Maturation Defects in Temperature-sensitive Mutants of Sindbis Virus

Abstract

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 third mutant may have defects in a minor structural protein or possibly a maturation protein which is involved in the assembly of Sindbis virus.

Intracellular Components Associated with Chikungunya Virus‐Specific Ribonucleic Acids in Infected BHK21 Cells1

“…Concentration of NaCl in both solutions did not seem to have any significant effect on the extraction efficiency, because the 65 S component was obtained with low NaCl solutions containing 0.001m EDTA as effectively, but not with 0.1m NaCl solutions containing 0.0015M MgCl2. It might be worth to recall that, with other group A arboviruses, the 65 S component was prominent when infected cells were extracted with buffer solutions containing EDTA [3] or at a low magnesium concentration [28], and that when cell homogenate was prepared in RSB, 26 S RNA was mainly associated with cytoplasmic reticulum sedimenting to the bottom of the sucrose gradient [2], although a small peak of the 65 S component was identified [2,8].…”

Viral Events Necessary for the Induction of Interferon in Chick Embryo Cells

“…Our data confirm those of Skehel and Burke (8) which showed that early viral events are needed to induce interferon production in chick cells. Also, viral RNA replication, or its accumulation in the cell (10), is insufficient to account for interferon production; if it is assumed that double-stranded RNA is a necessary intermediate for replication, these molecules are probably not the actual inducers of interferon production.…”

“…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 RNAand 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.962 Vol. 2,No. 10…”

Interferon Induction by Viruses

“…We also used cultures of chick embryo cells as our inducible system. As inducers we used several temperature sensitive mutants of Sindbis virus which differ with respect to their ability to replicate their viral RNA at 42°C (42,43). Sindbis virus replicates in chick embryo cells under our laboratory conditions to produce several thousand plaque-forming units per cell of virus in about 8-10 hr (43).…”

“…As inducers we used several temperature sensitive mutants of Sindbis virus which differ with respect to their ability to replicate their viral RNA at 42°C (42,43). Sindbis virus replicates in chick embryo cells under our laboratory conditions to produce several thousand plaque-forming units per cell of virus in about 8-10 hr (43). Viral RNA synthesis can be demonstrated by the use of uridine-3H and actinomycin D. Viral RNA synthesis commences at about 1 i hr after infection and reaches a maximum rate at about 6 hr.…”