Bryan T. Eaton

Heterogeneity in the poly(A) content of the genome of sindbis virus

Abstract The genome of Sindbis virus is a single-stranded infectious 42S RNA molecule which contains poly(A). Virion RNA molecules can be separated into two fractions by either filtration through Millipore membranes in 0.5 M KCl or by sequential phenol extraction of virions at pH 7.6 and pH 9.0. Eighty to ninety percent of 42S RNA molecules fail to bind to Millipore membranes and 97–98% remain in the aqueous phase during phenol extraction at pH 7.6. Both these nonbinding and pH 7.6 RNA species contain a poly(A) sequence 60–80 nucleotides in length. The remainder of the virion RNA molecules which bind to membranes and are extractable from virions by phenol at pH 9.0 contain a larger poly(A) species of 150–250 nucleotides. Membrane binding and nonbinding, pH 7.6 and pH 9.0 species of viral 42S RNA, are equally infectious. Thus, the size of the poly (A) sequence in a 60- to 250-nucleotide range is not a factor influencing the infectivity of Sindbis RNA.

Altered pattern of viral RNA synthesis in cells infected with standard and defective Sindbis virus

Abstract Serial passage of Sindbis virus on BHK cells results in the accumulation of defective interfering (DI) particles (Schlesinger et al. , 1972). Cells infected with preparations of Sindbis passaged extensively on BHK cells (Sindbis H) produce reduced amounts of infectious virus and contain low levels of 42 S and 26 S RNA and 20 S RNase-resistant replicative form (RF). In Sindbis H-infected cells the major single-stranded RNA species sediments at 20 S and the RF at 12 S. The interfering component in Sindbis H is sensitive to ultraviolet light and indistinguishable from standard virions in haemagglutination inhibition tests. Sindbis M was prepared by passage of Sindbis H in suckling mouse brain. The presence of 42 S and 26 S single-stranded RNA and 20 S RNase-resistant RF in Sindbis M infected cells (up to passage 5) indicates that mouse brain-passaged Sindbis lacks demonstrable interfering components.

Defective interfering particles of Semliki Forest virus generated in BHK cells do not interfere with viral RNA synthesis in Aedes albopictus cells

Abstract Defective interfering (DI) particles of Semliki Forest virus (SFV) were generated by serial high multiplicity passage of plaque-purified virus on BHK cells. Defective interfering passages of SFV depressed the synthesis of 42 and 26 S viral RNA and induced the formation of two new single-stranded RNA forms (molecular weight, 1.2 × 10 6 and 0.56 × 10 6 ) in BHK and Vero cells but not in Aedes albopictus cells. These results suggest that the invertebrate cells restricted replication of the alphavirus DI particles.

Evidence for the synthesis of defective interfering particles by Aedes albopictus cells persistently infected with sindbis virus

Abstract The species of double- and single-stranded viral RNA in Aedes albopictus cells persistently infected with Sindbis virus have been analyzed. In addition to small amounts of 42-S and 26-S single-stranded and 23-S double-stranded viral RNA, persistently infected mosquito cells contain several new species of single-stranded RNA with molecular weights ranging from 0.65 x 108 to 1.0 x 108. New double-stranded RNA forms with sedimentation coefficients in the 12- to 15-S range are also detected. Similar double- and single-stranded RNA forms are synthesized in BHK cells after infection with virus released from persistently infected cells. Treatment of the virus from persistently infected cells with anti-Sindbis antiserum inhibits the synthesis of all the viral RNA species in BHK cells. These results suggest that DI particles of Sindbis virus may be synthesized and released from persistently infected A. albopictus cells.

Sindbis virus variants from a persistently infected mosquito cell culture contain an altered E2 glycoprotein

Small plaque temperature-sensitive (SPTS) variants of Sindbis virus were isolated from persistently infected Aedes albopictus cells 73 days after infection with wild-type virus. The viral RNA and protein species synthesized in chick embryo fibroblast (CEF) cells infected by one variant, clone 73-2, were compared with their wild-type virus counterparts. T1 RNase fingerprints of 32P-labeled intracellular 42 S and 26 S viral RNA species revealed that clone 73-2 virus RNA differed from wild-type virus RNA in four oligonucleotide spots. Three of these were located in the region of the genome coding for structural proteins. Analysis of the virus structural proteins by polyacrylamide gel electrophoresis indicated that clone 73-2 virus glycoprotein E2 had a higher electrophoretic mobility than wild-type virus E2. Analysis of the proteins synthesized in infected cells in the presence and absence of tunicamycin revealed that both the glycosylated and unglycosylated forms of clone 73-2 PE2 migrated slightly faster than their wild-type virus counterparts.

Transient enhanced synthesis of a cellular protein following infection of Aedes albopictus cells with Sindbis virus

Abstract Sindbis or Chikungunya virus infection of Aedes albopictus cells led to the synthesis of maximum amounts of intracellular virus structural protein at approximately 24 hr postinfection. Subsequently, viral protein synthesis declined and by 3 days postinfection little capsid protein was synthesized in infected cells. Starting at 2 days postinfection and for a period of 2–3 days, infected cells synthesized large amounts of a predominantly nuclear protein with molecular weight 43K (P43). In addition, smaller quantities of four other nuclear proteins with molecular weights 80K, 71K, 41K, and 26K were also transiently synthesized. Several lines of evidence showed that P43 is very closely related to, if not identical to a 43K protein found in the nuclei of uninfected cells. First, the 43K protein from uninfected cells and P43 were indistinguishable on the basis of their electrophoertic mobilities in both one- and two-dimensional separations in denaturing poly-acrylamide gels. Second, both P43 and the 43K protein generated the same digestion products after treatment with Staphylococcus aureus V8 protease. Heterogeneous nuclear ribonucleoprotein (hnRNP) particles were isolated from the nuclei of both uninfected and virus infected A. albopictus cells 3 days postinfection. HnRNP particles sedimented at 39 S and contained, as their major constituent, a protein which comigrated in both one- and two-dimensional gels with P43. These data indicate that alphavirus infection of A. albopictus cells leads to a transient enhancement in the synthesis of a 43K hnRNA binding protein.