Reovirus variants selected during persistent infections of L cells contain mutations in the viral S1 and S4 genes and are altered in viral disassembly.

Reoviruses isolated from persistently infected cultures (PI viruses) can grow in the presence of ammonium chloride, a weak base that blocks acid-dependent proteolysis of viral outer-capsid proteins during viral entry into cells. We used reassortant viruses isolated from crosses of wild-type (wt) reovirus strain, type 1 Lang, and three independent PI viruses, L/C, PI 2A1, and PI 3-1, to identify viral genes that segregate with the capacity of PI viruses to grow in cells treated with ammonium chloride. Growth of reassortant viruses in ammonium chloride-treated cells segregated with the S1 gene of L/C and the S4 gene of PI 2A1 and PI 3-1. The S1 gene encodes viral attachment protein sigma1, and the S4 gene encodes outer-capsid protein sigma3. To identify mutations in sigma3 selected during persistent reovirus infection, we determined the S4 gene nucleotide sequences of L/C, PI 2A1, PI 3-1, and four additional PI viruses. The deduced amino acid sequences of sigma3 protein of six of these PI viruses contained a tyrosine-to-histidine substitution at residue 354. To determine whether mutations selected during persistent infection alter cleavage of the viral outer capsid, the fate of viral structural proteins was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after treatment of virions of wt and PI viruses with chymotrypsin in vitro. Proteolysis of PI virus outer-capsid proteins sigma3 and mu1C occurred with faster kinetics than proteolysis of wt virus outer-capsid proteins. These results demonstrate that mutations in either the S1 or S4 gene alter acid-dependent disassembly of the reovirus outer capsid and suggest that increased efficiency of proteolysis of viral outer-capsid proteins is important for maintenance of persistent reovirus infections of cultured cells.     

Genetic Variation During Persistent Reovirus Infection: Presence of Extragenically Suppressed Temperature-Sensitive Lesions in Wild-Type Virus Isolated from Persistently Infected L Cells

Persistent reovirus infection of L cells was established with a serially passaged stock of temperature-sensitive (ts) mutant C(447) containing greater than 90% defective interfering particles. Within a month after establishment of the carrier culture, the ts mutant was replaced by virus that expressed the wild-type (ts⁺) temperature phenotype (R. Ahmed and A. F. Graham, J. Virol. 23:250-262, 1977). To determine whether the ts⁺ phenotype of the virus was due to intragenic reversion or to the presence of an extragenic mutation suppressing the original ts defect, several clones were backcrossed to wild-type reovirus, and the progeny of each cross were screened for temperature sensitivity. The results indicated that the original tsC lesion had reverted. However, in two of the seven clones examined, new ts lesions were found. These new ts lesions appeared phenotypically as ts⁺ due to the presence of extragenic suppressor mutations. Temperature-sensitive mutants representing three different groups were rescued from one suppressed clone, indicating that this ts⁺ clone contained multiple ts lesions. Among the ts mutants rescued were the initial isolates of a new recombination group which we have designated H. Some of the ts mutants rescued from the suppressed clones are capable of interfering with the growth of wild-type reovirus and may play a role in maintaining the carrier state. The results of this study show that persistently infected L cells contain a genetically heterogeneous population of reovirus even though all virus clones express the ts⁺ phenotype. It is thus critical to distinguish between genotype and phenotype when analyzing viruses that emerge during persistent infection.     

Persistent reovirus infections of L cells select mutations in viral attachment protein sigma1 that alter oligomer stability.

