Search PubMed⌕ Search

Biomedical subjects

M Emerman

Publications and source records attributed to M Emerman.

At least 37 records · Page 2Linked to original sources
PubMed1996

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.

Animals↗
PubMed1996

HIV-1, Vpr and the cell cycle.

The human immunodeficiency virus 1 (HIV-1) is a complex retrovirus with more genes than most retroviruses. One of these extra genes codes for a protein called Vpr, which has recently been shown to prevent activation of the mitotic cyclin-dependent kinase and thereby prevent infected cells from undergoing mitosis and proliferating. Vpr also plays an important role in another property of HIV-1 that is unusual for a retrovirus - its ability to enter the nucleus of a nondividing cell. Understanding the interactions between HIV-1 and the cell cycle should lead to new insights into both viral pathogenesis and basic cell biology.

Cell Cycle↗
PubMed1996

Protein stability influences human immunodeficiency virus type 2 Vpr virion incorporation and cell cycle effect.

Using recombinant proteins as standards, we calculated the amount of Vpr and Vpx present in HIV-2ROD particles. We find 2000-3000 copies of Vpx per particle but only 40-50 copies of Vpr. We investigated the reasons for this discrepancy between Vpx and Vpr and found that viral factors, including HIV-2 Vpx, do not restrict its incorporation. Instead, the accumulation of HIV-2ROD Vpr during infection is restricted by a short protein half-life which acts to limit its virion incorporation. The half-life of HIV-2 Vpr was calculated to be about 90 min, while HIV-2 Vpx and HIV-1 Vpr had half-lifes of 36 and 20 hr, respectively. Moreover, while both HIV-1 Vpr and HIV-2 Vpr cause cells to accumulate in G2 of the cell cycle, the effect of HIV-2 Vpr is attenuated relative to HIV-1 Vpr. Thus, protein stability correlates with both the function of Vpr and its virion incorporation.

Cell Cycle↗
PubMed1996

Vpx association with mature core structures of HIV-2.

Vpx is a virion-incorporated protein encoded by HIV-2 and certain strains of SIV which is necessary for efficient infection of lymphocytes and macrophages. To determine the location of Vpx within the HIV-2 particle, core structures were obtained from mature HIV-2 virions. Analysis of purified cores reveal the presence of monomeric Vpx as well as a nondenaturable and nonreducible higher order Vpx complex. Characterization of the core-associated Vpx complex suggests it to be a Vpx homodimer, indicative of high concentrations of Vpx. The incorporation and concentration of Vpx in HIV-2 cores support the hypothesis that it functions within the viral preintegration complex.

Animals↗
PubMed1996

Human immunodeficiency virus type 1 cell cycle control: Vpr is cytostatic and mediates G2 accumulation by a mechanism which differs from DNA damage checkpoint control.

Vpr is a 96-amino-acid protein encoded by human immunodeficiency virus type 1 (HIV-1) that prevents proliferation of infected cells. We have established a system for infection of 100% of a T-cell population with HIV and use this system to show that within the context of HIV-1 infection, Vpr is primarily cytostatic rather than cytotoxic. Vpr acts upstream of dephosphorylation of the mitotic cyclin-dependent kinase, and causes infected cells to accumulate in the G2 stage of the cell cycle. However, some HIV-1 infected cells increase in ploidy and size, accumulating DNA to an 8N level. Furthermore, the mechanism of the Vpr mitotic block is qualitatively different from that of G2 DNA damage checkpoint control.

CDC2 Protein Kinase↗
PubMed1995

Molecular basis of cell cycle dependent HIV-1 replication. Implications for control of virus burden.

Research is beginning to yield insight into determinants which govern cell cycle dependence of provirus establishment by the onco-retroviruses. In the case of HIV-1, nucleophilic components associated with the viral preintegration complex facilitate mitosis independent nuclear localization of viral DNA and provirus establishment. Differences in the metabolic activity between G0 T cells and macrophages, the two primary targets for HIV-1 infection, lead to significantly different outcomes with regards to provirus establishment following infection of these cells. Thus, macrophages appear fully permissive to productive HIV-1 replication while non-dividing (G0 T cells) restrict virus replication at a step which proceeds nuclear import of viral DNA. The requirement for T cell activation in productive HIV-1 replication has important implications for the relationship between immune activation and virus burden. It remains to be determined whether modulating the immune activation status of the infected individual may provide an opportunity for modulating virus burden and influencing disease course.

