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Sendai virus defective interfering nucleocapsids, isolated from infected cell cytoplasm by equilibrium banding in CsCl gradients, contain only the viral N protein. Neither end of the genomic RNA within these nucleocapsids is accessible to RNase digestion.
Hamster embryo fibroblasts persistently infected with a derivative of the Schwarz vaccine strain of measles virus spontaneously released virus particles with an average buoyant density considerably lower than that of the parental virus. The released virus contained all of the measles virus structural proteins and interfered with replication of standard virus. All of the virus structural proteins were associated with a membrane-free cytoplasmic extract from the persistently infected cells. Membrane-free cytoplasmic extracts prepared from Vero cells lytically infected with Schwarz strain measles contained little or no virus envelope structural protein. Maintenance of persistent infection may involve both the presence of virus variants and a defect in the ability of the infected cell to replicate the virus efficiently.
We have established a persistent infection of BHK cells with a preparation of Sindbis virus heavily enriched in defective interfering (DI) particles. The small fraction of cells that survived the initial infection grew out to form a stable population of cells [BHK(Sin-1) cells], most of which synthesized viral RNA and viral antigens. The presence of DI particles in this virus stock was required to establish this persistent state. BHK(Sin-1) cells released a small-plaque, temperature-sensitive virus (Sin-1 virus) as well as DI particles containing DI RNAs larger than those present in the original stock used to establish the persistent state. A cloned stock of Sin-1 virus, free of detectable DI particles, was able to initiate a persistent infection more quickly and with greater cell survival than the original stock of Sindbis virus containing DI particles. About 2 weeks after the Sin-1 virus-infected cells were cultured, DI RNAs arose and soon became the dominant viral RNA species produced by these cells.
The results of experiments investigating T1 macromolecular synthesis under N-mediated excluding conditions failed to demonstrate a substantial alteration in the T1 mRNA production in excluding cultures at any stage in the T1 infectious cycle. The number of T1 DNA sequences in the excluding culture was found to be one-third to one-half that found in T1-infected cultures. The most severe reduction in T1-specific macromolecules was seen in protein synthesis. Total incorporation of labeled amino acids was reduced sixfold, and gel experiments confirmed that the T1-specific proteins capable of detection are reduced in excluding cells.
Infection of a chicken cell with avian sarcoma virus requires division of the infected cell before synthesis of infectious progeny is initiated. This requirement for a cell division for the complete expression of avian sarcoma virus has been examined further with chicken embryo fibroblasts infected with two distinct viruses. Chicken cells infected with and producing a mutant of Rous sarcoma virus temperature sensitive for transformation (tsLA24PR-A) were arrested in G0 by depletion of serum factors from growth medium. These stationary cells continued to produce infectious progeny in the absence of further cell division. Superinfection of the stationary cells with the wild-type Prague strain of Rous sarcoma virus (PR-RSV-C) produced a stable double infection in these cells. Progeny of the superinfecting PR-RSV-C, however, were not detected until these cells underwent division after stimulation with fresh serum-containing medium. The addition of colchicine to these serum-stimulated cells, although not affecting production of the tsLA24PR-A, inhibited the appearance of progeny of the superinfecting PR-RSV-C. These experiments indicate that each avian sarcoma virus infection of a chicken embryo fibroblast requires division of the infected cell for production of that virus regardless of whether or not the cell is already producing a similar virus. The results suggest, therefore, that the requirement for a cell division represents a requirement for an event that controls virus expression in a "cis-acting" fashion specific for the provirus.
Bunyamwera virus replication was examined in Aedes albopictus (mosquito) cell cultures in which a persistent infection is established and in cytopathically infected BHK cells. During primary infection of A. albopictus cells, Bunyamwera virus reached relatively high titers ( approximately 10(7) PFU/ml), and autointerference was not observed. Three virus-specific RNAs (L, M, and S) and two virion proteins (N and G1) were detected in infected cells. Maximum rates of viral RNA synthesis and viral protein synthesis were extremely low, corresponding to <2% of the synthetic capacities of uninfected control cells. Viral protein synthesis was maximal at 12 h postinfection and was shut down to barely detectable levels at 24 h postinfection. Virus-specific RNA and nucleocapsid syntheses showed similar patterns of change, but later in infection. The proportions of cells able to release a single PFU at 3, 6, and 54 days postinfection were 100, 50, and 1.5%, respectively. Titers fell to 10(3) to 10(5) PFU/ml in carrier cultures. Persistently infected cultures were resistant to superinfection with homologous virus but not with heterologous virus. No changes in host cell protein synthesis or other cytopathic effects were observed at any stage of infection. Small-plaque variants of Bunyamwera virus appeared at approximately 7 days postinfection and increased gradually until they were 75 to 95% of the total infectious virus at 66 days postinfection. Temperature-sensitive mutants appeared between 23 and 49 days postinfection. No antiviral activity similar to that reported in A. albopictus cell cultures persistently infected with Sindbis virus (R. Riedel and D. T. Brown, J. Virol. 29: 51-60, 1979) was detected in culture fluids by 3 months after infection. Bunyamwera virus replicated more rapidly in BHK cells than in mosquito cells but reached lower titers. Autointerference occurred at multiplicities of infection of approximately 10. Virus-specific RNA and protein syntheses were at least 20% of the levels in uninfected control cells. Host cell protein synthesis was completely shut down, and nucleocapsid protein accumulated until it was 4% of the total cell protein. We discuss these results in relation to possible mechanisms involved in determining the outcome of arbovirus infection of vertebrate and mosquito cells.