During maintenance of L-cell cultures persistently infected with reovirus, mutations are selected in viruses and cells. Cells cured of persistent infection support growth of viruses isolated from persistently infected cultures (PI viruses) significantly better than that of wild-type (wt) viruses. In a previous study, the capacity of PI virus strain L/C to grow better than wt strain type 1 Lang (T1L) in cured cells was mapped genetically to the S1 gene (R. S. Kauffman, R. Ahmed, and B. N. Fields, Virology 131:79-87, 1983), which encodes viral attachment protein sigma1. To investigate mechanisms by which mutations in S1 confer growth of PI viruses in cured cells, we determined the S1 gene nucleotide sequences of L/C virus and six additional PI viruses isolated from independent persistently infected L-cell cultures. The S1 sequences of these viruses contained from one to three mutations, and with the exception of PI 2A1 mutations in each S1 gene resulted in changes in the deduced amino acid sequence of sigma1 protein. Using electrophoresis conditions that favor migration of sigma1 oligomers, we found that sigma1 proteins of L/C, PI 1A1, PI 3-1, and PI 5-1 migrated as monomers, whereas sigma1 proteins of wt reovirus and PI 2A1 migrated as oligomers. These findings suggest that mutations in sigma1 protein affecting stability of sigma1 oligomers are important for the capacity of PI viruses to infect mutant cells selected during persistent infection. Since no mutation was found in the deduced amino acid sequence of PI 2A1 sigma1 protein, we used T1L X PI 2A1 reassortant viruses to identify viral genes associated with the capacity of this PI virus to grow better than wt in cured cells. The capacity of PI 2A1 to grow better than T1L in cured cells was mapped to the S4 gene, which encodes outer-capsid protein sigma3. This finding suggests that in some cases, mutations in sigma3 protein in the absence of sigma1 mutations confer growth of PI viruses in mutant cells. To confirm the importance of the S1 gene in PI virus growth in cured cells, we used T1L X PI 3-1 reassortant viruses to genetically map the capacity of this PI virus to grow better than wt in cured cells. In contrast to our results using PI 2A1, we found that growth of PI 3-1 in cured cells was determined by the sigma1-encoding S1 gene. Given that the sigma1 and sigma3 proteins play important roles in reovirus disassembly, findings made in this study suggest that stability of the viral outer capsid is an important determinant of the capacity of reoviruses to adapt to host cells during persistent infection.     

Efficiency of viral entry determines the capacity of murine erythroleukemia cells to support persistent infections by mammalian reoviruses.

To determine mechanisms by which persistent viral infections are established and maintained, we initiated persistent infections of murine erythroleukemia (MEL) cells by using reovirus strains type 3 Abney and type 3 Dearing. Establishment of persistent reovirus infections of MEL cells was not associated with a significant cytopathic effect despite the presence of high titers of infectious virus in the cultures (>10(5) PFU/ml of culture lysate). Maintenance of persistently infected MEL-cell cultures was associated with coevolution of mutant viruses and cells. Mutant viruses produced greater yields than the parental wild-type (wt) strains in MEL cells cured of persistent infection and in cells treated with ammonium chloride, a weak base that blocks viral disassembly. Mutant cells supported growth of wt infectious subvirion particles, which are disassembly intermediates generated in vitro by treatment of virions with chymotrypsin, substantially better than growth of wt virions. These findings indicate that viral and cellular mutations selected during maintenance of persistently infected MEL-cell cultures affect acid-dependent proteolysis of virions during entry into cells. We also found that wt infectious subvirion particles produce greater yields than wt virions in wt MEL cells, which suggests that inefficient viral disassembly in MEL cells favors establishment of persistent infection. Therefore, steps in reovirus replication leading to viral disassembly appear to be critical determinants of the capacity of MEL cells to support both establishment and maintenance of persistent reovirus infections.     

