Repair Replication of HeLa Cell Deoxyribonucleic Acid in Cells Infected with Newcastle Disease Virus or Mengovirus or Treated with Puromycin

We examined repair replication of HeLa cell deoxyribonucleic acid (DNA) in cells infected with mengovirus or Newcastle disease virus or treated with puromycin. Cellular DNA was damaged by ultraviolet light and then pulse-labeled with (3)H-thymidine. Autoradiographic analysis of non-S-phase DNA synthesis (repair replication) showed that there was no inhibition of this process at a time when overall cellular DNA synthesis was severely inhibited by either virus infection or puromycin treatment.     

Replication of Simian Virus 40 Deoxyribonucleic Acid: Analysis of the One-Step Growth Cycle

The time course of replication of simian virus 40 deoxyribonucleic acid (DNA) was investigated in growing monolayer cultures of subcloned CV1 cells. At multiplicities of infection of 30 to 60 plaque-forming units (PFU)/cell, first progeny DNA molecules (component 1) were detected by 10 hr after infection. During the following 10 to 12 hr, accumulation of virus DNA proceeded at ever increasing rates, albeit in a non-exponential fashion. The rate of synthesis then remained constant, until approximately the 40th hour postinfection, when DNA replication stopped. Under these conditions, the duration of the virus growth cycle was approximately 50 hr. The time needed for the synthesis of one DNA molecule was found to be approximately 15 min. At multiplicities of infection of 1 or less than 1 PFU/cell, the onset of the linear phase of DNA accumulation was delayed, but the final rate of DNA synthesis was the same, independent of the input multiplicity. This was taken as a proof that templates for the synthesis of viral DNA multiply in the cell during the early phase of replication. However, the probability for every replicated DNA molecule to become in turn replicative decreased constantly during that phase. This could be accounted for by assuming a limited number of replication sites in the infected cell.     

Replication of baboon endogenous virus in human cells. Kinetics of DNA synthesis and integration

For the baboon endogenous virus to infect human cells a specific region on chromosome 6 is required for viral DNA replication and integration. In studying the kinetics of baboon endogenous virus DNA replication we show that linear DNA was synthesized as the predominant species after infection and that unintegrated DNA persisted after many cell passages. Examination of integrated DNA revealed the failure of the virus to integrate at early passages. With continuous replication, however, virus integration was observed, but at multiple sites in the host cell.     

Cytomegalovirus-mediated induction of antisense mRNA expression to UL44 inhibits virus replication in an astrocytoma cell line: identification of an essential gene.

We have used an antisense RNA approach in the analysis of gene function in human cytomegalovirus (HCMV). An astrocytoma cell line (U373-MG) that is permissive for virus replication was permanently transfected with a construct bearing sequence from HCMV UL44 (coding for the major late DNA-binding protein, ppUL44, also known as pp52 or ICP36) in an antisense orientation and under the control of the immediate-early enhancer-promoter element. Upon HCMV infection at a high multiplicity, we found a marked reduction in UL44 protein products (the ICP36 family of proteins) in established cell transfectants and a strong inhibition of virus yield in infected-cell supernatants at two weeks postinfection, while herpes simplex virus replication was not affected. In infected cells, viral DNA replication was strongly inhibited. While gene products such as pUS22 and pUL32 were also inhibited, pUL123 and pUL82 accumulated in the infected cells over time. Our data suggest an essential role for the UL44 family of proteins in HCMV replication and represent a model of virus inhibition by virus-induced antisense RNA synthesis in genetically modified cells.     

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.     

Replication of the gonad-specific virus Hz-2V in Ld652Y cells mimics replication in vivo

A newly discovered, nonoccluded insect virus, known as gonad-specific virus or Hz-2V, was found to replicate differently in two insect cell lines derived from ovarian tissues (Tn-368 cells from Trichoplusia ni and Ld652Y from Lymantria dispar). Differences between these two cell lines were observed in virus plaque forming ability, rate of viral DNA replication, time course of infectious virus production, and the mechanism of virus release from infected cells. Replication of Hz-2V in Ld652Y cells was more productive and more closely resembled in vivo virus replication.     