We have investigated some of the biochemical events that accompany the abortive infection by T2 of Shigella dysenteriae lysogenized with the temperate phage P2. After infection with T2, protein and RNA synthesis continued for 3 to 5 min. The virus-induced enzyme, deoxycytidylate hydroxymethylase was produced in reduced amounts (15% of normal), and the extent of deoxyribonucleic acid (DNA) synthesis was 0.1% of that found with a nonlysogenic strain. Measurements of the production of acid-soluble fragments and sedimentation analyses failed to detect enzymatic degradation of the infecting viral DNA which could be specifically related to the presence of the prophage P2. Each interaction between T2 and a bacterium resulted in the death of the cell. This observation is consistent with results obtained with other types of bacteria which show that only when a nucleolytic attack occurs on T2 DNA does the cell have an increased capacity to survive after adsorption of T2.
Wild-type beta and gamma corynebacteriophages are heteroimmune and infect lysogens of each other productively. Unlike their wild-type counterparts, the bin mutants of each phage are excluded in lysogens carrying the heteroimmune phage. The wild-type phages overcome exclusion by means of the bin gene product which appears to act as an antirepressor. When repression is lifted, exclusion of bin mutants is abolished (N. Groman and M. Rabin, J. Virol. 28:28-33, 1978; J. Virol. 36:526-532, 1980). It has not been clear whether the excluding compound is the immune repressor itself or one whose synthesis is positively regulated by repressor. We have isolated beta exclusion mutants (xcl) that as prophage exhibited normal immune repression but no longer excluded gamma-bin mutants. Furthermore, we have shown that an xcl phage with an active immune repressor acted in trans to continue the positive regulation of exclusion by a second xcl+ prophage whose immune repressor was inactivated. From these results it was concluded that there is a gene distinct from the imm gene which is directly or indirectly responsible for exclusion. The xcl gene, mapped in prophage crosses, was located between imm and bin, that is, in the regulatory region of the phage genome. The simplest hypothesis compatible with the established observations is that beta immune repressor regulates the expression of the xcl and bin genes, the former positively and the latter negatively. It is likely that an analogous regulatory model applies to gamma phage since it has already been shown that both beta and gamma have bin alleles.
A new variant of simian virus 40 (EL SV40), containing the complete viral DNA separated into two molecules, was isolated. One DNA species contains nearly all of the early (E) SV40 sequences, and the other DNA contains nearly all of the late (L) viral sequences. Each genome was encircled by reiterated viral origins and termini and migrated in agarose gels as covalently closed supercoiled circles. EL SV40 or its progenitor appears to have been generated in human A172 glioblastoma cells, as defective interfering genomes during acute lytic infections, but was selected during the establishment of persistently infected (PI) green monkey cells (TC-7). PI TC-7/SV40 cells contained EL SV40 as the predominant SV40 species. EL SV40 propagated efficiently and rapidly in BSC-1, another line of green monkey cells, where it also formed plaques. EL SV40 stocks generated in BSC-1 cells were shown to be free of wild-type SV40 by a number of criteria. E and L SV40 genomes were also cloned in the bacterial plasmid pBR322. When transfected into BSC-1 cell monolayers, only the combination of E and L genomes produced a lytic infection, followed by the synthesis of EL SV40. However, transfection with E SV40 DNA alone did produce T-antigen, although at reduced frequency.
