Restitution of fibroblast-transforming ability in src deletion mutants of avian sarcoma virus during animal passage.

The Schmidt-Ruppin strain of Rous sarcoma virus subgroup D (SR-D) gives rise to transformation defective (td) mutants which have lost either all or almost all of the src gene (standard td or std viruses) or have only a partial deletion of src. These partial deletion mutants, designated ptd viruses, contain genomic RNA slightly larger than std isolates, and heteroduplex analyses suggest that ptd viruses retain approximately 25% of src from the 5′ end of that gene [Lai et al. (1977) Proc. Natl. Acad. Sci. USA74, 4781–4785]. Several ptd isolates of SR-D were injected into newly hatched chickens and after prolonged latent periods caused sarcomas in about 30% of the birds. The tumors occurred in internal organs away from the site of injection. Infectious sarcoma viruses isolated from these growths show the envelope markers of subgroup D are nondefective for replication and induce a transformation in vitro which is morphologically distinct from that of SR-D. Electrophoresis of 35 S genomic RNA from these recovered sarcoma viruses shows it to be of the size characteristic for nondefective sarcoma viruses. Fingerprint analysis of ³²P-labeled RNA from one of the new sarcoma viruses detected all oligonucleotides present in ptd viruses, the src-specific oligonucleotides of SR-D, and one new oligonucleotide not present in SR-D. This new RNase T₁-resistant oligonucleotide and the src-specific oligonucleotides identical to those of SR-D map close to the 3′ end in the genome of the recovered sarcoma virus, which is the position expected for the src gene. These studies suggest that recovered avian sarcoma viruses have acquired cellular sequences which are closely related in structure and function to the viral src gene.     

Mapping of nonconditional and conditional mutants in the src gene of Prague strain Rous sarcoma virus

Restriction endonuclease EcoRI digestion of the viral DNA of 12 nonconditional transformation defective (td) mutants of Prague strain Rous sarcoma virus (PR-RSV) has divided these mutants into two groups. Five mutants possess an EcoRI B (src gene-containing) fragment of the same size as that from wild type PR-RSV and thus these mutants have no detectable diminution in the transforming src gene. The other 7 mutants bear deletions of 1.0 to 1.8 kilobases in the 3.2-kilobase EcoRI B fragment. The extents of these deletions have been mapped using a number of restriction endonucleases and by comparing these results with studies on the nucleotide sequence of src(Czernilovsky et al., Nature (London)287, 198–203, 1980) we conclude that the td mutants have deleted sequences at the 5′ end of src, and in some cases also in regions between src and env, leaving intact at least some 3′ src sequences. These td mutants recombine in differing patterns with 14 temperature-sensitive (ts) src gene mutants. This enables many of the ts mutations to be localized in limited regions of src, 10 of them being clustered in the 3′ 40% of the gene, the remaining four bearing at least one mutation in the 5′ 60% of src. A nonconditional src gene mutant that transforms cells to a fusiform as opposed to round cell morphology (td SF/LO 104) also possesses a lesion that maps in the 5′ 60% of the src gene.     

Continuous production of Rous sarcoma virus free of transformation defective virus in clones of established RSV-transformed quail cells

Quail embryo fibroblasts were infected with a Schmidt-Ruppin strain RSV × chf recombinant virus. Virus-transformed cells were established as a permanent line and then cloned in methyl cellulose. Out of 140 clones isolated four clones were capable of indefinite growth. These clones were examined for (i) production of sarcoma and td virus particles, (ii) number of integrated virus genome equivalents, and (iii) deletions of the src gene in the provirus. We found that the clones yield about 106 focus-forming units of the sarcoma virus per milliliter of the culture medium. No td virus could be detected by plating of the virus at the endpoint dilution and no 35 S td virus RNA but only 38 S sarcoma virus RNA was found in virions. Hybridization kinetic studies indicated that three different clones contain about 2 virus genome equivalents, and one clone contains about 4 virus genome equivalents per diploid cell. Upon transfection the proviruses of different clones generated sarcoma viruses and no td viruses. Finally digestion with EcoRI restriction endonuclease released in all four clones a 1.9 × 106-dalton fragment characteristic of the complete src gene, while no 0.8 × 10⁶-dalton fragment characteristic of a td provirus could be detected. We concluded that the clones of RSV-transformed quail cells contain only nondefective sarcoma proviruses and produce from these proviruses nondefective focus-forming virions in the absence of any segregant td virions.     