Persistent Infections in L Cells with Temperature-Sensitive Mutants of Reovirus

Serial passage of reovirus temperature-sensitive (ts) mutant C(447) produced by passage 9 (P9) a heavily defective population of virus from which the double-stranded RNA genomic segments L₁, L₃, and M₁ were largely missing. Viral cores obtained from this P9 population were heterogeneous with respect to buoyant density in CsCl gradients, suggesting that particles were present with different combinations of deleted segments. Similar observations were made with the E(320) ts mutant of reovirus. By serial passage P15, 90% of the E(320) viral population was defective and the major missing genomic segments were L₁ and L₃. Persistent infections were readily established in monolayer cultures of L cells with P9 of C(447) virus and P15 of E(320) virus and in Vero cells with P9 of C(447) virus. Under similar conditions persistent infections could not be initiated with defective-free populations of C(447) or E(320) viruses. The greater the capacity of defective virus in the population to interfere with viral growth, the more readily persistent infection was initiated. During their maintenance persistently infected cells were subcultured approximately twice a week. More than 80% of the cells continuously produced virus. By subculture 6 the original ts infectious viral component had been replaced by a small-plaque mutant with a ts⁺ phenotype. Defective virus was always present in the carrier cells. In addition to the more commonly observed defectives whose cores banded at approximately ρ = 1.40 to 1.415 g/ml in CsCl gradients, a new class of defective core was seen banding in the region of 1.34 to 1.36 g/ml. This latter particle, which has not been thoroughly characterized as yet, is termed “light defective.” Persistently infected cells underwent periodic crises during their maintenance, during which the cultures partially lysed and then rapidly grew to confluence. Crises corresponded to a burst of infectious virus from the cells and a relatively low concentration of light defectives. During quiescent periods the concentration of light defectives amounted to as much as 98% of the total viral population. The function of light defectives is not yet clear, but it seems essential to assign major importance to defective virus in maintaining persistent infections in this system.     

Persistent Baculovirus Infection Results from Deletion of the Apoptotic Suppressor Gene p35

Infection with the wild-type baculovirus Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) results in complete death of Spodoptera frugiperda (Sf) cells. However, infection of Sf cells with AcMNPV carrying a mutation or deletion of the apoptotic suppressor gene p35 allowed the cloning of surviving Sf cells that harbored persistent viral genomes. Persistent infection established with the virus with p35 mutated or deleted was blocked by stable transfection of p35 in the host genome or by insertion of the inhibitor of apoptosis (iap) gene into the viral genome. These artificially established persistently virus-infected cells became resistant to subsequent viral challenge, and some of the cell lines carried large quantities of viral DNA capable of early gene expression. Continuous release of viral progenies was evident in some of the persistently virus-infected cells, and transfection of p35 further stimulated viral activation of the persistent cells, including the reactivation of viruses in those cell lines without original continuous virus release. These results have demonstrated the successful establishment of persistent baculovirus infections under laboratory conditions and that their establishment may provide a novel continuous, nonlytic baculovirus expression system in the future.     

Role of the host cell in persistent viral infection: Coevolution of L cells and reovirus during persistent infection

Mutant L cells, designated LR cells, were isolated after “curing” a persistently infected cell line (L/C) with antireovirus serum. The LR cells were shown to be virus-free; no reovirus was detectable by infectious center assays, plaque assays, presence of viral proteins, presence of viral dsRNA and immunofluorescence studies. Persistent infections were readily established in LR cells following infection with either cloned, low passage wild-type reovirus or cloned, low passage reovirus isolated from carrier cultures. Reovirus isolated from carrier cultures, however, grew much better than wild-type reovirus in LR cells and showed complete dominance over wild-type reovirus in coinfection experiments. Infection of LR cells with wild-type reovirus resulted in a low-level persistent infection with inefficient viral replication; these mutant L cells were partially resistant to infection with wild-type reovirus. In contrast, infection of the mutant L cells with virus isolated from the persistently infected cells resulted in a persistent infection accompanied with efficient viral replication. Infection of the original L cells with either wild-type reovirus or reovirus isolated from the persistently infected cells resulted in a lytic infection with no surviving cells. Thus the host cell plays a crucial role in the maintenance of persistent reovirus infection. Our results show that there is a coevolution of both mutant L cells and mutant reovirus during persistent infection.     

