Antagonism of Host Antiviral Responses by Kaposi's Sarcoma-Associated Herpesvirus Tegument Protein ORF45
Figure 5
Evaluation of type I interferon (IFN) and downstream IFN stimulatory gene (ISG) transcription.
HFF cells seeded in 6-well plates were infected with the KSHV wild-type (BAC36) or the ORF45-null recombinant (stop45) viruses. Six hours post-infection, cells were lysed with Trizol reagent and total RNAs isolated. Residual DNA contamination was eliminated by subsequent treatment with Turbo DNase I and the RNA was subsequently reverse transcribed to cDNA. The cDNA samples were then subjected to an absolute real-time PCR based quantification with specific primers for IFNA1, IFNB and selected downstream antiviral effector genes (ISG56, MxA, PKR and OAS). The amounts of mRNA were quantitated based on comparison with the standard templates of cloned cDNAs of known copy number following which the expression levels were normalized to GAPDH.