Homologous Viral Interference in Trout and Atlantic Salmon Cell Cultures Infected with Infectious Pancreatic Necrosis Virus

The occurrence of homologous interference in the replication of infectious pancreatic necrosis virus was demonstrated after successive passages of partially purified virus at high input multiplicities in trout and Atlantic salmon cell cultures. Pretreatment of cell cultures with interfering virus inhibited the replication of homologous standard infectious virus but not unrelated viruses. The ability of infectious pancreatic interfering virus to interfere with homologous virus was abolished with UV irradiation, immune serum, and freeze-thawing.     

Position effects on the timing of replication of chromosomally integrated simian virus 40 molecules in Chinese hamster cells.

Simian virus 40 (SV40) DNA molecules chromosomally integrated at different sites in three Chinese hamster lung fibroblast lines replicated during the middle portion of S phase but not precisely at the same time in all three cell lines. The time of replication was unrelated to the presence of T antigen or to its relative activity in promoting SV40 replication. SV40 sequences and chromosomal DNA sequences adjacent to the SV40 insert in one cell line expressing a temperature-sensitive T antigen showed a T-antigen-independent difference in replication timing from the homologous, allelic locus not linked to SV40. Our results indicate that the timing of replication of these integrated SV40 molecules is dependent upon the site of integration and is not determined by the level of T antigen replication-promoting activity.     

Modulation of glycosylation and transport of viral membrane glycoproteins by a sodium ionophore

Analysis of viral glycoprotein expression on surfaces of monensin- treated cells using a fluorescence-activated cell sorter (FACS) demonstrated that the sodium ionophore completely inhibited the appearance of the vesicular stomatitis virus (VSV) G protein on (Madin- Darby canine kidney) MDCK cell surfaces. In contrast, the expression of the influenza virus hemagglutinin (HA) glycoprotein on the surfaces of MDCK cells was observed to occur at high levels, and the time course of its appearance was not altered by the ionophore. Viral protein synthesis was not inhibited by monensin in either VSV- or influenza virus-infected cells. However, the electrophoretic mobilities of viral glycoproteins were altered, and analysis of pronase-derived glycopeptides by gel filtration indicated that the addition of sialic acid residues to the VSV G protein was impaired in monensin-treated cells. Reduced incorporation of fucose and galactose into influenza virus HA was observed in the presence of the ionophore, but the incompletely processed HA protein was cleaved, transported to the cell surface, and incorporated into budding virus particles. In contrast to the differential effects of monensin on VSV and influenza virus replication previously observed in monolayer cultures of MDCK cells, yields of both viruses were found to be significantly reduced by high concentrations of monensin in suspension cultures, indicating that cellular architecture may play a role in determining the sensitivity of virus replication to the drug. Nigericin, an ionophore that facilitates transport of potassium ions across membranes, blocked the replication of both influenza virus and VSV in MDCK cell monolayers, indicating that the ion specificity of ionophores influences their effect on the replication of enveloped viruses.     

Low dynamic state of viral competition in a chronic avian hepadnavirus infection

The dynamic state of infection of 11 ducks with the duck hepatitis B virus was investigated. Chronic infections were established in newly hatched ducklings by inoculation with a mixture of wild-type virus and a mutant virus with a partial replication defect. As expected, the wild-type virus was rapidly enriched in the virus population during the spread of infection. Enrichment thereafter was correlated with normal growth of the liver, with the average mutant-to-wild-type ratio stabilizing for at least 2 months beyond the time at which the liver mass stabilized. Using experimentally determined growth rates for the mutant and wild-type viruses, we estimated that after the spread of infection, competition between the two virus strains was limited by the amount of replication required to infect new hepatocytes in the growing livers. The results suggest that, in a chronically infected liver, the selection of variants with a replication rate advantage is inefficient and that the emergence of such variants would depend on induced liver cell turnover, such as that occurring during chronic hepatitis.     