Specific restriction of avian sarcoma viruses by a line of transformed lymphoid cells.

MSB-1 cells are a line of transformed chicken lymphoid cells derived from tumors induced by Marek's disease viruses and free of exogenous avian leukosis viruses (ALV). They can be infected by ALV of subgroups A and C including transformation-defective (td) deletion mutants of avian sarcoma viruses (ASV). In terms of virus titers in supernatant culture medium, proportion of virus-producing cells, and levels of viral RNA detected by hybridization with a cDNA probe, infection by td ASV of MSB-1 cells was indistinguishable from infection of chicken embryo fibroblasts. In contrast, wild type ASV was restricted in its growth on MSB-1 cells. Different clones of ASV varied in their restriction by all these parameters of viral growth by factors of 10^?1 to 10^?4 Studies of a severely restricted viral clone showed equal quantities of hybridizable viral DNA in Hirt supernatant fractions of both fibroblasts and MSB-1 cells at 10 hr after high multiplicity infection, and transfection assays indicated infectious viral DNA in both cell types. Viral DNA largely disappeared from Hirt supernatant fractions of MSB-1 cells by 48 hr after infection, and sarcoma virus-specific DNA was not detected in Hirt pellet fractions from MSB-1 cells at levels found in comparably infected fibroblasts. Infectious ASV DNA, while easily detected in fibroblasts, could not be detected on MSB-1 cells at 48 hr or later times after infection. Because replication of td ASV does not appear restricted in MSB-1 cells, the failure of ASV DNA to integrate normally in these cells seems to be related to the presence of src sequences in the viral genome.     

Analysis on the reproducing and cell-transforming capacities of a temperature sensitive mutant (ts 334) of avian sarcoma virus B77

Ts 334, a temperature sensitive mutant of avian sarcoma virus B77, cannot produce infectious progeny nor induce neoplastic transformation at the nonpermissive temperature. In order to clarify the relationship between these two functions of ts 334 we attempted to (1) isolate and characterize nonconditional transformation defective (td) mutants from ts 334, (2) isolate and characterize recombinants between ts 334 and RAV-1, and (3) reexamine rescue of ts 334 with RAV-1 at the nonpermissive temperature.All seven nonconditional td mutants isolated from ts 334 kept their temperature sensitive character in replication, although they had lost transforming capacity both at the permissive and at the nonpermissive temperatures. They appear to have a temperature sensitive step in virus maturation like ts 334.The helper function of these td mutants for the defective Bryan high titer strain of Rous sarcoma virus is also temperature-dependent.Two recombinants were isolated from cells coinfected with ts 334 and RAV-1. These recombinants combined the cell-transforming ability of ts 334 and of the envelope properties of RAV-1. These two recombinants were unable to induce cell-transformation but grew well at the nonpermissive temperature.In RAV-1 producing cells not only the genome of ts 334, but also the envelope property of ts 334 were rescued at the nonpermissive temperature, though cell transformation was not observed.These observations suggest that ts 334 has two mutations, one affecting reproduction and another cell-transformation capacities.     

Detection and enumeration of transformation-defective strains of avian sarcoma virus with molecular hybridization.