A stock of plaque-purified Pichinde virus, prepared under conditions designed to limit the amounts of defective interfering virus, was used to infect BHK cells. At daily intervals after infection, cells were examined for infectious and radiolabeled virus particle production and for the synthesis of virus-specific polypeptides. Quantitative comparisons were also made of the concentrations of genomic Pichinde virus L and S RNAs in the cytoplasm of infected cells on different days after infection. Our results showed that virus particle production, rates of protein synthesis, and the intracellular levels of viral genomic RNAs all increased and decreased with similar kinetics, and that this regulation was independent of the cell growth cycle. We were unable to relate these changes in viral macromolecule and virus production to the appearance of readily identifiable defective interfering particles. Our findings suggest that regulation of virus replication early during the replicative cycle of Pichinde virus may not be dependent upon the generation of defective interfering virus.
The replication of the RNA of vesicular stomatitis virus (VSV) defective interfering (DI) particles was established in a defined cell-free system. The transition from synthesis of only the DI-leader RNA to replication of the full-length DI RNA was effected in the system by newly synthesized VSV proteins and occurred in the absence of VSV helper virus. Both positive- and negative-polarity full-length DI RNA were synthesized. Furthermore, the products of RNA replication associated with newly synthesized viral proteins to form complexes that were indistinguishable from authentic DI particle nucleocapsids on the basis of buoyant density and resistance to ribonuclease digestion. The DI-leader RNA did not form ribonuclease-resistant structures. We conclude that this in vitro system successfully executes many of the reactions of VSV DI particle replication and assembly.
An antigenic determinant capable of inducing type-common herpes simplex virus (HSV)-neutralizing antibodies has been located on glycoprotein D (gD) of HSV type 1 (HSV-1). A peptide of 16 amino acids corresponding to residues 8 to 23 of the mature glycoprotein (residues 33 to 48 of the predicted gD-1 sequence) was synthesized. This peptide reacted with an anti-gD monoclonal antibody (group VII) previously shown to neutralize the infectivity of HSV-1 and HSV-2. The peptide was also recognized by polyclonal antibodies prepared against purified gD-1 but was less reactive with anti-gD-2 sera. Sera from animals immunized with the synthetic peptide reacted with native gD and neutralized both HSV-1 and HSV-2.
The intracellular defective RNAs generated during high-multiplicity serial passages of mouse hepatitis virus JHM strain on DBT cells were examined. Seven novel species of single-stranded polyadenylic acid-containing defective RNAs were identified from passages 3 through 22. The largest of these RNAs, DIssA (molecular weight [mw], 5.2 X 10(6)), is identical to the genomic RNA packaged in the defective interfering particles produced from these cells. Other RNA species, DIssB1 (mw, 1.9 X 10(6) to 1.6 X 10(6)), DIssB2 (mw, 1.6 X 10(6)), DIssC (mw, 2.8 X 10(6)) DIssD (mw, 0.82 X 10(6)), DIssE (mw, 0.78 X 10(6)), and DIssF (mw, 1.3 X 10(6)) were detected at different passage levels. RNase T1-resistant oligonucleotide fingerprinting demonstrated that all these RNAs were related and had multiple deletions of the genomic sequences. They contained different subsets of the genomic sequences from those of the standard intracellular mRNAs of nondefective mouse hepatitis virus JHM strain. Thus these novel intracellular viral RNAs were identified as defective interfering RNAs of mouse hepatitis virus JHM strain. The synthesis of six of the seven normal mRNA species specific to mouse hepatitis virus JHM strain was completely inhibited when cells were infected with viruses of late-passage levels. However, the synthesis of RNA7 and its product, viral nucleoprotein, was not significantly altered in late passages. The possible mechanism for the generation of defective interfering RNAs was discussed.
To determine whether defective interfering (DI) particles modulate virulence by initiating a cyclic pattern of virus growth in vivo, adult mice were infected with vesicular stomatitis virus (VSV), both with and without DI particles. A total of 184 mice divided into groups were inoculated intranasally. A majority of mice inoculated only with standard VSV developed paralysis, most of them between days 7 and 9. The addition of DI particles altered the development of paralysis in several ways. When there was significant protection, a few still became paralyzed on days 7 and 9. When overall mortality was unaffected or even slightly increased, the majority of mice became paralyzed between days 7 and 9 as well. Protection could not be predicted based on a single ratio of standard VSV to DI particles or on the absolute amount of DI particles inoculated. Infectious virus recovered from mouse brains at the time of paralysis and incipient death showed considerable variation, although the titer in a majority of the animals was between 10(5) and 10(7) PFU/ml. When the brains of these paralyzed mice were examined for hybridizable VSV RNA, the detection of standard VSV RNA correlated well with infectivity. The amount of DI RNA in the coinfected mice was more variable and independent of the amount of 40S RNA, although DI RNA was usually found when standard RNA was present. Survivors examined between days 14 and 21 did not contain infectious virus or any detectable viral RNA in their brains. Because these results were consistent with the hypothesis of viral cycling in vivo, rather than a gradual accumulation of total infectious virus, mice were coinfected with 10(8) PFU of standard VSV and 10(5) PFU equivalents of DI particles and sacrificed daily thereafter, irrespective of whether they developed paralysis. Infectivity measurements indicated a reproducible cycling pattern of VSV in the mouse brains with a periodicity of about 5 days. This cycling and the detection of DI RNA in brains several days after intranasal inoculation suggest that there is a dynamic continuous interaction between standard VSV and its DI particle beyond the initial site of replication as the virus population spreads into the host animal. Such cycling of virus production before the full development of specific immune responses from the host may have important implications for viral diagnostics and disease transmission.
