Fig 6. Excision of HIF1α decreases viral reactivation from latency in infected splenocytes in vivo.

C57BL/6J mice (n = 3–4) or B6.129-Hif1a^tm3Rsjo/J (n = 3–4) were infected with MHV68-Cre virus by intranasal infection. Splenocytes were harvested on day 16. (A) Limiting-dilution PCR was performed from infected WT and HIF1αLoxP splenocytes with two rounds of PCR were performed against MHV68 ORF50. There were no statistical differences (P = 0.4350) between the two groups of mice infected with MHV68-Cre virus. Data represent results for one experiment (n = 5 for each mice strain) out of two experiments (B) Ex vivo reactivation by limiting-dilution assay was performed to determine the frequency of infected WT and HIF1αLoxP splenocytes that harbor the viral genome. The frequency of cells reactivating the virus in HIF1αLoxP mice were less (1 in 73,181 splenocytes), when compared to C57Bl/6J mice (1 in 16,818 splenocytes) and was statistically significant (P = 0.0287). For both limiting-dilution assays, curve fit lines were derived from nonlinear regression analysis. Symbols represent the mean (n = 5 per mice strain) percentage of wells positive for virus CPE +/- the standard error of the mean. (The dotted line represents 63.2%, from which the frequency of cells reactivating virus was calculated based on the Poisson distribution. Data represents results of one experiment (n = 5 per mice strain) out of more than three independent experiments. Statistical significance was determined in Graph Pad Prism by Student’s t- test. *, p< 0.05. (C) RNA from 10⁷ splenocytes (25ng per PCR rxn) of WT and HIF1αLoxP mice were analyzed for excision of HIF1α exon 2 by PCR and the products were run on DNA agarose gels. Tissue from an uninfected naïve HIF1αLoxP mouse was used as negative control. A 400bp fragment was observed only HIF1αLoxp and not in parental WT mice when infected with MHV68-Cre virus.