Preferential Inhibition of Herpes-Group Viruses by Phosphonoacetic Acid: Effect on Virus DNA Synthesis and Virus-Induced DNA Polymerase Activity

In tissue culture phosphonoacetic acid (PAA) specifically inhibited DNA synthesis of human cytomegalovirus (CMV), murine CMV, simian CMV, Epstein-Barr virus, and Herpesvirus saimiri. Fifty to one hundred micrograms per milliliter PAA completely inhibited viral DNA synthesis with no significant damage to host cell DNA synthesis. In vitro DNA polymerization assays showed that 10 μg/ml of PAA specifically inhibited partially purified human CMV-induced DNA polymerase, while little inhibition of host-cell DNA polymerase activity was found. The specific inhibition of herpes-group virus DNA synthesis with little toxicity to host cells suggests that PAA has great potential as an antiherpesvirus therapeutic agent.     

Lithium chloride restores host protein synthesis in herpes simplex virus-infected endothelial cells

In previous studies we have shown that herpes simplex virus type 1 (HSV-1) infection suppresses host-cell protein synthesis in human endothelial cells (EC). It has been demonstrated that lithium salts prevent viral replication in HSV-1 infected cells. In the present study, we have measured host-cell protein synthesis in HSV-1 infected EC in the presence or absence of 20 and 30 mM LiCl. Although LiCl restored synthesis of almost all host-cell proteins, [35S]methionine incorporation was most pronounced in thrombospondin and plasminogen activator inhibitor 1 and least in fibronectin and type IV collagen. LiCl was more effective at the higher concentration (30 mM) and when the compound was added to the EC culture at the time of infection rather than after adsorption of HSV-1. Synthesis of virus proteins continued in LiCl-treated EC but at a reduced rate. The data suggest that LiCl not only interferes with virus replication, but may also, to some extent, interfere with the virion-associated inhibition of host protein synthesis.     

Effect of cytosine arabinoside on viral-specific protein synthesis in cells infected with herpes simplex virus.

The relationship between viral DNA and protein synthesis during herpes simplex virus type 1 (HSV-1) replication in HeLa cells was examined. Treatment of infected cells with cytosine arabinoside (ara-C), which inhibited the synthesis of HSV-1 DNA beyond the level of detection, markedly affected the types and amounts of viral proteins made in the infected cell. Although early HSV-1 proteins were synthesized normally, there was a rapid decline in total viral protein synthesis beginning 3 to 4 h after infection. This is the time that viral DNA synthesis would normally have been initiated. ara-C also prevented the normal shift from early to late viral protein synthesis. Finally, it was shown that the effect of ara-C on late protein synthesis was dependent upon the time after infection that the drug was added. These results suggest that inhibition of progeny viral DNA synthesis by ara-C prevents the "turning on" of late HSV-1 protein synthesis but allows early translation to be "switched off."     

Mengovirus-Induced Cytopathic Effect in L-Cells: Protective Effect of Interferon

The effect of interferon on mengovirus-induced cytopathic effect (CPE) in L cells, the cut-off of host-cell protein synthesis, and production of mature virus were found to be dependent on the concentration of interferon. CPE and inhibition of host protein synthesis were not affected until the concentration of interferon was increased 100-fold over that required to reduce viral yields by 90%.     

5' sequence of vesicular stomatitis virus N-gene confers selective translation of mRNA.

The infection of cells by vesicular stomatitis virus results in the rapid inhibition of host-cell protein synthesis, but not of viral protein synthesis. To determine if this translational selectivity might be conferred by the viral mRNA, we constructed a plasmid (pUCLN beta-4) containing the 5' end of the viral nucleocapsid (N)-gene, including the ribosome binding site, fused in frame with the gene encoding beta-galactosidase, and compared it to a control plasmid (pMC1924) containing the cellular rabbit beta-globin gene 5' end fused with the beta-galactosidase encoding gene. Both plasmids contained identical promoter and 3' nontranslated regions and expressed similar levels of beta-galactosidase in the indicator cell line 293. In cells transfected with either plasmid, viral infection resulted in a approximately 70% decrease in protein synthesis by five hours. The level of beta-galactosidase from cells transfected with pMC1924 also decreased concomitantly with the decrease in total protein synthesis. However, the level of beta-galactosidase from cells transfected with pUCLN beta-4 was not affected by viral infection. Our data suggest that sequences in the 5' end of the viral mRNA allow for the selective translation of the viral message in the presence of an inhibited translational machinery.     

Restricted replication of human hepatitis A virus in cell culture: intracellular biochemical studies.