Nora Virus Persistent Infections Are Not Affected by the RNAi Machinery

Drosophila melanogaster is widely used to decipher the innate immune system in response to various pathogens. The innate immune response towards persistent virus infections is among the least studied in this model system. We recently discovered a picorna-like virus, the Nora virus which gives rise to persistent and essentially symptom-free infections in Drosophila melanogaster. Here, we have used this virus to study the interaction with its host and with some of the known Drosophila antiviral immune pathways. First, we find a striking variability in the course of the infection, even between flies of the same inbred stock. Some flies are able to clear the Nora virus but not others. This phenomenon seems to be threshold-dependent; flies with a high-titer infection establish stable persistent infections, whereas flies with a lower level of infection are able to clear the virus. Surprisingly, we find that both the clearance of low-level Nora virus infections and the stability of persistent infections are unaffected by mutations in the RNAi pathways. Nora virus infections are also unaffected by mutations in the Toll and Jak-Stat pathways. In these respects, the Nora virus differs from other studied Drosophila RNA viruses.     

Persistent infection of human oligodendrocytic and neuroglial cell lines by human coronavirus 229E

Human coronaviruses (HuCV) cause common colds. Previous reports suggest that these infectious agents may be neurotropic in humans, as they are for some mammals. With the long-term aim of providing experimental evidence for the neurotropism of HuCV and the establishment of persistent infections in the nervous system, we have evaluated the susceptibility of various human neural cell lines to acute and persistent infection by HuCV-229E. Viral antigen, infectious virus progeny and viral RNA were monitored during both acute and persistent infections. The astrocytoma cell lines U-87 MG, U-373 MG, and GL-15, as well as neuroblastoma SK-N-SH, neuroglioma H4, and oligodendrocytic MO3.13 cell lines, were all susceptible to an acute infection by HuCV-229E. The CHME-5 immortalized fetal microglial cell line was not susceptible to infection by this virus. The MO3.13 and H4 cell lines also sustained a persistent viral infection, as monitored by detection of viral antigen and infectious virus progeny. Sequencing of the S1 gene from viral RNA after approximately 130 days of infection showed two point mutations, suggesting amino acid changes during persistent infection of MO3.13 cells but none for H4 cells. Thus, persistent in vitro infection did not generate important changes in the S1 portion of the viral spike protein, which was shown for murine coronaviruses to bear hypervariable domains and to interact with cellular receptor. These results are consistent with the potential persistence of HuCV-229E in cells of the human nervous system, such as oligodendrocytes and possibly neurons, and the virus's apparent genomic stability.     

Persistent Infection of L Cells with Vesicular Stomatitis Virus: Evolution of Virus Populations

A previous report (Youngner et al., J. Virol. 19:90-101, 1976) documented that noncytocidal persistent infection can be established with wild-type vesicular stomatitis virus (VSV) in mouse L cells at 37°C and that a rapid selection of RNA⁻, group I temperature-sensitive (ts) mutants consistently occurs in this system. To assess the selective advantage of the RNA⁻ts phenotype, evolution of the virus population was studied in persistent infections initiated in L cells by use of VSV ts 0 23 and ts 0 45, RNA⁺ mutants belonging to complementation groups III and V. In L cells persistently infected with ts 0 23, the ts RNA⁺ virus population was replaced gradually by viruses which had a ts RNA⁻ phenotype. VSV ts 0 45 (V) has another marker in addition to reduced virus yield at 39.5°C: a defective protein (G) which renders virion infectivity heat labile at 50°C. Persistent infections initiated with this virus (ts, heat labile, RNA⁺) evolved into a virus population which was ts, heat resistant, and RNA⁻. These findings suggest that the ts phenotype itself is not sufficient to stabilize the VSV population in persistently infected L cells and also indicate that the ts RNA⁻ phenotype may have a unique selective advantage in this system. In addition to the selection of ts RNA⁻ mutants, other mechanisms which also might operate in the maintenance of persistent VSV infections of L cells were explored. Whereas defective-interfering particles did not seem to mediate the carrier state, evidence was obtained that interferon may play a role in the regulation of persistent infections of L cells with VSV.     

Precise missense and silent point mutations are fixed in the genomes of poliovirus mutants from persistently infected cells.

