since the carbohydrate moiety of viral glycoproteins was the target of periodate attack, these observations strongly suggest that the structural integrity of the sugar residues is not a perequisite for adsorption and penetration. Nevertheless, no evidence for viral DNA or protein synthesis was detected in cells infected with oxidized virus, and even after 3 weeks in culture, no cytopathic effect was observed.     

Induction of cellular DNA synthesis by a temperature-sensitive mutant of herpes simplex virus type 2.

A temperature-sensitive mutant (ts 4) of herpes simplex virus type 2 (HSV-2), having the ability to transform hamster embryo (HaE) cells at the nonpermissive temperature of 38.5°, has been investigated in several aspects. It is defective in thymidine (TdR) kinase induction at both the permissive (34°) and the nonpermissive temperatures and defective in viral DNA synthesis at the nonpermissive temperature. However, stimulation of chromosomal DNA synthesis was detected at 16–28 hr after infection at the nonpermissive temperature in HaE cells arrested with low serum concentration. DNA synthesis was estimated by the incorporation of [³H]TdR or [³H]CdR (deoxycytidine) into DNA, and differentiation of cellular from viral DNA was performed by buoyant density gradient centrifugation in CsCl or by DNA-DNA hybridization. By autoradiography with [³H]TdR, it was found that the number of cells with grains in the nuclei increased in infected cultures at 16–28 hr after infection. Virus exposed to heat or uv light lost the ability to induce cellular DNA synthesis, indicating that active virus is responsible for stimulation of cellular DNA synthesis.     

Interactions of Pseudorabies virus with swine alveolar macrophages I: virus replication

Summary The replication of Pseudorabies virus (PRV) in cultured swine alveolar macrophages (AM) was studied using 6 different virus strains. AM were highly permissive to PRV infection, with progeny virus titres of 10⁷ TCID₅₀/ml from some strains. Virus progeny titres were higher in cultures infected with the field strains S-62 and 4892 than in cultures infected with the strains Bartha or PRV-C. Virus replication, viral DNA synthesis and the concomitant cell damage were dependent upon virus input m.o.i.s. and virus strain. Furthermore, cells from 7 day old pigs yielded higher virus progeny titres than cells of 6 week old pigs. The results from this study provide support to the premise that PRV infection may predispose anials to respiratory disease.     

Role of tight junctions of polarized epithelial MDCK cells in the replication of herpes simplex virus type 1

Before completion of polarization, Madin-Darby canine kidney (MDCK) cells showed high infectivity and progeny production of herpes simplex virus type 1 infection. After polarization or formation of tight junctions, the infectivity and virus replication in MDCK cells was restricted significantly. The disruption of tight junctions by depletion of Ca²⁺ resulted in increasing virus infectivity and productivity. Mechanical disruption of tight junctions by scratching the cell monolayers with injection needle allowed markedly the replication of HSV-I in the cells aligned along the injured area. In polarized MDCK cells the progeny were released preferentially from the apical surface of the cells. These data suggest that because polarized MDCK cells mimic the epithelial cell layers, this cell line is helpful for determining the factors which regulate viral transmission in the human body. © Wiley-Liss, Inc.     

TYLCSV DNA, but not infectivity, can be transovarially inherited by the progeny of the whitefly vector Bemisia tabaci (Gennadius)

The transovarial transmission of two species of begomovirus, Tomato yellow leaf curl virus (TYLCV) and Tomato yellow leaf curl Sardinia virus (TYLCSV), through generations of Bemisia tabaci of the B and Q biotypes has been investigated. Different life stages of the progeny of viruliferous female whiteflies have been analysed by PCR detection of viral DNA and infectivity tests. Our results indicate that TYLCSV DNA can be detected in eggs and nymphs, and to a lesser extent adults, of the first-generation progeny. Infectivity tests using a large number of adult progeny of the first, second, and third generation indicate that even when viral DNA is inherited, infectivity is not. For TYLCV, neither viral DNA nor infectivity were associated with the progeny of viruliferous female whiteflies. Because the inherited viral DNA is unable to give rise to infections, the transovarial transmission of TYLCSV DNA appears to have no epidemiological relevance.     

Capsid-targeted viral inactivation can destroy dengue 2 virus from within in vitro

Summary. Capsid-targeted viral inactivation (CTVI) has emerged as a conceptually powerful antiviral strategy that exploits viral structural proteins to target a destructive enzyme specifically into progeny virions. We have recently demonstrated the principle of CTVI against dengue virus infection and observed a modest therapeutic effect in vitro (Arch Virol 2005, 150: 659–669). Here we tested a prophylactic model of CTVI, in which mammalian cells stably expressing the dengue 2 virus capsid protein fused to a nuclease were infected with dengue virus and determined the effects on progeny virion infectivity. CTVI efficiently destroyed dengue 2 virus from within and decreased the infectious titers by 10³- to 10⁴-fold, suggesting that CTVI has potential in the prophylactic application for dengue virus infection.     

