GFP MMLV Retrovirus

During retroviral infection, histone-free DNA copies of the viral RNA genome are synthesized and rapidly loaded onto nucleosomes de novo upon entry into the nucleus. The potential role of viral accessory proteins in histone loading onto retroviral DNA has not been extensively investigated. The p12 protein of Moloney murine leukemia virus (MMLV) is a virion protein that is essential for tethering incoming viral DNA to host chromatin during early stages of infection. Infection with virions containing mutant p12 (PM14) results in the formation of viral DNA that does not accumulate in the nucleus. Here, researchers show that viral DNA from these mutants is not loaded with histones. Furthermore, DNA genomes delivered by mutant p12 exhibit prolonged association with the viral structural proteins nucleocapsid (NC) and capsid (CA). Viral DNA genomes lacking histones do not associate with the host RNA polymerase II machinery. These findings provide insights into fundamental aspects of retroviral biology, demonstrating that tethering of chromatin to the host cell and retention in the nucleus by p12 is required for histone loading onto viral DNA.

Previous studies have shown that PM14 mutant viruses express very poorly after infection. To test expression, HeLa cells were infected with VSV-G pseudotyped MMLV-WT GFP or p12-PM14 mutant GFP, and GFP expression was monitored by flow cytometric analysis 48 hours after infection (Figure 1A). The percentage of GFP-positive cells was reduced 60-fold to near background levels with the p12-PM14 mutant virus compared to the WT virus, and the average GFP fluorescence intensity was reduced by 3-fold (Figure 1A). The researchers also performed ChIP assays to measure the DNA fraction of RNA polymerase II (RNA Pol II) localized on viral DNA. For the WT virus, the MMLV-LTR showed a strong association with Pol II comparable to the constitutively expressed GAPDH gene (Figure 1B). Notably, 1 also detected a large fraction of the 2-LTR loop bound to Pol II (Figure 1B). This is surprising given that these circular DNAs are poorly expressed, indicating that Pol II is bound but not actively transcribed on these DNA templates. The p12-PM14 virus showed a dramatic decrease in the recovery of viral reporter DNA precipitated with Pol II antibodies compared to WT ( Figure 1B ), consistent with the very low GFP expression levels in this mutant ( Figure 1A ). Together, these findings suggest that the lack of histone loading on retroviral DNA is associated with very low Pol II accessibility and very low gene expression levels.

Figure 1. MMLV p12-PM14 mutant DNA is poorly expressed and has reduced binding to RNA polymerase II. (A) Flow cytometric analysis of HeLa cells 1 day after infection with VSV-G pseudotyped WT or p12-m14 mutant MMLV-GFP viruses. (B) RNA polymerase II ChIP analysis of chromatin harvested at 2 days following WT or p12-PM14 mutant MMLV-GFP infection of HeLa cells. (Wang G Z, Goff S P., 2021)