Poliovirus mutants selected in persistently infected human neuroblastoma cells have a modified cell tropism and can establish a secondary persistent infection in nonneural cells, such as HEp-2c cells. Nucleotide sequence analysis revealed that the genome of a persistent mutant, S11, differed from that of the parental lytic Sabin 1 poliovirus strain by 31 point mutations. Three mutations occurred in the noncoding regions. The other mutations resulted in 12 amino acid substitutions; 1 substitution occurred in a nonstructural protein (3A), while the other 11 substitutions were clustered in the capsid proteins VP2 and VP1. The same missense mutations, as well as many of the silent mutations that we observed in mutant S11, also accumulated in the genome of two other persistent viruses isolated from independent infections. This finding indicates that both missense and silent mutations are selected during the persistent infection of neuroblastoma cells and suggests that the secondary structure of RNA in the coding region may play a role in viral infection.     

Generation of defective virus after infection of newborn rats with reovirus.

When 2-day-old rats were inoculated subcutaneously with the R2 strain of reovirus type 3 or with a class B (352) or class C (447) temperature-sensitive (ts) mutant, 5 to 10% of the animals died from acute encephalitis within 12 days. Approximately half of the survivors recovered rapidly and grew normally, but the remainder became runted. Two phases of infection are distinguished in the animals: an acute phase during which infectious virus reaches a maximum titer in brain and other tissues by 10 days p.i. and thes runting of the rats and the slow disappearance of virus from their brains over a period of 2 months or so. Virus isolated from chronically infected brains generally retained the genetic character (ts or wild type) of the inoculated virus, but two exceptions to this are described. Defective virions lacking the L1 segment of the viral genome (L1 defectives) were generated in rat brains during the acute phase of infection. Defective virus was also generated during the chronic phase, but during this period defectives were found with multiple segments deleted from the genome in addition to L1 defectives. In another type of experiment defective virus exerted a marked protective effect when inoculated intracerebrally with R2 virus. In the absence of defectives all animals died, but in their presence 17 of 23 animals survived and 15 of 23 became runted and chronically infected. The formation and evolution of defective particles in the brains of these rats were similar to those found in rats chronically infected after subcutaneous inoculation of reovirus. We conclude that the formation of defective virus particles may play a role in the initiation and maintenance of chronic neutropic infections with reovirus.     

Identification of a region of the poliovirus genome involved in persistent infection of HEp-2 cells.

Poliovirus mutants were selected during the persistent infection of human neuroblastoma cells. These viruses could establish secondary persistent infections in HEp-2 nonneural cells. We report the identification of a region of the genome of a persistent virus (S11) that was sufficient to confer to a recombinant virus the phenotype that causes persistent infection in HEp-2 cells. This region, between nucleotides 1148 and 3481, contained 11 missense mutations mapping exclusively in the genes of capsid proteins VP1 and VP2. Because recombinant viruses carrying only one of these two mutated genes were not able to cause persistent infection, it seems very probable that two or more mutations in these genes are required for expression of the phenotype that causes persistent infection.     

Transgenic Tobacco Lines Expressing Defective CMV Replicase-Derived dsRNA Are Resistant to CMV-O and CMV-Y

Cucumber mosaic virus (CMV) is a tripartite, positive sense RNA virus causing infections and yield losses to many plant species. Here, we generated a construct containing inverted repeat of 1,793 bp fragment of defective CMV replicase gene derived from RNA2 of cucumber mosaic virus strain O (CMV-O). The replicase gene was modified by deleting a 9 bp region between nucleotides 1909–1918. This caused a deletion in the active centre motif of polymerases, producing defective translated product 9 nucleotides shorter than the full length protein. The RNAi construct containing inverted repeat of the defective gene was used to produce transgenic tobacco lines expressing CMV-derived double-stranded RNA via Agrobacterium-mediated transformation. Of the four transgenic lines inoculated with CMV-O or CMV-Y in vitro and ex vivo, three lines (T1, T4 and T5) showed immunity to both strains of CMV as no symptoms were detected, whereas one line (T7) exhibited high resistance with mild symptoms limited to inoculation portions. No virus could be detected in uninoculated new leaves of the transgenic lines after RT-PCR and Dot-immunobinding assay analyses. Small interfering RNAs present in transgenic lines before and after virus challenge indicates that the resistance was acquired through RNA silencing.     