Murine mammary tumor virus (MuMTV) infection of an epithelial cell line established from C57BL/6 mouse mammary glands.

An epithelioid cell line derived from the mammary glands of a C57BL/6 mouse and designated C57MG cell line was found to be susceptible to infection by routine mammary tumor virus (MuMTV). Although uninfected C57MG cells contain endogenous MuMTV-related DNA sequences, no RNA sequences homologous to MuMTV were detectable, even after treatment with the glucocorticoid, dexamethasone. However, after infection with MuMTV, these cells acquire additional MuMTV DNA, and viral RNA and proteins were readily detectable. Most, if not all, of the additional MuMTV DNA in infected C57MG cells appeared to be integrated. Synthesis of viral RNA and protein, and the release of virions into culture fluid by infected C57MG cells, was stimulated by incorporation of dexamethasone in the growth medium. The efficiency of infection by MuMTV in C57MG cells was similar to that in nonmurine cells. There was a direct relationship between multiplicity of infection (m.o.i.) and the average number of MuMTV RNA molecules detected per cell 5 weeks after infection. However, even at the highest m.o.i. used (4 × 10⁵ virions/cell), a plateau in the average number of viral RNA molecules per cell was not achieved. The origin of MuMTV synthesized by infected C57MG cells, whether it is the progeny of infecting MuMTV or of the endogenous C57BL/6 MuMTV or of both, remains undetermined. We have been unable to detect any morphological or growth pattern changes in C57MG cells following infection with MuMTV.     

Low infectivity of HSV-1 DNA caused by defective-interfering genomes

The infectivity of herpes simplex virus, type 1, strain ANG progeny DNA from standard virus infections and of progeny DNA from infections involving defective-interfering virus particles (DI DNA) was compared in transfection assays. No difference in infectivity of virus DNA isolated either from infected cells or from progeny virus was found for a given type of infection. However, the values for two types of infection differed markedly, with DI progeny DNA being less infectious by more than 2 log10. The low infectivity was mainly due to the presence of interfering DNA molecules in DI progeny DNA, regardless of whether intracellular DNA or DNA extracted from mature virions was analysed. The interfering capacity of DI progeny DNA did not depend on the integrity of the genomes. The physical proximity provided by simultaneous precipitation of infectious and of interfering DNA is an important factor influencing the degree to which DI DNA interferes. Interference by DI DNA in the transfection assay can be partly reversed by the addition of XbaI fragments of standard DNA; in control experiments this fragmented DNA was shown to lead to a reduction rather than to an enhancement of the infectivity of standard virus DNA.     

Macromolecular synthesis in cells infected by frog virus 3. I. Virus-specific protein synthesis and its regulation

Heat-inactivated frog virus 3 inhibited protein synthesis in fathead minnow and baby hamster kidney cells but did not affect the replication of superinfecting infectious frog virus 3 in these cells. This method of controlling host-cell protein synthesis enabled us to identify 20 proteins induced by frog virus 3 infection. All detectable virus-specific proteins were synthesized within 2 hr after infection. Three viral structural proteins (VSP)—7, 9, and 13—were synthesized at maximum rates early in infection, and two others, VSP 5 and 11, reached their peak rate of synthesis late in infection. All structural and nonstructural proteins were made in the presence of cytosine arabinoside, an inhibitor of DNA synthesis. In the absence of viral DNA replication, the kinetics of synthesis of VSP 5 and 11 were similar to those during normal infection, but there was no reduction in the synthesis of VSP 7, 9, and 13 late in the infection. These results suggest that synthesis of all viral structural proteins is an early event in the FV 3 replication cycle, with progeny viral DNA required to regulate the synthesis of certain viral proteins.     

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.     

Synthesis of polyoma virus structural polypeptides in mouse kidney cells

The polypeptide composition of highly purified polyoma virus was studied by SDS-polyacrylamide gel electrophoresis, and in agreement with the observations of Roblin et al. (1971), six structural polypeptides were found. An examination of the time course of synthesis of these polypeptides showed that the synthesis of VP4, 5 and 6, the small basic proteins, begins at about the same time as does the synthesis of viral DNA. The synthesis of VP1, the major viral polypeptide, was first detected 2 hr later, while that of the minor components VP2 and 3, whose appearance even in isolated nuclei, is obscured by a high background of cellular protein synthesis, could not be detected with confidence earlier than 5 to 6 hrs after the onset of DNA synthesis. Polypeptides VP4, 5, and 6 are not present in either naturally occurring or artificially produced empty capsids, but are structural components of pseudovirions. They have a relatively high lysine content, but contain no tryptophan, and in SDS-polyacrylamide gels were found to comigrate with certain of the histones isolated from uninfected mouse kidney cells. Histones, synthesized in mouse kidney cells prior to infection with polyoma virus, were shown to be incorporated into both progeny virions and pseudovirions.     