Visualization of DNA G-quadruplexes in herpes simplex virus 1-infected cells

We have previously shown that clusters of guanine quadruplex (G4) structures can form in the human herpes simplex-1 (HSV-1) genome. Here we used immunofluorescence and immune-electron microscopy with a G4-specific monoclonal antibody to visualize G4 structures in HSV-1 infected cells. We found that G4 formation and localization within the cells was virus cycle dependent: viral G4s peaked at the time of viral DNA replication in the cell nucleus, moved to the nuclear membrane at the time of virus nuclear egress and were later found in HSV-1 immature virions released from the cell nucleus. Colocalization of G4s with ICP8, a viral DNA processing protein, was observed in viral replication compartments. G4s were lost upon treatment with DNAse and inhibitors of HSV-1 DNA replication. The notable increase in G4s upon HSV-1 infection suggests a key role of these structures in the HSV-1 biology and indicates new targets to control both the lytic and latent infection.     

Neutralizing antibodies to visna lentivirus: mechanism of action and possible role in virus persistence.

Lentiviruses are nononcogenic retroviruses that cause persistent infections and slowly progressive diseases. Visna virus, a lentivirus of sheep, persists in cells of the macrophage lineage despite the presence of neutralizing antibodies in the animal. These antibodies are measured by prevention of virus replication in sheep fibroblast cell cultures. In this study we have compared the antiviral properties of the antibodies in sheep fibroblast and macrophage cell cultures, the latter being more relevant to infection in the animal. Using infectivity assays, binding of radiolabeled virus to cell membranes, cellular processing of labeled virus into acid-precipitable and acid-soluble components, and in situ hybridization of viral nucleic acid, we show that the antibodies prevented virus replication in both fibroblasts and macrophages. However, the site of neutralization differed between the two cell types. In fibroblasts, the site of virus neutralization was at the cell membrane, when the antibodies prevented virus attachment. In macrophages, virus incubated with the antibodies was phagocytized rapidly, followed by uncoating of the virions. However, virus RNA was not transcribed. Despite this ability of the antibodies to abort virus replication in macrophages, the kinetics of binding of the antibodies to the virus was much slower than the binding of virus to the macrophages. Therefore, persistent virus replication in immune sheep may be the result of virus spreading from macrophage to macrophage before the agent can be neutralized by antibodies in the plasma.     

Human Immunodeficiency Virus Vpx Is Required for the Early Phase of Replication in Peripheral Blood Mononuclear Cells

Functional importance of Vpx protein of human immunodeficiency virus type 2 was evaluated in various types of cells. In 8 lymphocytic or monocytic cell lines tested, vpx mutant virus grew as well as wild-type virus. Only in primary peripheral blood mononuclear cell cultures, severely retarded growth of mutant virus was observed. No replication of vpx-minus virus was detected in primary macrophage cells. A highly sensitive single-round replication assay system was used to determine the defective replication phase in primary mononuclear cells of vpx mutant virus. In all cell lines examined, vpx mutant displayed no abnormality. In contrast, the vpx mutant was demonstrated to be defective at an early stage of the infection cycle in primary cell cultures. No evidence of a replication-defect at a late phase in primary cells of the vpx mutant was obtained by a transfection-coculture method. These results indicate that the virion-associated Vpx protein is essential for early viral replication process in natural target cells such as primary macrophages.     

Anti-viral activity of red microalgal polysaccharides against retroviruses

Red microalgal polysaccharides significantly inhibited the production of retroviruses (murine leukemia virus- MuLV) and cell transformation by murine sarcoma virus(MuSV-124) in cell culture. The most effective inhibitory effect of these polysaccharides against both cell transformation and virus production was obtained when the polysaccharide was added 2 h before or at the time of infection. Although, addition of the polysaccharide post-infection significantly reduced the number of transformed cells, but its effect was less marked than that obtained when the polysaccharide was added before or at the time of infection.The finding that the inhibition of cell transformation by MuSV-124 was reversible after removal of the polysaccharide suggested that microalgal polysaccharides inhibited a late step after provirus integration into the host genome. In conclusion, our findings could support the possibility that the polysaccharide may affect early steps in the virus replication cycle, such as virus absorption into the host cells, in addition to its effect on a late step after provirus integration.     

Replication timing control can be maintained in extrachromosomally amplified genes.