When hepatitis A virus was inoculated into Vero cells, virus-specified protein and RNA synthesis was detected. Production of viral protein was detected by electrophoretic analysis in polyacrylamide gels by using a double-label coelectrophoresis and subtraction method which eliminated the contribution of host protein components from the profiles of virus-infected cytoplasm. Eleven virus-specified proteins were detected in the net electrophoretic profiles of hepatitis A virus-infected cells. The molecular weights of these proteins were very similar to those detected in cells infected with poliovirus type 1. Virus-specified protein synthesis could be detected at 3 to 6 h and continued for at least 48 h postinfection, but no significant effect on host-cell macromolecular synthesis was observed. Limited viral RNA replication occurred between 2 and 6 h postinfection. The genomic RNA of hepatitis A virus was extracted and shown to be capable of infecting cells and inducing the same set of proteins as intact virus, indicating that the RNA genome is positive stranded. Progeny virus was never detected in the supernatant fluids of infected cell cultures, and the cells showed no observable cytopathology, even though hepatitis A virus-specific proteins and antigens were being produced. The nature of the defect in the replicative cycle of hepatitis A virus in this system remains unknown.     

Mechanisms of infection with Epstein-Barr virus. I. Viral DNA replication and formation of noninfectious virus particles in superinfected Raji cells.

Human lymphoblastoid Raji cells, which do not produce virus, supported replication of Epstein-Barr virus (EBV) upon superinfection. Early antigen, viral capsid antigen, and virions were produced in Raji cells superinfected with EBV. Viral DNA replicated under complete inhibition of host cell DNA synthesis to the extent that a few micrograms of EBV DNA were recovered from 107 superinfected Raji cells, corresponding to 5,000 viral genomes/cell. Homology of the synthesized viral DNA to parental EBV DNA was more than 90%. Virions produced by the Raji cells contained a 55S DNA but failed to induce early antigen, viral capsid antigen, and viral DNA synthesis after a second superinfection of Raji cells.     

Inhibition of Host-Cell Protein and Ribonucleic Acid Synthesis by Newcastle Disease Virus

The mechanisms of Newcastle disease virus-(NDV) induced inhibition of cell protein and ribonucleic acid (RNA) synthesis were investigated. It was observed that the ability of NDV to inhibit cell RNA synthesis is dependent on the virus strain. The inhibitors, azauridine and cycloheximide, were added to cell cultures at different times after infection to study the roles of protein and RNA synthesis in the viral inhibition process. Viral inhibition of cell RNA synthesis and viral inhibition of cell protein synthesis become resistant to cycloheximide at a different time after infection than that in which they become resistant to azauridine. The results indicate that the inhibition of cell RNA synthesis by the Texas strain involves the synthesis of inhibitory proteins which are coded by the viral genome. The Texas and Beaudette strains of NDV appear to employ different mechanisms for the inhibition of host-cell protein synthesis. Viral inhibition of cell protein synthesis does not appear to cause, or be the result of, viral inhibition of cell RNA synthesis.     

Replication Process of the Parvovirus H-1 III. Factors Affecting H-1 RF DNA Synthesis

Replication of the single-stranded DNA parvovirus H-1 involves the synthesis of a double-stranded DNA replicative form (RF). In this study, the metabolism of RF DNA was examined in parasynchronous hamster embryo cells. The initiation of RF DNA replication was found to occur late in S phase, as was the synthesis of the DNA upon which subsequent viral hemagglutinin synthesis is dependent. Evidence is presented which indicates that initiation of RF replication requires proteins synthesized in late S phase, but that concomittant protein synthesis is not required for the continuation of RF replication. The data also suggest a requirement for viral protein(s) for progeny strand synthesis. Incorporation of 5-bromo-2'-deoxyuridine (BUdR) into viral DNA resulted in an "all-or-none" inhibition of viral hemagglutinin and viral antigen synthesis. BUdR inactivation of viral protein function was used to explore the time of synthesis of viral DNA serving as template for viral RNA synthesis and the effect of viral protein on RF replication and progeny strand synthesis. Results of this study suggest that parental RF DNA is synthesized shortly after infection, and that viral mRNA is transcribed from only a few copies of the viral genome in each cell. They also support the conclusion that viral protein is inhibitory to RF DNA replication. Density labeling of RF DNA with BUdR, allowing separation of viral strand DNA (V) from viral complementary strand (C), provided additional data in support of the above findings.     