Serial propagation of avian sarcoma viruses generates deletions in the viral gene responsible for cellular transformation (src). We have devised an assay for these deletion mutants which utilizes molecular hybridization and exploits the availability of DNA (cDNA_sarc) complementary to the nucleotide sequences affected by the deletion in src. Our procedure is also applicable to deletions in other viral genes and offers several advantages over conventional bioassays for the deletion mutants; moreover, it can be used to detect deletions in virus-specific intracellular nucleic acids. In order to illustrate the utility of the assay, we demonstrate that all 20 copies of the proviral DNA for avian sarcoma viruses in XC cells contain src, and we show that single avian cells can contain functioning proviruses for both avian sarcoma virus and a congenic deletion mutant. It should now be possible to use molecular hybridization to study the mechanism by which deletions in src are generated.     

Structural and nonstructural proteins of strain Colburn cytomegalovirus

The growth of most Rous sarcoma viruses (RSV) is severely restricted on MSB-1 cells (a line of chicken T lymphoblasts) in comparison to growth on chicken embryo fibroblast (CEF). Nonconditional transformation defective mutants of RSV from which the complete src region has been deleted (td RSV) are not subject to growth restriction. We examined the formation and integration of RSV and td RSV in MSB-1 cells following high multiplicity infection. Nearly equivalent quantities of the linear form of unintegrated RSV and td RSV DNA were formed in these cells during the first 10 hr after infection. Linear RSV DNA from MSB-1 cells could not be distinguished from linear RSV from CEF by restriction endonuclease analysis and by previously described transfection assays (P. E. Neiman, C. McMillin-Helsel, and G. M. Cooper, 1978, Virology 89,360–371). Beyond 10 hr after infection, and with progressive cell growth in the MSB-1 cultures, the level of RSV linear DNA rapidly decreased. Presumptive circular RSV DNA was detected only transiently, and at very low levels, about 15 hr after infection. Association of RSV DNA with high-molecular-weight chromosomal DNA, i.e., integration, was not detected in this study. In contrast, nearly constant levels of td RSV unintegrated linear DNA and, after 20 hr, circular DNA persisted in MSB-1 cells for at least 7 days after infection. Integration of td RSV proviral DNA was inefficient, occurring in only about 5% of MSB-1 cells (even at very high multiplicities of infection) in the first round of infection, and in 25–40% of cells by 3 days after infection. Almost all MSB-1 cells containing td RSV DNA produced virus. Analysis of eight nonconditional transformation defective mutants of RSV which retain the src region to different extents showed that all of these mutants replicated to the same normal titer on MSB-1 cells as on CEF without further deletion of the src region. Two temperature sensitive src mutants that thermal inactivation of the scr gene on MSB-1 cells at both 35° and 41°, indicating that thermal inactivation of the src gene product could not abrogate the replication block. These studies clearly demonstrate that the presence of the src region in RSV impedes the formation and/or integration of provirus in some types of host cells.     

Restriction fragment analysis of the temporal program of bacteriophage SPO1 transcription and its control by phage-modified RNA polymerases.

Wild-type WSN of influenza A virus which had been inactivated by ultraviolet (uv) irradiation was capable, to various extents, of rescuing temperature-sensitive (ts) mutants of the same virus, resulting in the production of wild-type recombinants at the nonpermissive temperature. The capability of wild-type virus to rescue ts mutants decreased with increasing uv dose following single-hit kinetics. Inactivation of rescue capability varied when mutants of different recombination-complementation (recombination) groups were compared, but was nearly identical to different members of one recombination group, except for Group IV mutants. The inactivation rate was significantly greater when it was determined with two mutants (ts-11 and ts-60) than when determined with the rest of the Group IV mutants. Because the inactivation rate was expected to be greater for a double mutant than for virus strains having either one of two mutational lesions alone, it was suspected that ts-11 and ts-60 had an additional mutational lesion in a still undefined gene. Segregation of a new mutant from ts-60 was, therefore, attempted by backcrossing it with wild-type WSN and screening progeny clones. A clone which had lost Group IV mutation during the process but still retained temperature sensitivity had been obtained. Genetic crossing of this segregant, ts-60S, with mutants of all seven other recombination groups gave rise to recombinants of wild-type character, indicating that ts-60S was a mutant of unknown recombination group (Group VIII). Both ts-11 and ts-60 were confirmed to be double mutants (Groups IV + VIII). During the process of segregation, a number of progeny clones with a novel genotype were obtained which had not been manifest in the parent viruses, i.e., the mutation in either the group I or Group III gene, in addition to the one in the Group VIII gene. It was found that these paradoxical phenomena were caused by mixed aggregates present in the parent viruses, ts-11 and ts-60. Mixed aggregates contained, at least, triple mutants (either Groups I + IV + VIII or Groups III + IV + VIII) and the double mutant (Groups IV + VIII). The preparation of ts-11 predominantly showed aggregates which sedimented faster than wild-type virus particles and which could be dispersed by the treatment with neuraminidase. Aggregates were formed most likely as a result of incomplete desialylation due to low neuraminidase activity of Group IV mutants.     

