Polyamine depletion of cells reduces the infectivity of herpes simplex virus but not the infectivity of Sindbis virus

The effect of polyamines on the viral growth was examined using cell strains that could be effectively depleted of polyamines. In order to avoid the polyamines present in serum we used a polyamine auxotrophic Chinese hamster ovary cell line P22 growing in serum-free medium and Vero cells growing in low serum medium. The final yield of an enveloped RNA virus, Sindbis, in P22 cells was not decreased by depletion of cellular polyamines although the onset of the viral replication was delayed. In contrast the final yield of an enveloped DNA virus, Herpes simplex virus (HSV), was considerably reduced in Vero cells, depleted of polyamines by alpha-difluoromethylornithine, an inhibitor of polyamine synthesis. However, the number of HSV particles detected by electronmicroscopy was not decreased. Southern blot analysis of HSV-DNA from the polyamine depleted and the control cells showed changes in the relative abundance of the DNA fragments suggesting that impairment in DNA synthesis may have caused the decreased infectivity of HSV.     

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.     

Adeno-associated virus rep protein synthesis during productive infection.

Adeno-associated virus (AAV) Rep proteins mediate viral DNA replication and can regulate expression from AAV genes. We studied the kinetics of synthesis of the four Rep proteins, Rep78, Rep68, Rep52, and Rep40, during infection of human 293 or KB cells with AAV and helper adenovirus by in vivo labeling with [35S]methionine, immunoprecipitation, and immunoblotting analyses. Rep78 and Rep52 were readily detected concomitantly with detection of viral monomer duplex DNA replicating about 10 to 12 h after infection, and Rep68 and Rep40 were detected 2 h later. Rep78 and Rep52 were more abundant than Rep68 and Rep40 owing to a higher synthesis rate throughout the infectious cycle. In some experiments, very low levels of Rep78 could be detected as early as 4 h after infection. The synthesis rates of Rep proteins were maximal between 14 and 24 h and then decreased later after infection. Isotopic pulse-chase experiments showed that each of the Rep proteins was synthesized independently and was stable for at least 15 h. A slower-migrating, modified form of Rep78 was identified late after infection. AAV capsid protein synthesis was detected at 10 to 12 h after infection and also exhibited synthesis kinetics similar to those of the Rep proteins. AAV DNA replication showed at least two clearly defined stages. Bulk duplex replicating DNA accumulation began around 10 to 12 h and reached a maximum level at about 20 h when Rep and capsid protein synthesis was maximal. Progeny single-stranded DNA accumulation began about 12 to 13 h, but most of this DNA accumulated after 24 h when Rep and capsid protein synthesis had decreased.     

Photodynamic treatment of herpes simplex virus during its replicative cycle.

Photodynamic treatment of herpes simplex virus type 1-infected hamster embryo fibroblasts (LSH strain) with a low concentration of proflavine (0.08 mug/10(5) cells per ml), a 3-9-diamine acridine dye, inhibited production not only of infectious progeny but also of virion particles. However, there was no appreciable inhibition of viral or cellular DNA synthesis, even when the infected cells were repeatedly exposed to this low concentration of dye and light during the replication cycle of the virus. It thus appears that photodynamic treatment of infected cells interferes with the processes involved in virus maturation.     

Productive replication of adeno-associated virus can occur in human papillomavirus type 16 (HPV-16) episome-containing keratinocytes and is augmented by the HPV-16 E2 protein

We used a sensitive assay to test whether an adeno-associated virus (AAV) productive replication cycle can occur in immortalized human keratinocytes carrying episomal human papillomavirus type 16 (HPV-16) DNA. Following transfection with cloned AAV DNA, infectious AAV was produced, and the infectivity was blocked by anti-AAV antiserum. The HPV-16 E2 protein substantially increased the yield of AAV. Other HPV early proteins did not, in our experiments, show this ability. E2 has been shown to be able to affect p53 levels and to block cell cycle progression at mitosis. We tested the effect of changes in p53 expression on AAV replication and found that large differences in the level of p53 did not alter AAV DNA replication. In extension of this, we found that cellular help for AAV in response to stress was also independent of p53. To test if a mitotic block could trigger AAV DNA replication, we treated the cells with the mitotic inhibitor nocodazole. AAV DNA replication was stimulated by the presence of nocodazole in these and a number of other cell types tested. Yields of infectious virus, however, were not increased by this treatment. We conclude that the HPV-16 E2 protein stimulates AAV multiplication in these cells and propose that this occurs independently of the effects of E2 on p53 and cell cycle progression. Since the effect of E2 was not seen in keratinocytes lacking the HPV-16 episome, we suggest that E2 can help AAV by working in concert with other HPV-16 proteins.     