Acquired Immunodeficiency Syndrome↗
PubMed1995

The human immunodeficiency virus type 1 vpr gene prevents cell proliferation during chronic infection.

Human immunodeficiency virus type 1 (HIV-1) is a retrovirus that can cause extensive cytopathicity in T cells. However, long-term productive infection of T-cell lines has been described. Here we show that although Vpr has no effect on the initial cytopathic effect of HIV-1, viruses that contain an intact vpr gene are unable to establish a chronic infection of T cells. However, virus with a mutated vpr gene can readily establish such long-term cultures. The effect of Vpr is independent of the env gene and the nef gene. Furthermore, expression of Vpr alone affects the progression of cells in the cell cycle. These results suggest that HIV-1 has evolved a viral gene to prevent chronic infection of T cells.

Cell Cycle↗
PubMed1994

The Vpr protein of human immunodeficiency virus type 1 influences nuclear localization of viral nucleic acids in nondividing host cells.

The replication of human immunodeficiency virus type 1 (HIV-1) in nondividing host cells such as those of macrophage lineage is an important feature of AIDS pathogenesis. The pattern of HIV-1 replication is dictated, in part, by the nucleophilic property of the viral gag matrix (MA) protein, a component of the viral preintegration complex that facilitates nuclear localization of viral nucleic acids in the absence of mitosis. We now identify the accessory viral protein Vpr, as a second nucleophilic component that influences nuclear localization of viral nucleic acids in nondividing cells. Reverse transcription and nuclear localization of viral nucleic acids following infection of cells by viruses lacking Vpr or viruses containing mutations in a gag MA nuclear localization sequence were indistinguishable from the pattern observed in cells infected by wild-type HIV-1. These viruses retained the ability to replicate in both dividing and nondividing host cells including monocyte-derived macrophages. In contrast, introduction of both gag MA and Vpr mutations in HIV-1 attenuated nuclear localization of viral nucleic acids in nondividing cells and virus replication in monocyte-derived macrophages. These studies demonstrate redundant nucleophilic determinants of HIV-1 that independently permit nuclear localization of viral nucleic acids and virus replication in nondividing cells such as monocyte-derived macrophages. In addition, these studies provide a defined function for an accessory gene product of HIV-1.

Base Sequence↗
PubMed1994

Cellular latency in human immunodeficiency virus-infected individuals with high CD4 levels can be detected by the presence of promoter-proximal transcripts.

We have investigated the molecular basis of human immunodeficiency virus type 1 (HIV-1) latency in a tissue culture model and in HIV-infected people. We show that increased levels of Tat, but not Rev, can release the proviruses from latency in U1 cells. The absence of Tat in these cells is manifested by the accumulation of promoter-proximal viral transcripts, whereas the presence of Tat correlates with increased expression of viral proteins and an increase in promoter-distal transcripts. The presence of promoter-proximal transcripts also serves as a marker for latency in humans. We observed the exclusive presence of promoter-proximal viral transcripts in peripheral mononuclear cells from the majority (10/11) of asymptomatic HIV-infected individuals examined. Activation of these cells in vitro, and viremia in vivo, correlated with a switch from promoter-proximal transcription to promoter-distal transcription. These results suggest that the control between latency and replication of HIV in vivo is at the level of transcription elongation.

Base Sequence↗
PubMed1994

Passage through mitosis is required for oncoretroviruses but not for the human immunodeficiency virus.

The human immunodeficiency virus productively infects and integrates into cells that have been arrested in the cell cycle with either gamma irradiation or aphidicolin. Integration by oncoretroviruses such as the murine leukemia virus (MuLV), on the other hand, depends on cell proliferation. Although the entire cell cycle is not necessary for MuLV infection, it is essential that the infected cells pass through mitosis. The long terminal repeat circle junction, a marker for nuclear entry, is first observed in MuLV-infected cells immediately after mitosis. These results suggest that mitosis is necessary for nuclear entry of MuLV, but not human immunodeficiency virus, unintegrated proviral DNA.

Aphidicolin↗
PubMed1994

Functional exchange of an oncoretrovirus and a lentivirus matrix protein.