The structure and protein kinase activity of proteins encoded by nonconditional mutants and back mutants in the sec gene of avian sarcoma virus

We have characterizedsrc proteins encoded by approximately 30 nonconditional transformation-defective mutants of avian sarcoma virus (ASV) and by several back mutants which reestablish a transformed phenotype. We used gel electrophoresis of immunoprecipitated proteins labeled with³²PO₄ or [³⁵S]methionine to assess size, stability, and phosphorylation; partial digestion with staphylococcal V8 protease to determine structure; and an immune complex assay to measure protein kinase activity. The mutants were all isolated as phenotypic revertants of the B31 line of B77-ASV transformed rat cells, each revertant cell bearing a single provirus without appreciable deletions, as described in the accompanying report (Varmuset al., 1980). In several instancesm the mutant proteins were examined both in the revertant rat cells and in chicken cells infected with transformation-defective viruses rescued from the nonpermissive rat cells. In addition, secondary mutations to restore a transformed phenotype (back mutations) occurred in some cases, in the original rat cells and/or chicken cells infected with rescued viruses. Three categories of mutants were identified by this survey. The largest group (Class I) encodedsrc proteins of normal size (60,000M_r); these proteins were hypophosphorylated and exhibited little or no protein kinase activity.Class II mutants displayed immunoprecipitablesrc proteins of less than normal size. In three cases, the shortsrc related proteins were mapped to the amino terminus of wild-type pp60^src and may be the result of nonsense mutations; in two cases, the short proteins were mapped to the car?yl terminus. Most of Class II mutants lacked protein kinase activity, but the 45,000M_r protein in line 000 exhibited moderate levels of activity, thereby mapping the enzymatically active site to the car?yl terminal three-fourths of pp60^src. The smallest group of mutants (Class III) did not produce detectablesrc proteins. Some of the mutant proteins behaved differently in permissive and nonpermissive hosts; in particular, the product of mutant L produced fusiform transformation and was highly phosphorylated and associated with wild-type levels of protein kinase activity in chicken cells, but was nontransforming, hypo-phosphorylated, and associated with low levels of protein kinase activity in rat cells. In all cases, back mutation to a transformed phenotype was accompanied by a restoration of wild-type (or near wild type) levels of protein kinase activity, further documenting the functional significance of the enzymatic activity. Some of the back mutants, however, encoded proteins of atypical size, either smaller or larger than pp60^src. The active proteins larger than pp60^src ranged up to 68,000M_r in size and were altered at or near the amino terminus. In one case (a retransformed derivative of the Class II revertant 000), the generation of a functionalsrc protein of 68,000M_r coincided with the appearance of an insert of ca. 200 base pairs into the ASV provirus, within or adjacent to the coding region for the amino terminus ofsrc. The diversity of reagents, both mutants and back mutants, derived from the single provirus in B31 cells indicates that this system will be useful for correlation of functional and structural attributes ofsrc.     