Extrachromosomal elements are common early intermediates of gene amplification in vivo and in cell culture. The time at which several extrachromosomal elements replicate was compared with that of the corresponding amplified or unamplified chromosomal sequences. The replication timing analysis employed a retroactive synchrony method in which fluorescence-activated cell sorting was used to obtain cells at different stages of the cell cycle. Extrachromosomally amplified Syrian hamster CAD genes (CAD is an acronym for the single gene which encodes the trifunctional protein which catalyzes the first three steps of uridine biosynthesis) replicated in a narrow window of early S-phase which was approximately the same as that of chromosomally amplified CAD genes. Similarly, extrachromosomally amplified mouse adenosine deaminase genes replicated at a discrete time in early S-phase which approximated the replication time of the unamplified adenosine deaminase gene. In contrast, the multicopy extrachromosomal Epstein-Barr virus genome replicated within a narrow window in late S-phase in latently infected human Rajii cells. The data indicate that localization within a chromosome is not required for the maintenance of replication timing control.     

Evaluation of Factors Related to Growth of Rift Valley Fever Virus in Suspended Cell Cultures

The effect of several controlled variables on the peak titer and fold increase of Rift Valley fever virus grown in suspension culture on two variants of Earle's L cell, L-DR and L-MA clone 1-1, was studied. No significant amount of cell-associated virus was found at 24 hr, indicating a release of virus soon after its formation. Mild sonic treatment of the virus produced in serum-free medium increased the infective titer about 10x. This difference was not observed with virus produced in medium supplemented with serum. Peak titer was not affected by medium used during the infection period, by multiplicity of inoculum (MOI), or by initial cell concentration within the test range of 10(4) to 2 x 10(6) cell/ml. Cell strain employed influenced titer, because the L-DR cell did not produce virus efficiently at low MOI and low initial cell concentration. The time of peak titer and fold replication was dependent on MOI and initial cell concentration. Differences in virus propagation in monolayer and suspension systems are discussed.     

Axin expression delays herpes simplex virus‐induced autophagy and enhances viral replication in L929 cells

Axin, a negative regulator of the Wnt signaling pathway, plays a critical role in various cellular events including cell proliferation and cell death. Axin‐regulated cell death affects multiple processes, including viral replication. For example, axin expression suppresses herpes simplex virus (HSV)‐induced necrotic cell death and enhances viral replication. Based on these observations, this study investigated the involvement of autophagy in regulation of HSV replication and found axin expression inhibits autophagy‐mediated suppression of viral replication in L929 cells. HSV infection induced autophagy in a time‐ and viral dose‐dependent manner in control L929 cells (L‐EV), whereas virus‐induced autophagy was delayed in axin‐expressing L929 cells (L‐axin). Subsequent analysis showed that induction of autophagy by rapamycin reduced HSV replication, and that inhibiting autophagy by 3‐methyladenine (3MA) and beclin‐1 knockdown facilitated viral replication in L‐EV cells. In addition, preventing autophagy with 3MA suppressed virus‐induced cytotoxicity in L‐EV cells. In contrast, HSV replication in L‐axin cells was resistant to changes in autophagy. These results suggest that axin expression may render L929 cells resistant to HSV‐infection induced autophagy, leading to enhanced viral replication.     

Cytomegalovirus Replication in Cells Pretreated with 5-Iodo-2′-Deoxyuridine

The replication of human cytomegalovirus (CMV) in cells pretreated with 5-iodo-2′-deoxyuridine (IUdR) was studied. Pretreatment of cells with IUdR enhanced several parameters of virus replication. Virus grown in drug-treated cells exhibited a shorter eclipse period and the cells produced more infectious virus sooner than did untreated cells. There was an approximate fivefold increase in virus yield per cell in the drug-treated samples when compared to control cultures. The time required for plaque development was shortened by 6 days in drug-treated cultures. Pretreatment of cells with IUdR also increased plaquing efficiency of the virus by approximately 10-fold. The enhancement of virus replication by IUdR was further demonstrated by varying the multiplicity of infection. In a 7-day period there was a 100-fold increase in sensitivity of the cultures for virus detection when the cells had been previously exposed to IUdR. The data presented indicate the possibility that IUdR interferes with the production of a cellular product inhibitory for CMV replication.