Mengovirus Replication in Novikoff Rat Hepatoma and Mouse L Cells: Effects on Synthesis of Host-Cell Macromolecules and Virus-specific Synthesis of Ribonucleic Acid

Novikoff cells (strain N1S1-67) and L-67 cells, a nutritional mutant of the common strain of mouse L cells which grows in the same medium as N1S1-67 cells, were infected with mengovirus under identical experimental conditions. The synthesis of host-cell ribonucleic acid (RNA) by either type of cell was not affected quantitatively or qualitatively until about 2 hr after infection, when viral RNA synthesis rapidly displaced the synthesis of cellular RNA. The rate of synthesis of protein by both types of cells continued at the same rate as in uninfected cells until about 3 hr after infection, and a disintegration of polyribosomes occurred only towards the end of the replicative cycle, between 5 and 6 hr. The time courses and extent of synthesis of single-stranded and double-stranded viral RNA and of the production of virus were very similar in both types of cells, in spite of the fact that the normal rate of RNA synthesis and the growth rate of uninfected N1S1-67 cells are about three times greater than those of L-67 cells. In both cells, the commencement of viral RNA synthesis coincided with the induction of viral RNA polymerase, as measured in cell-free extracts. Viral RNA polymerase activity disappeared from infected L-67 cells during the period of production of mature virus, but there was a secondary increase in activity in both types of cells coincidental with virus-induced disintegration of the host cells. Infected L-67 cells, however, disintegrated and released progeny virus much more slowly than N1S1-67 cells. The two strains of cells also differed in that replication of the same strain of mengovirus was markedly inhibited by treating N1S1-67 cells with actinomycin D prior to infection; the same treatment did not affect replication in L-67 cells.     

Process of Infection with Bacteriophage φX174: XXXVI. Measurement of Virus-Specific Proteins During a Normal Cycle of Infection

Double-labeling techniques in which (14)C-labeled, phiX174-infected cells and (3)H-labeled, uninfected cells were used permitted the identification of the virus-specific proteins after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis without prior inhibition of host-cell protein synthesis by ultraviolet irradiation. It was also possible to detect previously undescribed components of high molecular weight which may represent induced host proteins. The gel regions specifically corresponding to cistron II protein and the chloramphenicol-resistant VI protein were identified, and a third new, small peak of unknown origin was detected. Studies of the rate of synthesis of virus-specific proteins at various times after infection indicated that the product of cistron I (lysis) is made only late in infection, but the other proteins seemed to be synthesized at the same relative rates throughout infection (although in different amounts). Studies of the proteins obtained from uniformly labeled phiX virus particles indicated that all of the spikes are identical and allowed a formulation of the structure of the phage capsid.     

Reovirus: Effect of Noninfective Viral Components on Cellular Deoxyribonucleic Acid Synthesis

We determined the effects of noninfective reovirus components on cellular deoxyribonucleic acid (DNA) synthesis. Reovirus inactivated by ultraviolet light inhibited cellular DNA synthesis, whereas reovirus cores and empty capsids did not. Both cores and empty capsids were adsorbed to cells. Adenine-rich ribonucleic acid (RNA) from reovirus, adsorbed to cells in the presence of diethyl-aminoethyl-dextran, produced a partial inhibition of DNA synthesis. RNA was synthesized in the presence of actinomycin D after infection with ultraviolet light-irradiated reovirus, and this RNA synthesis was not due to multiplicity reactivation of virus infectivity. These data suggest that viral structural proteins do not inhibit DNA synthesis and that the inhibition produced by ultraviolet-irradiated virus may be mediated in part or in toto by a newly synthesized viral product.     

Integration of progeny simian virus 40 DNA into the host cell genome

A procedure was developed for the separation of cellular DNA of productively infected monkey kidney cells from free simian virus 40 DNA. The application of this procedure allowed the investigation of progeny viral DNA integration into the host cell DNA by nucleic acid hybridization techniques. The purification consisted of precipitation of the cellular DNA by Hirt's (1967) method, velocity centrifugation in alkaline sucrose gradients, equilibrium centrifugation in ethidium bromide/CsCl solution, and an additional velocity centrifugation in an alkaline sucrose gradient. The efficiency of each step of the procedure was determined by monitoring the amount of contaminating free viral DNA. Purified cellular DNA, isolated from cells late after infection, contained approximately 0/sd006% free viral DNA, but as much as 2% integrated simian virus 40 DNA. This corresponds to more than 20,000 integrated virus genome equivalents per cell, as determined by DNA-DNA reassociation kinetics. Integration of simian virus 40 DNA into the cellular DNA became detectable at 24 hours after infection, and increased with the increase in the rate of viral DNA synthesis.     

