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Evolution of virus and defective-interfering RNAs in BHK cells persistently infected with Sindbis virus
Abstract:
We analyzed a BHK cell line persistently infected with Sindbis virus for 16 months and a virus (Sin-16) cloned from these cells. Sin-16 virus was resistant to the defective interfering particles present in the original infection. We found that (i) cells infected with Sin-16 were impaired in the processing of a viral precursor glycoprotein, (ii) high-multiplicity passaging of Sin-16 gave rise to a variant that was able to generate and be inhibited by defective-interfering particles to which the original Sin-16 virus was resistant, and (iii) the persistently infected culture contained a heterogeneous mixture of defective Sindbis virus RNAs which were not packaged into extracellular particles. To determine whether these intracellular RNAs could interfere with the replication of Sin-16, we analyzed cells that were cloned from the persistently infected culture. One clone (A3) synthesized a single defective viral RNA which was lost with continued passaging in culture. Infection of A3 cells with Sin-16 showed that the presence of the defective RNA greatly enhanced cell survival and led to enrichment of this RNA. In contrast, cured cells were highly susceptible to killing by Sin-16, and survivors did not synthesize this RNA. Thus, A3 cells were not genetically altered in their response to Sin-16, but were protected from the cytopathic effects of infection by an RNA with the characteristics of a defective-interfering RNA.
Insights
Persistent Sindbis virus infection in BHK cells generates defective RNAs that protect cells from viral cytopathic effects. These intracellular defective RNAs enhance cell survival during Sin-16 virus infection.
Area of Science:
- Virology
- Molecular Biology
- Cell Biology
Background:
- Persistent Sindbis virus infection in BHK cells was studied over 16 months.
- A cloned virus, Sin-16, was resistant to defective interfering particles (DIPs) from the original infection.
Purpose of the Study:
- To investigate the role of intracellular defective Sindbis virus RNAs in persistent infections.
- To understand the mechanisms of viral resistance and cell survival during Sindbis virus infection.
Main Methods:
- Analysis of a BHK cell line persistently infected with Sindbis virus.
- Cloning and characterization of the Sin-16 virus variant.
- Isolation and analysis of defective Sindbis virus RNAs from infected cells.
- Cell cloning and infection assays to assess viral interference and cell survival.
Main Results:
- Sin-16 virus exhibited impaired viral precursor glycoprotein processing.
- High-multiplicity passaging of Sin-16 generated a variant sensitive to DIPs.
- Persistently infected cultures contained heterogeneous intracellular defective Sindbis virus RNAs.
- A specific defective RNA in clone A3 enhanced cell survival and was enriched upon Sin-16 infection.
- Cured cells were susceptible to Sin-16-induced killing, lacking the protective defective RNA.
Conclusions:
- Intracellular defective Sindbis virus RNAs, not packaged into extracellular particles, can interfere with viral replication.
- The presence of a specific defective RNA protected cells from the cytopathic effects of Sindbis virus infection.
- Cellular protection was mediated by the defective RNA, not by genetic alterations in the host cell's response to the virus.