Michael Emerman

Genome organization and transactivation of the human immunodeficiency virus type 2.

Analysis of the nucleotide sequence of the human retrovirus associated with AIDS in West Africa, HIV-2, shows that it is evolutionary distant from the previously characterized HIV-1. We suggest that these viruses existed long before the current AIDS epidemics. Their biological properties are conserved in spite of limited sequence homology; this may help the determination of the structure–function relationships of the different viral elements.

Visualization of the intracellular behavior of HIV in living cells

To track the behavior of human immunodeficiency virus (HIV)-1 in the cytoplasm of infected cells, we have tagged virions by incorporation of HIV Vpr fused to the GFP. Observation of the GFP-labeled particles in living cells revealed that they moved in curvilinear paths in the cytoplasm and accumulated in the perinuclear region, often near the microtubule-organizing center. Further studies show that HIV uses cytoplasmic dynein and the microtubule network to migrate toward the nucleus. By combining GFP fused to the NH2 terminus of HIV-1 Vpr tagging with other labeling techniques, it was possible to determine the state of progression of individual particles through the viral life cycle. Correlation of immunofluorescent and electron micrographs allowed high resolution imaging of microtubule-associated structures that are proposed to be reverse transcription complexes. Based on these observations, we propose that HIV uses dynein and the microtubule network to facilitate the delivery of the viral genome to the nucleus of the cell during early postentry steps of the HIV life cycle.

Human immunodeficiency virus infection of cells arrested in the cell cycle.

Cell proliferation is necessary for proviral integration and productive infection of most retroviruses. Nevertheless, the human immunodeficiency virus (HIV) can infect non‐dividing macrophages. This ability to grow in non‐dividing cells is not specific to macrophages because, as we show here, CD4+ HeLa cells arrested at stage G2 of the cell cycle can be infected by HIV‐1. Proliferation is necessary for these same cells to be infected by a murine retrovirus, MuLV. HIV‐1 integrates into the arrested cell DNA and produces viral RNA and protein in a pattern similar to that in normal cells. In addition, our data suggest that the ability to infect non‐dividing cells is due to one of the HIV‐1 core virion proteins. HIV infection of non‐dividing cells distinguishes lentiviruses from other retroviruses and is likely to be important in the natural history of HIV infection.

HIV-1 Vpr increases viral expression by manipulation of the cell cycle : a mechanism for selection of Vpr in vivo

The human immunodeficiency virus type 1 (HIV-1) encodes a protein, called Vpr, that prevents proliferation of infected cells by arresting them in G2 of the cell cycle. This Vpr-mediated cell-cycle arrest is also conserved among highly divergent simian immunodeficiency viruses, suggesting an important role in the virus life cycle. However, it has been unclear how this could be a selective advantage for the virus. Here we provide evidence that expression of the viral genome is optimal in the G2 phase of the cell cycle, and that Vpr increases virus production by delaying cells at the point of the cell cycle where the long terminal repeat (LTR) is most active. Although Vpr is selected against when virus is adapted to tissue culture, we show that selection for Vpr function in vivo occurs in both humans and chimpanzees infected with HIV-1. These results suggest a novel mechanism for maximizing virus production in the face of rapid killing of infected target cells.

Retroviral DNA Integration: Viral and Cellular Determinants of Target-Site Selection

Retroviruses differ in their preferences for sites for viral DNA integration in the chromosomes of infected cells. Human immunodeficiency virus (HIV) integrates preferentially within active transcription units, whereas murine leukemia virus (MLV) integrates preferentially near transcription start sites and CpG islands. We investigated the viral determinants of integration-site selection using HIV chimeras with MLV genes substituted for their HIV counterparts. We found that transferring the MLV integrase (IN) coding region into HIV (to make HIVmIN) caused the hybrid to integrate with a specificity close to that of MLV. Addition of MLV gag (to make HIVmGagmIN) further increased the similarity of target-site selection to that of MLV. A chimeric virus with MLV Gag only (HIVmGag) displayed targeting preferences different from that of both HIV and MLV, further implicating Gag proteins in targeting as well as IN. We also report a genome-wide analysis indicating that MLV, but not HIV, favors integration near DNase I–hypersensitive sites (i.e., +/− 1 kb), and that HIVmIN and HIVmGagmIN also favored integration near these features. These findings reveal that IN is the principal viral determinant of integration specificity; they also reveal a new role for Gag-derived proteins, and strengthen models for integration targeting based on tethering of viral IN proteins to host proteins.

Capsid is a dominant determinant of retrovirus infectivity in nondividing cells.

ABSTRACT A major difference between lentiviruses such as human immunodeficiency virus (HIV) and most other retroviruses is their ability to productively infect nondividing cells. We present here genetic evidence for involvement of the capsid protein (CA) in the infectious phenotype in nondividing cells. A chimeric HIV type 1 (HIV-1) in which the MA and CA of HIV-1 are replaced with the MA, p12, and CA encoding sequences from murine leukemia virus (MLV) loses the ability to efficiently infect nondividing cells. Analysis of the accumulation of two-long-terminal-repeat circles implies that the impairment of nuclear transport of preintegration complexes is responsible for the restricted infection of this chimeric virus in nondividing cells. Incorporation of MLV MA and MLV p12 into HIV virions alone does not exert any adverse effects on viral infection in interphase cells. These results suggest that CA is the dominant determinant for the difference between HIV and MLV in the ability to transduce nondividing cells.

Nuclear import and cell cycle arrest functions of the HIV-1 Vpr protein are encoded by two separate genes in HIV-2/SIV(SM).