Persistent RNA virus infection of lepidopteran cell lines: Interactions with the RNAi machinery

RNAi is broadly used as a technique for specific gene silencing in insects but few studies have investigated the factors that can affect its efficiency. Viral infections have the potential to interfere with RNAi through their production of viral suppressors of RNAi (VSRs) and the production of viral small RNAs that can saturate and inactivate the RNAi machinery. In this study, the impact of persistent infection of the RNA viruses Flock house virus (FHV) and Macula-like virus (MLV) on RNAi efficiency was investigated in selected lepidopteran cell lines. Lepidopteran cell lines were found to be readily infected by both viruses without any apparent pathogenic effects, with the exception of Bombyx-derived Bm5 and BmN4 cells, which could not be infected by FHV. Because Sf21 cells were free from both FHV and MLV and Hi5-SF were free from FHV and only contained low levels of MLV, they were tested to evaluate the impact of the presence of the virus. Two types of RNAi reporter assays however did not detect a significant interference with gene silencing in infected Sf21 and Hi5-SF cells when compared to virus-free cells. In Hi5 cells, the presence of FHV could be easily cleared through the expression of an RNA hairpin that targets its VSR gene, confirming that the RNAi mechanism was not inhibited. Sequencing indicated that the B2 RNAi inhibitor gene of FHV and a putative VSR gene from MLV were intact in persistently infected cell lines, indicating that protection against RNAi remains essential for virus survival. It is proposed that infection levels of persistent viruses in the cell lines are too low to have an impact on RNAi efficiency in the lepidopteran cell lines and that encoded VSRs act locally at the sites of viral replication (mitochondrial membranes) without affecting the rest of the cytoplasm.     

Reovirus infection in rat lungs as a model to study the pathogenesis of viral pneumonia.

We undertook the present study to elucidate the pathogenesis of the pathologic response to reovirus infection in the lungs and further understand the interactions of reoviruses with pulmonary cells. We found that reoviruses were capable of causing acute pneumonia in 25- to 28-day-old Sprague-Dawley rats following intratracheal inoculation with the reoviruses type 1 Lang (T1L) and type 3 Dearing (T3D). The onset of the pneumonia was rapid, marked by type I alveolar epithelial cell degeneration, type II alveolar epithelial cell hyperplasia, and the infiltration of leukocytes into the alveolar spaces. More neutrophils were recruited into the lungs during T3D infection than during T1L infection, and the serotype difference in the neutrophil response was mapped to the S1 gene of reovirus. Viral replication in the lungs was required for the development of pneumonia due to T1L and T3D infections, and replication occurred in type I alveolar epithelial cells. T1L grew to higher titers in the lungs than did either T3D or type 3 clone 9, and the S1 gene was found to play a role in determining the level of viral replication. We propose that experimental reovirus infection in the lungs can serve as a model for the pathogenesis of viral pneumonia in which pulmonary inflammation results following direct infection of lung epithelial cells.     

Phylogenetic Evidence of Long Distance Dispersal and Transmission of Piscine Reovirus (PRV) between Farmed and Wild Atlantic Salmon