Phenotypic heterogeneity among temperature-sensitive mutants of rous sarcoma virus. Studies with inhibitors of protein synthesis

Fourteen temperature-sensitive (ts), transformation-defective mutants have been isolated from mutagenized Schmidt-Ruppin Rous sarcoma virus. We report that, while in cells infected with most of the mutants all parameters of cell transformation were coordinately suppressed, certain ts mutants induced the ability of infected cells to multiply in soft agarose and to express the tumor-specific surface antigen (TSSA), in the absence of morphological conversion. Studies with inhibitors of protein synthesis have shown that cycloheximide (Ch) and pactamycin (Pac) act differently on focus formation from Pu and that the heat-labile src gene product of these mutants may be spontaneously reactivated following a downshift to permissive temperature. Our data also indicate the existence of two classes of mutants regarding the response of infected cells to Pu. In most cases, focus formation is inhibited by Pu when infected cells are shifted to 37°. However, cells infected with three ts mutants resume morphological transformation at permissive temperature in the presence of this inhibitor. In addition, the fact that the same mutants induce high levels of TSSA expression at restrictive temperatures suggests that the two properties may be linked.     

Revertants of an ASV-transformed rat cell line have lost the complete provius or sustained mutations in src

We have isolated and characterized 12 revertants of a clonal line (B31) of avian sarcoma virus (ASV)-transformed rat-1 cells. The B31 cells contain a single normal ASV provirus, display the classical features of virally transformed cells, and revert to normal phenotype at low frequency. Revertants isolated after selective killing of transformed cells resemble uninfected rat-1 cells morphologically, fail to grow in suspension, and are at least 100-fold less tumorigenic than B31 cells. Two mechanisms of reversion have been identified in these cells. (i) Three of the revertant lines have lost the entire provirus, including both copies of the sequences repeated at the ends of the provirus; the manner in which the provirus is lost is not known. (ii) The other nine revertants retain a provirus of normal size and unaltered flanking cellular DNA: contain the same species of viral RNA at the same concentrations as in the parental line, B31; are susceptible to retransformation by wild-type ASV; and yield transformation-defective (td) virus after fusion with chicken cells. In one case, the rescued virus transforms chicken cells, but produces fusiform rather than normal foci and does not retransform rat cells morphologically. Hence these revertants arise as a consequence of nonconditional mutations (base substitutions or small deletions) in the viral transforming gene,src. In several cases, the revertant cells retransform spontaneously, or transforming virus appears in stocks of rescued td virus after passage through chicken cells, indicating back mutations to wild-type. Several of the rescued td viruses can also recombine to restore a wild-type phenotype. Analysis of the structure and enzymatic activity of products ofsrc confirms that the revertant cells bear various mutations insrc.     

Temperature-sensitive mutants of influenza virus. VI. Transfer of TS lesions from the Asian subtype of influenza A virus (H2N2) to the Hong Kong subtype (H3N2).

Temperature-sensitive genetic lesions were transferred from the ts-1 (H2N2) and ts-2 (H2N2) mutants of influenza A virus to wild-type influenza A (H3N2) virus by genetic reassortment. The ts-2 (H3N2) recombinants appeared to be homogeneous and did not undergo recombination with one another, suggesting that the original ts-2 mutant of influenza A (H2N2) contained a ts lesion(s) on only one RNA segment of its genome. In contrast the ts-1 (H3N2) recombinants fell into three phenotypic subsets which differed in degree of temperature sensitivity. Initially, the three subsets of ts-1 (H3N2) recombinants were thought to represent distinct complementation-recombination groups. However, complementation-recombination between the three subsets of ts-1 (H3N2) recombinants subsets was variable. Subsequent study indicated that mutants in each of the three subsets shared one ts lesion and two of the subsets shared an additional ts lesion. The mechanism whereby viruses of the three subsets which share one or two ts lesions, and nevertheless undergo apparent complementation-recombination on occasion is not understood.     

