HIV-1 establishes immediate latency in T cells expressing the viral Nef protein

A variety of dual reporter HIV-1 derivatives have been developed that enable detection of infected cells independently from transcription initiated from the 5' LTR promoter. These reporters enable isolation of cells that are latently infected with HIV-1 provirus. We have previously described several dual reporter derivatives, including Red Green HIV-1 (RGH), which expresses mCherry from a constitutive internal promoter and GFP from the 5' LTR. A limitation of most dual HIV-1 reporter derivatives, including RGH, is that the Nef ORF is often disrupted to accommodate insertion of the internal reporter. Consequently, the potential role of Nef for establishment of latency using these reporters has not been clarified. To address this issue, we created three different RGH derivatives (RGHI), which express Nef from IRES elements downstream of the mCherry internal promoter. We found that each of these derivatives produced Nef protein at higher levels than wild-type LAI virus and that Nef expressed from each of the IRES elements was localized to the cell membrane. All of the Nef-expressing RGHI reporter viruses formed latent and productive infections and could be reactivated from latency at similar levels as the parental RGH derivative. These results indicate that expression of Nef does not affect the capability of HIV-1 to form latent infections. Furthermore, we propose that the RGHI derivatives described here represent novel tools to examine the role of Nef for HIV-1 replication.