Interferons alpha and beta induce an efficient antiviral state against influenza virus in mouse cells that possess the Mx gene, but not in mouse cells that lack this gene. In Mx-containing cells treated with interferon the amount of viral mRNA synthesized as a result of primary transcription is drastically reduced. Only two viral mRNAs could be detected by Northern analysis and by translating the poly(A)+ RNA from infected cells in wheat germ extracts: a reduced amount of the mRNA for nonstructural protein 1 and an even lower amount of the mRNA for the matrix protein. The other viral mRNAs were not made in detectable amounts. In addition, the rate of viral mRNA synthesis catalyzed by the inoculum transcriptase, measured by in vitro RNA synthesis catalyzed by permeabilized cells, was severely inhibited. In contrast, interferon treatment of cells lacking the Mx gene had little or no effect on either the steady-state level or the rate of synthesis of viral mRNAs made by the inoculum transcriptase. These results indicate that the interferon-induced Mx gene product, a 75,000-molecular-weight protein that accumulates in the nucleus, inhibits influenza viral mRNA synthesis which occurs in the nucleus. No Mx-specific effect acting directly on viral protein synthesis in the cytoplasm was observed.
We have attempted to characterize the molecular defect in a temperature-sensitive mutant of vaccinia virus, ts22, which has an abortive late phenotype. At the nonpermissive temperature, ts22 displays normal viral protein synthesis until 8 h postinfection. Between 8 and 10 h after infection all viral protein synthesis ceases abruptly. Characterization of ts22 revealed that (i) primary transcription of late viral genes was not grossly impaired, (ii) late viral mRNA was biologically inactive since it could not stimulate in vitro protein synthesis, and (iii) extensive cleavage of rRNA and late viral mRNA occurred at the time that viral protein synthesis aborted in vivo. These data suggest that ts22 is defective in a function which prevents host rRNA and viral mRNA from being degraded. Inhibitor studies with cytosine arabinoside and cycloheximide showed that induction of and protection from rRNA breakdown occurred at approximately the same time during infection and required late viral gene expression. The viral protein synthesis pattern observed in vaccinia virus-infected cells treated with the drug isatin-beta-thiosemicarbazone was strikingly similar to that observed in ts22-infected cells at the nonpermissive temperature (J. Cooper, B. Moss, and E. Katz, Virology 96:381-392, 1979). Analysis of rRNA integrity in isatin-beta-thiosemicarbazone-treated, vaccinia virus-infected cells revealed extensive cleavage of rRNA, suggesting that the ts22 and drug inhibitor may function in the same pathway.
Pretreatment of human fibroblast cells with 100 U of either cloned human alpha-2 or beta interferon per ml for 24 h reduced the release of infectious herpes simplex virus type 1 by more than 99%. This inhibition in infectivity correlated well with the total number of extracellular virus particles released from treated cells as determined by DNA dot blot hybridization analysis. Electron microscopic observations of interferon-treated human fibroblast cells clearly demonstrated typical assembly of nucleocapsids inside the nucleus, even though very few mature extracellular particles were seen. Analysis of virus-specific proteins by the immunoblot technique showed that neither species of interferon had a significant inhibitory effect on the synthesis of major nucleocapsid proteins. However, the synthesis of specific glycoproteins (D and B) was drastically reduced or delayed in beta-interferon-treated cells. The results presented in this communication suggest that cloned human interferons block herpes simplex virus morphogenesis at a late stage and inhibit the release of particles from the treated cells.
Wild-type adenovirus was coinfected with a mutant temperature sensitive for the immediate-early gene of pseudorabies virus. At the nonpermissive temperature, this mutant, tsG, strongly inhibited the transcription of all adenovirus early genes, including E1A. This inhibition was not observed with wild-type pseudorabies virus coinfection or with tsG coinfection at the permissive temperature. The level of repression was dependent upon the ratio of tsG to adenovirus in the infection. The results suggest that the temperature-sensitive protein may be interacting with transcription factors on the viral DNA or with the DNA itself to inhibit adenovirus transcription.