Fv-1 host cell restriction of friend leukemia virus: microinjection of unintegrated viral DNA

The murine gene Fv-1 has been shown to exert a major influence over the replication of ecotropic murine leukemia viruses. Studies of the replication of Friend murine leukemia virus have shown that the restriction of viral replication occurs intracellularly after the initiation of viral DNA synthesis. The precise mechanism of the block imposed by the Fv-1 gene product is not completely understood. Our studies of Fv-1 restrictive infection have shown a variable decrease in the accumulation of intracellular unintegrated form I viral DNA. Analysis by microinjection of the viral DNA formed in nonpermissively infected BALB/c cells indicates that this DNA is infectious. These studies indicate that the form I DNA accumulated in nonpermissively infected BALB/c cells contains the complete viral sequences necessary for the production of viral progeny, and therefore, they suggest that the Fv-1 host restrictive mechanism recognizes viral factors other than form I DNA alone. These results support the possibility that Fv-1 host restriction occurs after formation of infectious viral DNA, perhaps at the integration step itself.     

Infectious linear DNA sequences replicating in simian virus 40-infected cells.

A new class of linear duplex DNA structures that contain simian virus 40 (SV40) DNA sequences and that are replicated during productive infection of cells with SV40 is described. These structures comprise up to 35% of the radioactively labeled DNA molecules that can be isolated by selective extraction. These molecules represent a unique size class corresponding to the length of an open SV40 DNA molecule (FO III), and they contain a heterogeneous population of DNA sequences either of host or of viral origin, as shown by restriction endonuclease analysis and nucleic acid hybridization. Part of the FO III DNA molecules contain viral-host DNA sequences covalently linked with each other. They start to replicate with the onset of SV40 superhelix replication 1 day after infection. Their rate of synthesis is most pronounced 3 days after infection when superhelix replication is already declining. Furthermore, they cannot be chased into other structures. At least a fraction of these molecules is infectious when administered together with DEAE-dextran to permissive cells. After intracellular circularization, superhelical DNA FO I with an aberrant cleavage pattern accumulates. In addition, tumor and viral capsid antigen are induced, and infectious viral progeny is obtained. Infection of cells with purified SV40 FO I DNA does not result in FO III DNA molecules in the infected cells or in the viral progeny. It is suggested, therefore, that these FO III DNA molecules are perpetuated within SV40 virus pools by encapsidation into pseudovirions.     

Latent infection of KB cells with adeno-associated virus type 2.

Adeno-associated virus (AAV) is a prevalent human virus whose replication requires factors provided by a coinfecting helper virus. AAV can establish latent infections in vitro by integration of the AAV genome into cellular DNA. To study the process of integration as well as the rescue of AAV replication in latently infected cells after superinfection with a helper virus, we established a panel of independently derived latently infected cell clones. KB cells were infected with a high multiplicity of AAV in the absence of helper virus, cloned, and passaged to dilute out input AAV genomes. AAV DNA replication and protein synthesis were rescued from more than 10% of the KB cell clones after superinfection with adenovirus type 5 (Ad5) or herpes simplex virus types 1 or 2. In the absence of helper virus, there was no detectable expression of AAV-specific RNA or proteins in the latently infected cell clones. Ad5 superinfection also resulted in the production of infectious AAV in most cases. All mutant adenoviruses tested that were able to help AAV DNA replication in a coinfection were also able to rescue AAV from the latently infected cells, although one mutant, Ad5hr6, was less efficient at AAV rescue. Analysis of high-molecular-weight cellular DNA indicated that AAV sequences were integrated into the cell genome. The restriction enzyme digestion patterns of the cellular DNA were consistent with colinear integration of the AAV genome, with the viral termini present at the cell-virus junction. In addition, many of the cell lines appeared to contain head-to-tail concatemers of the AAV genome. The understanding of the integration of AAV DNA is increasingly important since AAV-based vectors have many advantages for gene transduction in vitro and in vivo.     