Arenavirus recombination: The formation of recombinants between prototype pichinde and pichinde munchique viruses and evidence that arenavirus S RNA codes for N polypeptide

The ability of arenaviruses to form recombinant viruses by viral RNA segment reassortment has been investigated using a cloned, high-temperature adapted strain of prototype Pichinde virus (PIC), and a cloned, alternate Pichinde virus topotype named Pichinde Munchique (designated MUC). The oligonucleotide fingerprints of the large and small viral RNA segments of the two viruses can be easily distinguished. The virion RNA species of cloned wild-type progeny viruses recovered from dual wild-type PIC and MUC coinfections of BHK-21 cells have been analyzed by oligonucleotide fingerprinting. Other than PIC and MUC genotypes, progeny virus clones were found with PIC/MUC (large/small) RNA segment genotypes, indicating that these recombinant arenaviruses were formed by RNA segment reassortment. Dual infections of BHK-21 cells with a temperature-sensitive (ts), conditional lethal, mutant of MUC and the Group I ts mutants of PIC (but not the PIC Group II ts mutants), have yielded wild-type progeny viruses at high frequency. Genotype analysis of one of these recombinant progeny showed that it had a PIC/MUC genotype. This result indicated that the Group I mutants of PIC have a defective small viral RNA segment, and that the MUC ts mutant (and probably the PIC Group II ts mutants) has a defective large viral RNA segment. Plaque size analyses of the parental wild-type and reassortment viruses have shown that the plaque size of the reassortants was determined by the large viral RNA segment. Tryptic peptide analyses of the nucleocapsid (N) polypeptide of the PIC/MUC recombinant by comparison to similar analyses for PIC and MUC indicate that the recombinant has a MUC N polypeptide, i.e., that the viral S RNA codes for N polypeptide.     

The expression of pp60src and its associated protein kinase activity in cells infected with different transformation-defective temperature-sensitive mutants of Rous sarcoma virus

A set of five transformation-defective temperature-sensitive mutants of Rous sarcoma virus has been used to investigate the relation between pp60^src its associated protein kinase activity, and expression of the transformed phenotype. In radioimmune competition experiments, the levels of pp60^src induced by the mutants did not vary by more than a factor of two, either among the mutants at a given temperature or between nonpermissive and permissive temperatures for a given mutant. The mutants fell into two distinct classes with respect to the temperature conditional expression of pp60^src-associated kinase activity. Three mutants (GI 201, GI 202, and GI 251) induced two- to fivefold higher levels of pp60^src-associated kinase activity at the permissive temperature. The other mutants GI 203 and 253 induced only very low levels of pp60^src-associated kinase at either temperature. The pp60^src-associated kinase activity induced by GI 201, 202, and 251 at the permissive temperature was significantly more heat labile in vitro than that of the wild type. Furthermore, downshift of the mutant-infected cells to the permissive temperature resulted in a rapid increase (within 15 min) in the pp60^src-associated kinase activity only with mutants GI 201, GI 202, and GI 251, i.e., only with those mutants having an elevated activity at the permissive temperature. The results taken as a whole suggest that there is not a simple relationship between pp60^src, pp60^src-associated kinase activity, and transformation and support the idea of multifunctionality of the src gene product.     

Isolation of polyoma viruses lacking endonuclease HindIII sites.

Spontaneous mutants of polyoma virus have been isolated which lack either one or both of the two endo HindIII cleavage sites present in polyoma DNA. Some of the mutants contain single base changes or small deletions at the HindIII site in the early region of the polyoma genome. Most of these mutants have a normal phenotype; however, one member of this group grows poorly in comparison with wild-type virus. Other mutants contain large deletions, have lost either of the HindIII sites, and appear to require complementation by helper viruses in order to complete their growth cycle.     

Mutants of Rous sarcoma virus with extensive deletions of the viral genome.

