Mutants Of Sindbis Virus Research Articles

Protein Synthesis Directed by an RNA − Temperature-Sensitive Mutant of Sindbis Virus

Protein Synthesis Directed by an RNA ⁻ Temperature-Sensitive Mutant of Sindbis Virus

Viral RNAs associated with ribosomes in Sindbis virus-infected HeLa cells.

Virus specific RNA ribosome complexes were isolated by sucrose density gradient centrifugation of cytoplasmic extracts from HeLa cells infected at 42 C with an RNA(+) mutant (ts2) of Sindbis virus. Viral RNA-ribosome complexes were accumulated by infected cells treated with sodium fluoride and cycloheximide. The RNA-ribosome complexes were characterized by (i) their sensitivity to the action of ribonuclease or ethylenediaminetetraacetic acid, (ii) their density in cesium chloride gradients, and (iii) presence of host ribosomes and viral RNAs. The viral RNAs were isolated and characterized. The results showed that two species of single-stranded RNAs (a 28s and 18 to 15s species) were associated with the complexes. Base composition analysis of the viral RNAs indicated that both species had a higher adenine content than the 42s or 26s forms of viral RNAs. The RNAs associated with the ribosome complexes were virus specific since they annealed with denatured double-stranded RNAs from the infected cells. Little or no 42S RNA was associated with the RNA-ribosome complexes. The results suggest that the 28s and 18 to 15s forms of RNAs may represent viral messenger RNAs.

Protein synthesis directed by an RNA temperature-sensitive mutant of Sindbis virus.

The structural proteins of wild-type Sindbis virus were shown to arise by posttranslational cleavage of larger precursors. The proteins synthesized in wildtype infection were compared with those specified by ts-11, a temperature-sensitive mutant unable to synthesize viral RNA at the restrictive temperature. Abnormally large, virus-specific proteins were found in the mutant-infected cells after the shift from 28 C to 41.5 C. These large polypeptides were presumably precursors which were cleaved too rapidly to be detected in the wild-type infection. The largest had a molecular weight of 133,000 and was the same size as the apparent precursor detected during infection with a group of Sindbis mutants which could not form nucleocapsids at the nonpermissive temperature. The stability of ts-11-specific RNA synthesis, after shift from permissive to restrictive conditions, differed from that in cells infected by wild-type virus, indicating that the virus had a genetic lesion in an enzyme involved in RNA synthesis. This mutation might have caused the precursor to fold incorrectly so that it could not be cleaved. The possibility cannot be excluded, however, that a second lesion in an uncharacterized viral function, such as a protease, was the cause of the accumulation of the precursors.

Temperature Sensitivity of a Sindbis Virus Mutant Isolated from Persistently Infected <i>Aedes aegypti</i> Cell Culture

SV-S, a cloned small plaque mutant of Sindbis virus derived from persistently infected <i>Aedes aegypti</i> cell cultures, was shown also to be an RNA+ temperature-sensitive mutant. It interfered with the replication of SV-W at the nonpermissive temperature as well as the permissive temperature, but did not depress net viral RNA synthesis in cells coinfected with SV-W.

A simple and sensitive method for assay of chick interferon.

SummaryA new method for the assay of chick interferon (IF) was devised. Serial dilutions of IF were inoculated into wells made in the agar overlay covering chick embryo cells, and after 1 day's incubation at 37° the wells received a constant dose of the u mutant of Sindbis virus. Three days later a large plaque whose size was almost independent of the challenge virus dose appeared around the control well. This plaque formation was inhibited by the IF pretreatment. Thus, one monolayer dish, 90 mm in diameter, sufficed to titrate each IF sample. The highest IF dilution which reduced the normalized plaque diameter (the plaque diameter minus the cup diameter) to less than half that of control was comparable to the end point determined by the ordinary 50% plaque reduction test. The standard deviation of end points was approximately ±2-fold. A 1000-fold fluctuation in the challenge virus dose did not influence the IF titer determined. When a small-plaque clone of Sindbis virus was used and DEAE-dextran incorpor...

Association of viral ribonucleic acid with cellular membranes in chick embryo cells infected with Sindbis virus.

Membranes from cells infected with Sindbis virus had associated with them viral ribonucleic acid (RNA) polymerase and about 60 to 70% of the viral RNA labeled when short pulses were used. This RNA contained most of the replicative intermediate and replicative form of viral RNA found in the infected cells. The use of "Mg(2+) sarkosyl crystals" permitted the isolation of membrane-bound nucleic acids and allowed the demonstration that Sindbis virus RNA was synthesized on a membrane-viral RNA complex. Viral RNA from the infecting virions first became associated with the membranes during the latent period and, subsequently, slowly detached. The attachment of the viral RNA to the membranes did not require active viral RNA polymerase, since RNA from ts6, an RNA(-) temperature-sensitive mutant of Sindbis virus, associated with cellular membranes at a nonpermissive temperature. However, the subsequent detachment of the RNA from the membranes was restricted in the absence of viral RNA synthesis. The results indicate that association of viral RNA with cellular membranes may represent an early step occurring during the replication of Sindbis virus RNA.