Mechanisms of human immunodeficiency virus type 1 inhibition by hydroxyurea

Virus life cycles depend on cellular factors. Therefore, targeting cellular in combination with viral enzymes could be an effective control in virus replication. In contrast to viral proteins, cellular proteins are not prone to mutations; therefore, viral escape is not expected from drugs inhibiting cellular factors. Hydroxyurea inhibits the cellular enzyme ribonucleotide reductase, thus reducing DNA synthesis. Furthermore, this drug potentiates the activity of nucleoside analogues, inhibits the escape of A-analogue resistant mutants, and increases the phosphorylation of T-analogues. Besides its antiviral activity, hydroxyurea effects the immune system by decreasing immune activation, inhibiting the expansion of CD8 cells and the depletion of CD4 cells. Hydroxyurea has been used in medicine for 40 years, is well tolerated, and it is the least expensive available anti-HIV-1 drug. These characteristics make hydroxyurea a primary candidate for use in combination therapies for the treatment of HIV-1 infection.     

Genetic analysis of adeno-associated virus: properties of deletion mutants constructed in vitro and evidence for an adeno-associated virus replication function.

Transfection of a pBR322-based, recombinant plasmid, pAV2, containing the entire adeno-associated virus (AAV) type 2 genome into human 293 cells in the presence of helper adenovirus resulted in rescue and replication of AAV to yield infectious particles. We constructed mutants of pAV2 containing deletions within the AAV sequence. We describe here the phenotypes of these AAV deletion mutants. The results can be summarized as follows. Mutants (cap-) with deletions between map positions 53 and 85 did not synthesize capsid antigen or progeny single-stranded DNA but accumulated normal levels of duplex replicating form DNA. Mutants (rep-) with deletions between map positions 17 and 36 failed to rescue or replicate any AAV DNA. The rep- mutants could be complemented for replicating form DNA synthesis by a cap- mutant. This clearly demonstrates an AAV-coded replication function which is different from the capsid antigen. Other mutants (inf-) with deletions in the region between map positions 40 and 52 synthesized abundant amounts of replicating form DNA and capsid antigen but gave a low yield of infectious particles. This suggests that there may be an additional region of AAV, perhaps within the intron, which is required for efficient particle assembly. This work shows that AAV is genetically complex and expresses at least three clearly different functions.     

Genetics of adeno-associated virus: isolation and preliminary characterization of adeno-associated virus type 2 mutants.

We constructed insertion and deletion mutants with mutations within the adeno-associated virus (AAV) sequences of the infectious recombinant plasmid pSM620. Studies of these mutants revealed at least three AAV phenotypes. Mutants with mutations between 11 and 42 map units were partially or completely defective for rescue and replication of the AAV sequences from the recombinant plasmids (rep mutants). The mutants could be complemented by mutants with replication-positive phenotypes. The protein(s) that is affected in rep mutants has not been identified, but the existence of the rep mutants proves that at least one AAV-coded protein is required for viral DNA replication. Also, the fact that one of the rep mutant mutations maps within the AAV intron suggests that the intron sequences code for part of a functional AAV protein. Mutants with mutations between 63 and 91 map units synthesized normal amounts of AAV duplex DNA but could not generate single-stranded virion DNA (cap mutants). The cap phenotype could be complemented by rep mutants and is probably due to a defect in the major AAV capsid protein, VP3. This suggests that a preformed capsid or precursor is required for the accumulation of single-stranded AAV progeny DNA. Mutants with mutations between 48 and 55 map units synthesized normal amounts of AAV single-stranded and duplex DNA but produced substantially lower yields of infectious virus particles than wild-type AAV (lip mutants). The lip phenotype is probably due to a defect in the minor capsid protein, VPI, and suggests the existence of an additional (as yet undiscovered) AAV mRNA. Evidence is also presented for recombination between mutant AAV genomes during lytic growth.     

