Background: Through incorporation into virus particles, the HIV‑1 Vpr protein participates in the early steps of the virus life cycle by influencing the reverse transcription process. We previously showed that this positive impact on reverse transcription was related to Vpr binding to the uracil DNA glycosylase 2 enzyme (UNG2), leading to enhance‑ ment of virus infectivity in established CD4‑positive cell lines via a nonenzymatic mechanism. Results: We report here that Vpr can form a trimolecular complex with UNG2 and the p32 subunit (RPA32) of the replication protein A (RPA) complex and we explore how these cellular proteins can influence virus replication and dissemination in the primary target cells of HIV‑1, which express low levels of both proteins. Virus infectivity and replication in peripheral blood mononuclear cells and monocyte‑derived macrophages (MDMs), as well as the efficiency of the viral DNA synthesis, were significantly reduced when viruses were produced from cells depleted of endogenous UNG2 or RPA32. Moreover, viruses produced in macrophages failed to replicate efficiently in UNG2‑ and RPA32‑depleted T lymphocytes. Reciprocally, viruses produced in UNG2‑depleted T cells did not replicate efficiently in MDMs confirming the positive role of UNG2 for virus dissemination. Conclusions: Our data show the positive effect of UNG2 and RPA32 on the reverse transcription process leading to optimal virus replication and dissemination between the primary target cells of HIV‑1.