Reversible restriction of vesicular stomatitis virus in permissive cells treated with inhibitors of prostaglandin biosynthesis

Indomethacin, a potent nonsteroidal inhibitor of prostaglandin synthetase (cyclooxygenase) reduced yields of infectious vesicular stomatitis virus in HEp-2 cells more than 99% if added to cultures at levels of 10^?3M either before or after infection. Other permissive cell lines differed according to the treatment period and drug level required for restricting productive infections. The inhibitory effect of indomethacin was progressively reduced if infection of cells was delayed for increasing times after drug removal. Strong inhibition of viral replication also occurred in cells treated with the cyclooxygenase antagonists naproxen, phenylbutazone, and oxyphenylbutazone whereas phenacetin, which does not block cyclooxygenase function, was inactive. Enhanced viral replication occurred in indomethacin-treated HEp-2 cultures when these cells were subsequently exposed to such substances as prostaglandin El, cyclic AMP, or insulin. Conversely, indomethacin-treated cells remained restrictive for VSV if they were subsequently exposed to metabolic inhibitors of functional DNA (actinomycin D or mitomycin C), messenger RNA synthesis (α-amanitin), or protein synthesis (cycloheximide) at concentrations that normally do not compromise viral replication. Pretreatment of HEp-2 cells with mitomycin C markedly shifted the dose response for indomethacin-mediated inhibition of VSV from a 90% inhibitory dose of about 10^?4 M to one of 10^?9 M or lower. These findings suggest that preexisting host factors essential for replication of VSV, although rendered nonfunctional by the drug indomethacin, can be replenished unless their synthesis is blocked by various classes of metabolic inhibitors.     

Studies on the in vivo methylation of replicating herpes simplex virus type 1 DNA.

Intracellular replicating DNA molecules (g9_CsC1 = 1.725 g/cm³) of herpes simplex virus type 1 (HSV-1) were found to be transiently methylated. Primary rabbit kidney cells infected with HSV-1 (KOS strain) were labeled with l-[methyl-³H]methionine at different times during the virus growth cycle. Viral DNA was deproteinized, separated from cellular DNA by centrifugation in CsCl density gradients, and treated with NaOH to avoid any RNA contamination. Analysis of this intracellular viral DNA indicated that it was maximally methylated during 4–9 hr postinfection and the methylation of viral DNA started to decrease during later stages of infection. DNA from mature virions was not found to be methylated. The methylated base was identified as 5-methylcytosine. Nicotinamide, a potent acceptor of methyl groups, effectively inhibited the production of infectious virus particles at a concentration of 50 mM. These results suggest that methylation of HSV-1 DNA during active viral DNA synthesis is a prerequisite for infectious virus production.     

Sarcoma and transformation-defective viruses produced with infectious DNA(s) from Rous sarcoma virus (RSV)-transformed chicken cells

Chicken cells were transformed with helper-independent avian sarcoma viruses, subgroup C and D, respectively. The DNA extracted from these cells gave rise, in infectivity assays, to both transforming and transformation-defective viruses. Both viruses were recovered with a similar frequency at the end-point dilution (0.05 μg DNA per 10⁷ cells) of infectious DNA. Increasing the DNA concentration had little influence on the probability of successful recovery of a sarcoma virus. On the other hand, the probability of recovering a transformation-defective virus increased when chicken cells received higher concentrations of DNA.Transformation-defective viruses here recovered for the first time after transfection with DNA from RSV-transformed cells were further investigated. Interference assays showed that these transformation-defective viruses interfered with sarcoma viruses recovered with the same DNA or belonging to the same antigenic subgroup as the DNA parent. No interference was encountered between viruses produced with infectious DNAs of antigenically different parents. Interference patterns of a cloned transforming virus suggested that the transforming viral DNA progeny was independent of a helper virus. Both transformation-defective and transforming viruses possessed the same buoyant density of 1.168 g/cm³. Viral RNA of the transformation-defective viruses sedimented slower than that of the transforming virus, the sedimentation coefficients being 65 S and 71 S, respectively. In distinction to the sarcoma virus, a transformation-defective isolate did not give rise to tumors in bioassays. Chicken cells infected with the transformation-defective virus harbored infectious DNA which produced transformation-defective viruses in infectivity assays but was unable to generate a sarcoma virus. A transformation-defective virus isolated in these infections interfered with its sarcomatogenous ancestor.To explain these results it is suggested that RSV-transformed chicken cells may carry two species of infectious DNA belonging to the sarcoma virus and its transformation-defective segregant, respectively. RSV-transformed mammalian cells appear to harbor only sarcoma viral DNA.