Adeno-associated virus replication. The effect of L-canavanine or a helper virus mutation on accumulation of viral capsids and progeny single-stranded DNA

We have studied the relationship between adeno-associated virus (AAV) DNA replication and virus particle assembly. Formation of empty or full particles and accumulation of AAV capsid proteins was prevented in the presence of the arginine analogue, L-canavanine, or when a temperature-sensitive helper adenovirus was used at the nonpermissive temperature. In each case there was a concomitant inhibition of AAV single-stranded (progeny) DNA accumulation but little or no effect upon synthesis of AAV duplex, replicating form DNA. These results indicate that AAV protein, perhaps in the form of assembled capsids, is required for AAV single-stranded progeny DNA accumulation.     

Isolation and characterization of a Chinese hamster ovary mutant cell line with altered sensitivity to vaccinia virus killing.

The Chinese hamster ovary (CHO) cell line is nonpermissive for vaccinia virus, and translation of viral intermediate genes was reported to be blocked (A. Ramsey-Ewing and B. Moss, Virology 206:984-993, 1995). However, cells are readily killed by vaccinia virus. A vaccinia virus-resistant CHO mutant, VV5-4, was isolated by retroviral insertional mutagenesis. Parental CHO cells, upon infection with vaccinia virus, die within 2 to 3 days, whereas VV5-4 cells preferentially survive this cytotoxic effect. The survival phenotype of VV5-4 is partial and in inverse correlation with the multiplicity of infection used. In addition, viral infection fails to shut off host protein synthesis in VV5-4. VV5-4 was used to study the relationship of progression of the virus life cycle and cell fate. We found that in parental CHO cells, vaccinia virus proceeds through expression of viral early genes, uncoating, viral DNA replication, and expression of intermediate and late promoters. In contrast, we detect only expression of early genes and uncoating in VV5-4 cells, whereas viral DNA replication appears to be blocked. Consistent with the cascade regulation model of viral gene expression, we detect little intermediate- and late-gene expression in VV5-4 cells. Since vaccinia virus is known to be cytolytic, isolation of this mutant therefore demonstrates a new mode of the cellular microenvironment that affects progression of the virus life cycle, resulting in a different cell fate. This process appears to be mediated by a general mechanism, since VV5-4 is also resistant to Shope fibroma virus and myxoma virus killing. On the other hand, VV5-4 remains sensitive to cowpox virus killing. To examine the mechanism of VV5-4 survival, we investigated whether apoptosis is involved. DNA laddering and staining of apoptotic nuclei with Hoechst 33258 were observed in both CHO and VV5-4 cells infected with vaccinia virus. We concluded that the cellular pathway, which blocks viral DNA replication and allows VV5-4 to survive, is independent of apoptosis. This mutant also provides evidence that an inductive signal for apoptosis upon vaccinia virus infection occurs prior to viral DNA replication.     

Host range restriction of vaccinia virus in Chinese hamster ovary cells: relationship to shutoff of protein synthesis.

Chinese hamster ovary cells were found to be nonpermissive for vaccinia virus. Although early virus-induced events occurred in these cells (RNA and polypeptide synthesis), subsequent events appeared to be prevented by a very rapid and nonselective shutoff of protein synthesis. Within less than 2 h after infection, both host and viral protein syntheses were arrested. At low multiplicities of infection, inhibition of RNA synthesis with cordycepin resulted in failure of the virus to block protein synthesis. Moreover, infection of the cells in the presence of cycloheximide prevented the immediate onset of shutoff after reversal of cycloheximide. Inactivation of virus particles by UV irradiation also impaired the capacity of the virus to inhibit protein synthesis. These results suggested that an early vaccinia virus-coded product was implicated in the shutoff of protein synthesis. Either the nonpermissive Chinese hamster ovary cells were more sensitive to this inhibition than permissive cells, or a regulatory control of the vaccinia shutoff function was defective.