The vpr genes of human and simian immunodeficiency viruses (HIV/SIV) encode proteins which are packaged in the virus particle. HIV‐1 Vpr has been shown to mediate the nuclear import of viral reverse transcription complexes in non‐dividing target cells (e.g. terminally differentiated macrophages), and to alter the cell cycle and proliferation status of the infected host cell. Members of the HIV‐2/SIV(SM) group encode, in addition to Vpr, a related protein called Vpx. Because these two proteins share considerable sequence similarity, it has been assumed that they also exhibit similar functions. Here, we report that the functions of Vpr and Vpx are distinct and non‐redundant, although both proteins are components of the HIV‐2/SIV(SM) virion and reverse transcription complex. Characterizing SIV(SM) proviruses defective in one or both genes, we found that Vpx is both necessary and sufficient for the nuclear import of the viral reverse transcription complex. In contrast, Vpr, but not Vpx, inhibited the progression of infected host cells from the G2 to the M phase of the cell cycle. Thus, two independent functions of the HIV‐1 Vpr protein are encoded by separate genes in HIV‐2/SIV(SM). This segregation is consistent with the conservation of these genes in HIV‐2/SIV(SM) evolution, and underscores the importance of both nuclear transport and cell cycle arrest functions in primate lentivirus biology.

The Ability of Primate Lentiviruses to Degrade the Monocyte Restriction Factor SAMHD1 Preceded the Birth of the Viral Accessory Protein Vpx

The human SAMHD1 protein potently restricts lentiviral infection in dendritic cells and monocyte/macrophages but is antagonized by the primate lentiviral protein Vpx, which targets SAMHD1 for degradation. However, only two of eight primate lentivirus lineages encode Vpx, whereas its paralog, Vpr, is conserved across all extant primate lentiviruses. We find that not only multiple Vpx but also some Vpr proteins are able to degrade SAMHD1, and such antagonism led to dramatic positive selection of SAMHD1 in the primate subfamily Cercopithecinae. Residues that have evolved under positive selection precisely determine sensitivity to Vpx/Vpr degradation and alter binding specificity. By overlaying these functional analyses on a phylogenetic framework of Vpr and Vpx evolution, we can decipher the chronology of acquisition of SAMHD1-degrading abilities in lentiviruses. We conclude that vpr neofunctionalized to degrade SAMHD1 even prior to the birth of a separate vpx gene, thereby initiating an evolutionary arms race with SAMHD1.

HIV-1 Infection Requires a Functional Integrase NLS

HIV-1 is able to infect nondividing cells productively in part because the postentry viral nucleoprotein complexes are actively imported into the nucleus. In this manuscript, we identify a novel nuclear localization signal (NLS) in the viral integrase (IN) protein that is essential for virus replication in both dividing and nondividing cells. The IN NLS stimulates the efficient nuclear accumulation of viral DNA as well as virion-derived IN protein during the initial stages of infection but is dispensable for catalytic function. Because this NLS is required for infection irrespective of target cell proliferation, we suggest that interactions between uncoated viral nucleoprotein complexes and the host cell nuclear import machinery are critical for HIV-1 infection of all cells.

HIV-1 Sequence Variation: Drift, Shift, and Attenuation

Toward the low-end of the sliding scale of virus fitness is the generation of viruses with attenuated phenotypes. Such viruses are expected to exist in any virus population due to the high mutation rate of the virus. In an infected person with high viral burden, such viruses would normally be out-competed by the descendents of viruses with greater fitness. However, attenuated viruses could occasionally become fixed if the virus passes through an extreme “bottleneck” as low numbers of infectious particles establish new transmissions (Duarte et al., 1992xDuarte, E., Clarke, D., Moya, A., Domingo, E., and Holland, J. Proc. Natl. Acad. Sci. USA. 1992; 89: 6015–6019Crossref | PubMed | Scopus (200)See all References(Duarte et al., 1992). Studies of HIV-1 infected people who progress to disease very slowly or not at all (called long-term nonprogressors or survivors) show that a minority of these cases are, in fact, attributable to obviously attenuated viruses. One of the most prominent examples of this is a cohort of transfusion recipients in Sydney who were infected with a virus that lacked an intact nef gene before 1985, and who have remained mostly asymptomatic (Learmont et al., 1999xNew Engl. Learmont, J.C., Geczy, A.F., Mills, J., Ashton, L.J., Raynes-Greenow, C.H., Garsia, R.J., Dyer, W.B., McIntyre, L., Oelrichs, R.B., Rhodes, D.I. et al. J. Med. 1999; 340: 1715–1722See all References(Learmont et al., 1999). It is likely that continued analyses of full-length virus sequences will identify additional cases; studying the defects in these viruses has the potential to be very revealing of viral functions that are important in vivo.It is important to reemphasize that viruses do not necessarily evolve toward greater pathogenicity. Indeed, the SIVs that naturally infect non-human primates in Africa discussed above do not cause observable disease in their natural hosts, yet can clearly induce fatal diseases when transferred to other species. Since some of these SIVs have resided in their host species for (at least) thousands of years, they might be examples of viruses that have become attenuated for disease in their natural hosts. There are numerous examples of viruses that are ubiquitous in the human population that are usually not pathogenic. Given time, is it possible that HIV-1 might similarly evolve? In essence, we do not know if HIV-1 diversification is yielding more, less, or equally pathogenic strains. Only difficult studies that combine extensive sequence data, standardized natural history, and carefully measured biological determinants will be able to begin to answer this question.