The extent and effect of disease interaction and pathogen exchange between wild and farmed fish populations is an ongoing debate and an area of research that is difficult to explore. The objective of this study was to investigate pathogen transmission between farmed and wild Atlantic salmon (Salmo salar L.) populations in Norway by means of molecular epidemiology. Piscine reovirus (PRV) was selected as the model organism as it is widely distributed in both farmed and wild Atlantic salmon in Norway, and because infection not necessarily will lead to mortality through development of disease. A matrix comprised of PRV protein coding sequences S1, S2 and S4 from wild, hatchery-reared and farmed Atlantic salmon in addition to one sea-trout (Salmo trutta L.) was examined. Phylogenetic analyses based on maximum likelihood and Bayesian inference indicate long distance transport of PRV and exchange of virus between populations. The results are discussed in the context of Atlantic salmon ecology and the structure of the Norwegian salmon industry. We conclude that the lack of a geographical pattern in the phylogenetic trees is caused by extensive exchange of PRV. In addition, the detailed topography of the trees indicates long distance transportation of PRV. Through its size, structure and infection status, the Atlantic salmon farming industry has the capacity to play a central role in both long distance transportation and transmission of pathogens. Despite extensive migration, wild salmon probably play a minor role as they are fewer in numbers, appear at lower densities and are less likely to be infected. An open question is the relationship between the PRV sequences found in marine fish and those originating from salmon.     

Evasion of Antiviral Innate Immunity by Theiler's Virus L* Protein through Direct Inhibition of RNase L

Theiler''s virus is a neurotropic picornavirus responsible for chronic infections of the central nervous system. The establishment of a persistent infection and the subsequent demyelinating disease triggered by the virus depend on the expression of L*, a viral accessory protein encoded by an alternative open reading frame of the virus. We discovered that L* potently inhibits the interferon-inducible OAS/RNase L pathway. The antagonism of RNase L by L* was particularly prominent in macrophages where baseline oligoadenylate synthetase (OAS) and RNase L expression levels are elevated, but was detectable in fibroblasts after IFN pretreatment. L* mutations significantly affected Theiler''s virus replication in primary macrophages derived from wild-type but not from RNase L-deficient mice. L* counteracted the OAS/RNase L pathway through direct interaction with the ankyrin domain of RNase L, resulting in the inhibition of this enzyme. Interestingly, RNase L inhibition was species-specific as Theiler''s virus L* protein blocked murine RNase L but not human RNase L or RNase L of other mammals or birds. Direct RNase L inhibition by L* and species specificity were confirmed in an in vitro assay performed with purified proteins. These results demonstrate a novel viral mechanism to elude the antiviral OAS/RNase L pathway. By targeting the effector enzyme of this antiviral pathway, L* potently inhibits RNase L, underscoring the importance of this enzyme in innate immunity against Theiler''s virus.     

Cells and viruses with mutations affecting viral entry are selected during persistent infections of L cells with mammalian reoviruses.

Previous studies demonstrated that both cellular and viral mutants are selected during maintenance of persistent infections established in murine L cells with high-passage stocks of mammalian reoviruses. In particular, when one culture was cured of persistent infection, the resulting cells were found to support the growth of viruses isolated from persistently infected cultures (termed PI viruses here) better than that of wild-type (wt) viruses (R. Ahmed, W. M. Canning, R. S. Kauffman, A. H. Sharpe, J. V. Hallum, and B. N. Fields, Cell 25:325-332, 1981). To address the nature of cellular and viral mutations selected during maintenance of persistent reovirus infections, we established independent, persistently infected cultures with L cells and high-passage stocks of wt reovirus. These cultures served as sources of new PI viruses and cured cells for study. We found that although wt viruses grew poorly in cured cells when infection was initiated with intact virions, they grew well in cured cells when infection was initiated with infectious subvirion particles generated from virions by in vitro treatment with chymotrypsin. This finding indicates that the block to growth of wt viruses in cured cells involves an early step that is unique to infection by virions, such as proteolytic processing in an endocytic compartment. We also found that PI viruses grew better than wt viruses in L cells treated with ammonium chloride, a weak base that inhibits the pH decrease in endosomes and lysosomes. Because ammonium chloride blocks an early step in infection by intact virions, probably the proteolytic processing of viral outer capsid proteins by acid-dependent cellular proteases in late endosomes or lysosomes, this finding indicates that PI viruses differ from wt viruses with respect to viral entry into cells. Therefore, these results indicate that both cells and viruses evolve mutations that affect one or more early steps in the viral growth cycle during maintenance of L-cell cultures persistently infected with reoviruses.