1103 J. Exp. Med.  The R ockefeller University Press • 0022-1007/ 98/ 04/ 1103/ 09 $2.00 Volume 187, Number 7, April 6, 1998 1103–1111 http:/ / www.jem.org Human Immunodeficiency Virus Type 1 Vpr Is a Positive Regulator of Viral Transcription and Infectivity in Primary Human Macrophages By R amu A. Subbramanian,* Allegria Kessous-Elbaz, ‡ R obert Lodge,* Janique Forget,* Xiao-Jian Yao,* Dominique Bergeron,* and Eric A. Cohen* From the *Laboratory of Human R etrovirology, Department of Microbiology and Immunology, and the ‡ Department of Pathology, Faculty of Medicine, University of Montreal, Montreal, Q uebec, Canada H3C3J7 Summary It is currently well established that HIV-1 Vpr augments viral replication in primary human macrophages. In its virion-associated form, Vpr has been suggested to aid efficient translocation of the proviral DNA into the cell nucleus. Although Vpr growth-arrests dividing T cells, the relevance of this biological activity in nondividing macrophages is unclear. Here we use Vpr- mutants to demonstrate that the molecular determinants involved in G2-arresting T cells are also involved in increasing viral transcription in macrophages, even though these cells are re- fractive to the diploid DNA status typical of G2 phase. Our results suggest that the two pheno- types, namely the nuclear localization and the G2-arrest activity of the protein, segregate func- tionally among the late and early functions of Vpr. The nuclear localization property of Vpr correlates with its ability to effectively target the proviral DNA to the cell nucleus early in the infection, whereas the G2-arrest phenotype correlates with its ability to activate viral transcrip- tion after establishment of an infection. These two functions may render Vpr’s role essential and not accessory under infection conditions that closely mimic the in vivo situation, that is, primary cells being infected at low viral inputs. T he Vpr protein is one of the regulatory gene products coded by HIV-1, the etiological agent of AIDS, and is expressed late in the infection cycle (1, 2). All primate len- tiviruses including HIV-1 code for a Vpr-like product. However, some lentiviruses such as HIV-2 and most sim- ian immunodeficiency viruses (SIVs) 1 code not only for Vpr but also for a second protein named Vpx, which shares considerable sequence homology with Vpr (3, 4). Pheno- typically, several studies demonstrate that viruses that code for a Vpr protein replicate to much higher levels in mac- rophages than do their Vpr-negative counterparts (5–9). However, exactly how the protein contributes to this effect is not clearly understood. The fact that Vpr is packaged in the progeny virions at high copy numbers strongly suggests that the protein could play a role early in the infection (10). Experimental support for an early functional role comes from studies in which Vpr contributed to the nuclear import of proviral DNA in nondividing cells such as macrophages (11). In addition, a late function is also suggested by experiments in which ex- cluding the late de novo expression of Vpr effectively abol- ished the augmented replication even though the protein was made available in the virion early in the infection (7). However, the ability of Vpr to target the proviral DNA to the nuclear compartment early in the infection was not monitored in this study, and therefore the authors could not formally preclude an additional role for the protein early in the infection (7). Hence, several issues remain unclear about our under- standing of the ability of Vpr to increase viral replication in macrophages. Does Vpr play a role late in the viral life cy- cle that can be segregated from its function early in the in- fection, and if so, at what replication step does this late function manifest itself? Given the fact that other virion- associated proteins such as Gag matrix p17 (11, 12) and vi- ral integrase (13) also appear to facilitate proviral nuclear import, is Vpr’s nuclear targeting function essential? If not, what is the relevance of evolutionary conservation of mul- tiple nucleophilic determinants? If so, why has it not been detected in earlier studies? Finally, is Vpr’s ability to 1 A bbreviations used in this paper: 2-AR , 2-adrenergic receptor; SIV, sim- ian immunodeficiency virus. on June 25, 2015 jem.rupress.org Downloaded from Published April 6, 1998