Virus-specific proteins synthesized in cells infected with RNA+ temperature-sensitive mutants of Sindbis virus.

All Sindbis virus temperature-sensitive mutants defective in "late" functions were systematically surveyed by acrylamide-gel electrophoresis for similarities and differences in the intracellular pattern of virus-specific proteins synthesized at the permissive and nonpermissive temperatures. Only cells infected with mutants of complementation group C showed an altered pattern. At the nonpermissive temperature, these mutants failed to induce the synthesis of a polypeptide corresponding to the nucleocapsid protein and instead overproduced a protein of higher molecular weight than either viral structural protein. This defect was shown to be irreversible by the finding that (3)H-leucine incorporated at 41.5 C specifically failed to appear in the nucleocapsid of virions subsequently released at 29 C. Attempts to demonstrate a precursor protein in wild-type infections were inconclusive.

Maintenance of the u mutant of Sindbis virus by 40 degree passage.

The unusually large-plaque (u) mutant previously isolated from a small-plaque clone of Sindbis virus was maintained first by cloning passage, because the ordinary passage resulted in replacement of the u mutant by the more interferon-resistant parent type virus. After 9 cloning passages, it was found that the virus did not form such large plaques at 40 ° as it did before, and the pfu titer determined at 40 ° was lower than that determined at 37 °. However, serial passage of this virus in a standard passage manner adopting incubation at 40 ° allowed it to regain the original plaque morphology, i.e., formation of very large plaques at 40 ° as well as at 37 ° with about equal plating efficiencies. It seems likely that back mutation toward the parent type seldom occurs at 40 °. The virus so maintained was found to be slightly more sensitive to interferon, less pathogenic for intracerebrally inoculated baby mice, and more resistant to agar inhibitor than the parent type virus.

Viral events necessary for the induction of interferon in chick embryo cells.

Temperature-sensitive mutants of Sindbis virus were employed to investigate the nature of the viral event(s) which induces chick-embryo cells to produce interferon. Chick embryo cells induced by the parental heat-resistant strain of Sindbis virus produced essentially equal amounts of interferon at 29 and 42 C. An RNA(-) and three RNA(+) strains [temperature-sensitive mutants unable (RNA(-)) and able (RNA(+)) to make ribonucleic acid] produced interferon at 29 C but not at 42 C. It is concluded that viral RNA per se and the replication of viral RNA do not induce interferon production by chick embryo cells.

Maturation defects in temperature-sensitive mutants of Sindbis virus.

Temperature-sensitive mutants of Sindbis virus, which synthesize viral ribonucleic acid (RNA) but not mature virus at the nonpermissible temperature, were selected for the study of viral maturation. Of these, three mutants which complement each other genetically were used. Two major proteins, the nucleocapsid and membrane proteins, located, respectively, in the viral nucleoid and membrane, were found in intact virions. In cells infected with wild-type Sindbis virus, four distinct types of viral RNA with sedimentation coefficients of 40S, 26S, 20S, and 15S were detected in constant distribution. The 20S RNA was ribonuclease-resistant, whereas the other types were ribonuclease-sensitive. The 40S RNA, identical to that obtained from the virion, was found associated with nucleocapsid protein as a subviral particle, which was assumed to be the nucleoid. Viral materials from cells infected with the mutants under nonpermissive conditions were compared with those from cells infected with wild-type virus, in terms of (i) the distribution of the different types of RNA, (ii) the association of infectious viral RNA into subviral particles, and (iii) the ability of infected cells to hemadsorb goose erythrocytes. According to these criteria, each of the three mutants demonstrated different maturation defects. Defective nucleocapsid proteins and membrane proteins may each account for one of the above mutants. The thrid mutant may have defects in a minor structural protein or possibly a maturation protein which is involved in the assembly of Sindbis virus.

Functional defects of temperature-sensitive mutants of Sindbis virus

Certain temperature-sensitive mutants of Sindbis virus are able to induce the synthesis of viral RNA at a temperature that does not permit production of complete virus. Some of the mutants probably specify a defective viral structural protein. Mutants with such defects fall into three complementation groups. We have determined the ability of these mutants to carry out two steps of viral morphogenesis known to occur in wild-type infections: (1) intracellular formation of viral cores (nucleocapsids), and (2) alteration of the membrane of infected cells that results in the phenomenon of hemadsorption. Some mutants were observed to be defective in step (1), others in step (2); no mutant was defective in both steps. All mutants defective in a given function were members of the same previously defined complementation group. Thus a direct correlation was established between the genetic classification of these mutants and their functional defects.

