Summary auto-generated
Researchers created a HeLa cell line (HeAntiL) that constitutively expresses antisense RNA targeting the vesicular stomatitis virus (VSV) large polymerase gene (L) to test whether this approach could inhibit viral infection. VSV is a negative-sense RNA virus that replicates rapidly in the cytoplasm. The HeAntiL cells were generated by stable transfection with an expression plasmid containing the first 550 nucleotides of the VSV L gene in antisense orientation. When infected with VSV at low multiplicity of infection (m.o.i. 0.01 or 0.1), HeAntiL cells showed delayed cytopathic effects compared to control cells, postponing cell death by 5-9 hours and reducing viral titers by at least one log-fold. Ribonuclease protection assays demonstrated a 10-20 fold reduction in viral L mRNA levels in HeAntiL cells at various timepoints before cell death. However, complete inhibition was not achieved because VSV replicates so rapidly that viral L mRNA accumulation eventually overwhelmed the available antisense RNA, particularly as VSV-induced shutdown of host cell transcription reduced antisense RNA production. This represents the first reported successful reduction of VSV infectivity using stably integrated antisense RNA, though the fast replication rate of VSV prevented sustained complete inhibition.
Key findings
- Antisense RNA expression targeting the VSV large polymerase gene reduced viral L mRNA by 10-20 fold and delayed virus-induced cell death by 5-9 hours depending on multiplicity of infection
- Viral titers were reduced by at least one log-fold in cells expressing antisense RNA compared to controls at the time of cell death
- Complete inhibition of VSV infection could not be achieved because rapid viral RNA accumulation and VSV-mediated shutdown of host cell transcription eventually overwhelmed the supply of antisense RNA
- This study demonstrates the first successful reduction of VSV infectivity using stably integrated antisense RNA in mammalian cells, though VSV's rapid replication rate limits the duration of antiviral effect
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Abstract
To study the effect of virus-specific antisense RNA expression on vesicular stomatitis virus (VSV) infectivity in cultured cells, a HeLaS3 cell line constitutively expressing antisense RNA complimentary to a portion of the VSV large RNA-dependent RNA polymerase gene (L) was established (HeAntiL). At an m.o.i. of 0.01 or 0.1, the HeAntiL cell line was able to reduce virus titre and delay virus-induced cell death by 9 or 5 h, respectively, when compared to a HeLa cell line stably transfected with the expression vector devoid of antisense sequence. Ribonuclease protection experiments showed a 10-20-fold reduction of hybridizable virus L mRNA in infected HeAntiL cells compared to infected control cells at various times before cell death. These results indicate that the antisense RNA approach can significantly reduce VSV mRNA transcription and virus production for a reasonable period of time. The robust growth rate of VSV eventually overwhelms the available antisense RNA and leads to delayed cell death.