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These reporters enable isolation of cells that are latently infected with HIV\u20101 provirus. We have previously described several dual reporter derivatives, including Red Green HIV\u20101 (RGH), which expresses mCherry from a constitutive internal promoter and GFP from the 5\u2032 LTR. A limitation of most dual HIV\u20101 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\u2010type LAI virus and that Nef expressed from each of the IRES elements was localized to the cell membrane. All of the Nef\u2010expressing 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\u20101 to form latent infections. Furthermore, we propose that the RGHI derivatives described here represent novel tools to examine the role of Nef for HIV\u20101 replication.<\/jats:p>","DOI":"10.1002\/2211-5463.70186","type":"journal-article","created":{"date-parts":[[2025,12,20]],"date-time":"2025-12-20T12:20:26Z","timestamp":1766233226000},"page":"1132-1141","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["<scp>HIV<\/scp>\n                    \u20101 establishes immediate latency in T\u00a0cells expressing the viral Nef protein"],"prefix":"10.1002","volume":"16","author":[{"given":"Cindy","family":"Lam","sequence":"first","affiliation":[{"name":"Department of Biochemistry and Molecular Biology Molecular Epigenetics Group, Life Sciences Institute, University of British Columbia  Vancouver 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