Unusually Large-Plaque Mutant of Sindbis Virus

SummaryWhen Sindbis virus plaques were formed in chick embryo fibroblasts (CEF) at 40°C, plaques varying in size were observed. Plaques measuring less than 2 mm (sp) were subjected to cloning. Out of 51 sp plaques so examined, three yielded predominantly, on passage through CEF, unusually large-plaque (u) mutants. This mutant as well as the parent large-plaque (lp) and sp viruses were purified by consecutive clonings. Mixed infection of CEF with the sp and u viruses tended to exclude the sp virus. Contrarily, mixed infection with the lp virus and the u mutant tended to exclude the latter. Thus for maintenance of the u mutant, frequent clonings had to be done, because otherwise an incidental appearance of the parent type virus by back mutation seemed to result in exlusion of the u mutant virus. The u mutant as well as the sp virus was more sensitive to interferon than the lp virus. The reason why the interferon-sensitive u mutant can grow more rapidly and yield a higher amount of virus than the parent lp v...

Temperature-sensitive mutants of Sindbis virus: biochemical correlates of complementation.

Temperature-sensitive mutants of Sindbis virus fail to grow at a temperature that permits growth of the wild type, but when certain pairs of these mutants, mixed together, infect cells at that temperature, viral growth (i.e., complementation) occurs. The yield from this complementation, however, is of the same order of magnitude as the infectivity in the inoculum. Since in animal virus infections the protein components of the virion probably enter the cell with the viral nucleic acid, it was necessary to demonstrate that the observed complementation required synthesis of new viral protein and nucleic acid rather than some sort of rearrangement of the structural components of the inoculum. To demonstrate that complementation does require new biosynthesis, three biochemical events of normal virus growth have been observed during complementation and correlated with the efficiency of viral growth seen in complementation. These events include: (i) entrance of parental viral ribonucleic acid (RNA) into a double-stranded form; (ii) subsequent synthesis of viral RNA; and (iii) synthesis and subsequent incorporation of viral protein(s) into cell membranes where they were detected by hemadsorption. Although the infecting single-stranded RNA genome of the wild type was converted to a ribonuclease-resistant form, the genome of a mutant (ts-11) incapable of RNA synthesis at a nonpermissive temperature was not so converted. However, during complementation with another mutant also defective in viral RNA synthesis, some of the RNA of mutant ts-11 was converted to a ribonuclease-resistant form, and total synthesis of virus-specific RNA was markedly enhanced. The virus-specific alteration of the cell surface, detected by hemadsorption, was also extensively increased during complementation. These observations support the view that complementation between temperature-sensitive mutants and replication of wild-type virus are similar processes.

Isolation and characterization of conditional-lethal mutants of Sindbis virus

Twenty-three temperature-sensitive conditional-lethal mutants of Sindbis virus, a group A arbovirus, have been isolated and characterized. These appear to be single-step mutants whose replication at high temperatures is blocked by the temperature-sensitive defect of a protein determined by the viral genome. Two categories of mutants are found: those that synthesize infectious RNA but not infectious virus at high temperatures ( RNA + ), and those that produce neither infectious RNA nor infectious virus ( RNA −). RNA + mutants are in general more heat labile at 60 ° than either RNA − mutants or the Sindbis strain from which they were isolated. The most probable explanation for this behavior is discussed.

Complementation between temperature-sensitive mutants of Sindbis virus

Eleven temperature-sensitive mutants of Sindbis virus were classified according to their ability to complement other mutants in pairwise mixed infections at a non-permissive temperature. Five nonoverlapping complementation groups were thus defined. In two groups the temperature-sensitive function is required for viral RNA synthesis, while mutants of the other three groups are unable to carry out some essential step occurring after viral RNA synthesis. The progeny produced by complementation were found, in general, to include both the genomes used to initiate the mixed infection; these progeny particles were phenotypically mixed in a manner that suggests exclusion of mutationally altered protein from rescued particles.

Phenotypic Mixing between Group A Arboviruses

WHEN two different viruses infect the same cell some of the progeny may exhibit phenotypic mixing: that is, the genome of one parent may be enclosed in a coat determined, at least in part, by the genome of the other parent. Thus the phenotype may not correspond to the genotype. This anomaly disappears after one cycle of growth if further mixed infections are avoided. Phenotypic mixing was first detected in bacteriophage1 and has subsequently been observed in polio viruses2 and myxoviruses3. We shall describe a double mutant of Sindbis virus (a group A arbovirus) that can be used in a simple test for phenotypic mixing with other wild-type arboviruses. Such a test for phenotypic mixing may be useful in confirming or suggesting relationships among these viruses. The methods used for the growth and titration of arboviruses in primary chick embryo cultures have been described4.

Conditional-Lethal Mutants of an Animal Virus: Identification of Two Cistrons

Two different temperature-sensitive conditional-lethal mutants of Sindbis virus, an animal virus that contains RNA, have been isolated. When cultured in chick fibroblast monolayers at 42 degrees C, these mutants yield less than 0.05 percent as much virus as does the wild type, whereas at 27 degrees C they grow normally. One mutant appears to be altered in the synthesis of a protein that is produced early in the infection and is required for viral RNA synthesis. The other mutant produces as much infectious RNA as the wild type at 42 degrees C and appears to be altered in the synthesis of a protein produced late in the infection.