Deletion mutants of Rous sarcoma virus (RSV) have been isolated from a stock of Prague RSV which had been irradiated with ultraviolet light. Quail fibroblasts were infected with irradiated virus and transformed clones isolated by agar suspension culture. Three clones were obtained which did not release any virus particles. Analysis of DNA from these non-producer clones with restriction endonucleases and the Southern DNA transfer technique indicated that the clones carry defective proviruses with deletions of approximately 4 × 10⁶ daltons of proviral DNA. The defective proviruses, which retain the viral transformation (src) gene, contain only 1.7–2.0 × 10⁶ daltons of DNA. Multiple species of viral RNA containing the sequences of the src gene were detected in these clones; some of these RNAs may contain both viral and cellular sequences. The protein product of the src gene, p60^src (Brugge and Erikson, 1977), was also synthesized in the nonproducer clones. However these clones did not contain the products of the group-specific antigen (gag), DNA polymerase (pol), or envelope glycoprotein (env) genes, nor did they contain the 35 and 28 S RNA species which are believed to represent the messengers for these viral gene-products. The properties of these mutants indicate that expression of the src gene is sufficient to induce transformation. These clones may represent useful tools for the study of the expression of this region of the genome.     

Neurotropic Virus-Host Relationship Alterations Due to Variation in Viral Genome as Studied by Electron Microscopy

A series of experiments has been described in which litters of suckling rats were inoculated either with wild-type reovirus type III or one of two of its temperature-sensitive (ts) mutants. While the wild-type virus produced an acute, fatal syndrome, the ts mutants were substantially less neurovirulent. Of the ts mutant-inoculated animals, a large percentage of the surviving (chronic) animals given ts mutant B showed an unobstructive hydrocephalus ex vacuo whereas chronic ts mutant C animals showed no visible nervous system disease. The ts mutants persisted within the central nervous system (CNS) for 6 to 8 weeks, after which they could not be detected either virologically, immunologically or morphologically. In another set of experiments, organized CNS explants were studied following infection with either measles virus or the neuroadapted Mantooth strain of SSPE virus, a variant of measles. Wild measles (Edmonston strain) exerted an acute destructive effect, but SSPE virus had a tendency to enter into coexistence with the tissue without destroying its organotypic nature. These relationships are somewhat reminiscent of the neuropathologic conditions caused by these two viruses in man. Since the reovirus type III ts mutants possess both genetic and morphologic defects and in many instances cause CNS conditions different from that induced by the wild-type virus, it has been proposed that a comparable situation may exist after measles and SSPE virus infection. SSPE virions of the strain studied were found to be defective in certain viral components which may have contributed to the lower neurovirulence and its entering into a chronic relationship with the CNS, in contrast to the acute destructive nature of measles infection. The findings are discussed in terms of relevance to other chronic CNS diseases, particularly multiple sclerosis, in which the possiblity exists that a mutant virus is operative.     

Persistent infections of green monkey kidney cells initiated with temperature-sensitive mutants of simian virus 40

An early SV40 temperature-sensitive (ts) mutant, tsA58, and a late mutant, tsB11, each expressed homotypie interference in CV-1 cells coinfected with WT. Interference required the functional activities of the mutant genomes and resulted from a competitive interaction between the mutants and WT. Another late mutant, tsD101 did not display interference activity. Nevertheless, each of the is mutants promoted the survival of CV-1 cells coinfected with WT at 37° and, consequently, facilitated the establishment of persistent infections. Each of the mutants was also more able than WT to establish persistent infections under conditions of single infection. Only a few percent of the cells in these persistent infections produced the SV40 T or V antigens and clonal isolates from these systems contained both antigen-producing and nonproducing cells. These systems were resistant to superinfection with WT SV40 but were completely susceptible to vesicular stomatitis virus. There was a rapid selection for ts⁺ virus in the persistent infections initiated by mixed infection with the ts mutants and WT. The proportion of ts⁺ virus also increased, although somewhat more slowly, in the systems initiated by infection with the mutants alone. Defective interfering (DI) particles were detected in some stocks by yield reduction assays. Many viral genomes in stocks displaying DI particle activity contained multiple BglI cleavage sites, indicating that they possess reiterations of the origin for DNA replication.