Restricted Replication of Frog Virus 3 in Selected Variants of BHK Cells

Synthesis and maturation of frog virus 3 deoxyribonucleic acid (DNA) in BHK cells and selected variants, in chick fibroblasts, and in minnow cells were compared. Wide ranges in rates of DNA synthesis and assembly of virions were found. At least three variants of BHK cells can be obtained: (i) fully permissive, characterized by rapid DNA synthesis and assembly to give a high yield of infective virus; (ii) semipermissive, in which viral DNA is synthesized slowly for extended periods of time, and the yield of infective virus is poor; and (iii) nonpermissive, in which virus adsorbs to cells and arrests host functions but viral DNA is not synthesized. Kinetics of appearance of virions and procedures for their separation from cell extracts are described.     

Kinetics of Viral Deoxyribonucleic Acid, Protein, and Infectious Particle Production and Alterations in Host Macromolecular Syntheses in Equine Abortion (Herpes) Virus-infected Cells

Infection of exponential-phase suspension cultures of mouse fibroblast cells (L-M) with equine abortion virus (EAV) resulted in inhibition of cell growth and marked alterations in host metabolic processes. The synthesis of deoxyribonucleic acid (DNA) and ribonucleic acid was inhibited within 4 hr after infection and was suppressed by more than 90% by the time of maximal virus replication (14 to 18 hr). The overall rate of protein synthesis, however, was similar in uninfected and virus-producing cells as determined by measurements of net protein and isotope incorporation. The time course of viral DNA and protein synthesis and assembly into mature virus was determined with the inhibitors 5-fluorodeoxyuridine (FUdR) and cycloheximide, respectively. Thus, viral DNA synthesis was essentially completed at 14 hr, and viral protein and infectious virus synthesis was completed at 18 hr. Although the number of plaque-forming units (PFU) produced by FUdR-treated cells (10(3) to 10(4) PFU/ml) was at least 3 logs less than that produced by untreated cells, the yield of physical particles (as determined by electron microscopy) was approximately the same at 30 hr after infection. Besides being relatively non-infective, the particles produced in FUdR-treated cells appeared morphologically incomplete as they contained little or no nucleoid material.     

Inhibitory proteins in the Newcastle disease virus-induced suppression of cell protein synthesis

Bolognesi, D. P. (Rensselaer Polytechnic Institute, Troy, N.Y.), and D. E. Wilson. Inhibitory proteins in the Newcastle disease virus-induced suppression of cell protein synthesis. J. Bacteriol. 91:1896-1901. 1966.-Infection by Newcastle disease virus brings about a rapid and marked inhibition of cell protein synthesis (CPS) in chick embryo fibroblast monolayers. The block to CPS is initiated about 5 hr after infection, and by 9 hr about 85% of the host protein synthesis is shut off. Azauridine (3 mg/ml), a ribonucleic acid (RNA) synthesis inhibitor, prevents the virus-induced inhibition of CPS when added at the time of infection; but it does not prevent the inhibition when added at 3 hr after infection. When puromycin (60 mug/ml), a protein synthesis inhibitor, was added at 3.5 hr after infection, viral RNA was synthesized in normal amounts, but the virus-induced inhibition of CPS was prevented. Actinomycin D added at the time of infection does not, however, prevent the virus-induced inhibition of CPS. The results of these experiments indicate that proteins synthesized during Newcastle disease virus replication are responsible for the inhibition of host-cell protein synthesis. The synthesis of these inhibitory proteins depends on the prior synthesis of viral RNA.     

Phospholipid Synthesis in Sindbis Virus-Infected Cells

We investigated the metabolic requirements for the decrease in phospholipid synthesis previously observed by Pfefferkorn and Hunter in primary cultures of chick embryo fibroblasts infected with Sindbis virus. The incorporation of (32)PO(4) into all classes of phospholipids was found to decline at the same rate and to the same extent; thus, incorporation of (14)C-choline into acid-precipitable form provided a convenient measure of phospholipid synthesis that was used in subsequent experiments. Experiments with temperature-sensitive mutants suggested that some viral ribonucleic acid (RNA) synthesis was essential for the inhibition of choline incorporation, but that functional viral structural proteins were not required. The reduction in phospholipid synthesis was probably a secondary effect of infection resulting from viral inhibition of the cellular RNA and protein synthesis. All three inhibitory effects required about the same amount of viral RNA synthesis; the inhibition of host RNA and protein synthesis began sooner than the decline in phospholipid synthesis; and both actinomycin D and cycloheximide inhibited (14)C-choline incorporation in uninfected cells. In contrast, incorporation of (14)C-choline into BHK-21 cells was not decreased by 10 hr of exposure to actinomycin D and declined only slowly after cycloheximide treatment. Growth of Sindbis virus in BHK cells did not cause the marked stimulation of phospholipid synthesis seen in picornavirus infections of other mammalian cells; however, inhibition was seen only late in infection.