To map functional domains in the retroviral Gag protein we have constructed chimeric viruses where regions of the murine leukemia virus (MuLV) Gag protein have been replaced with analogous sequences from human immunodeficiency virus type 1 (HIV-1). Here we describe the chimeric virus MuLV(MAHIV) which contains the HIV-1 matrix (MA) protein in place of the MuLV MA. MuLV(MAHIV) is infectious but grows at a reduced rate compared with wild-type MuLV. We found that the partial defect in replication of the chimeric virus is at a late stage in the viral life cycle. The MuLV(MAHIV) Gag proteins are distributed aberrantly within cells and are not associated with cellular membranes. Unlike MuLV, HIV-1 is able to integrate into growth-arrested cells. Incorporation of the HIV-1 MA, which is known to play a role in infection of nondividing cells, does not enable MuLV(MAHIV) to be expressed in growth-arrested cells. While it possesses no amino acid homology, we found that the HIV-1 MA can efficiently replace the MuLV matrix protein in infection.

3T3 Cells↗
PubMed1993

A nuclear localization signal within HIV-1 matrix protein that governs infection of non-dividing cells.

Permissiveness of the host cell to productive infection by oncoretroviruses is cell-cycle dependent, and nuclear localization of viral nucleoprotein preintegration complexes will occur only after cells have passed through mitosis. In contrast, establishment of an integrated provirus after infection by the lentivirus HIV-1 is independent of host cell proliferation. The ability of HIV-1 to replicate in non-dividing cells is partly accounted for by the karyophilic properties of the viral preintegration complex which, after virus infection, is actively transported to the host cell nucleus. Here we report that the gag matrix protein of HIV-1 contains a nuclear localization sequence which, when conjugated to a heterologous protein, directs its nuclear import. In addition, HIV-1 mutants containing amino-acid substitutions in this nuclear localization signal integrate and replicate within dividing but not growth-arrested cells, and thus display a phenotype more representative of an oncoretrovirus.

Amino Acid Sequence↗
PubMed1993

Incorporation of human immunodeficiency virus type 1 Gag proteins into murine leukemia virus virions.

The retroviral Gag polyprotein is necessary and sufficient for assembly and budding of viral particles. However, the exact inter- and intramolecular interactions of the Gag polyproteins during this process are not known. To locate functional domains within Gag, we generated chimeric proviruses between human immunodeficiency virus type 1 (HIV-1) and murine leukemia virus (MuLV). In these chimeric proviruses, the matrix or capsid proteins of MuLV were precisely replaced with the matrix or capsid proteins of HIV-1. Although the chimeric proviruses were unable to efficiently assemble into mature viral particles by themselves, coexpression of wild-type MuLV Gag rescued the HIV proteins into virions. The specificity of the rescue of HIV proteins into MuLV virions shows that specific interactions involving homologous matrix or capsid regions of Gag are necessary for retroviral particle formation.

3T3 Cells↗
PubMed1992

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.

Base Sequence↗
PubMed1992

Functional mapping of the rev-responsive element of human immunodeficiency virus type 2 (HIV-2): influence of HIV-2 envelope-encoding sequences on HIV-1 gp120 expression in the presence or absence of Rev.

The human immunodeficiency virus type 1 (HIV-1) regulatory protein Rev stimulates expression of structural viral proteins via a target response element (RRE) located within gag-pol and env mRNAs. To analyse the HIV-2 Rev trans-activation effect on the expression of the envelope protein, we cloned a functionally active HIV-2 rev cDNA and showed that it contained four exons. Using transient expression assays, we mapped a 353 bp RRE fragment within the env gene of HIV-2 on which both HIV-1 and HIV-2 Rev could act. Interestingly, smaller fragments suppressed the use of additional splice sites within the env gene and caused envelope protein expression independent of Rev.

Base Sequence↗
PubMed1992

Detection of replication-competent and pseudotyped human immunodeficiency virus with a sensitive cell line on the basis of activation of an integrated beta-galactosidase gene.

We have constructed a HeLa cell line that both expresses high levels of CD4 and contains a single integrated copy of a beta-galactosidase gene that is under the control of a truncated human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR). This cell line, called CD4-LTR/beta-gal, can be used to determine quantitatively the titer of laboratory-adapted HIV strains, and the method used to do so is as sensitive as the determination of viral titers in a T-cell line by end point dilution. Using this cell line as a titer system, we calculated that HIV-1 stocks contain only one infectious particle per 3,500 to 12,000 virions. Virus derived from a molecular clone of a macrophagetropic provirus will not infect this cell line. We have also cocultivated peripheral blood lymphocyte cultures from HIV-infected individuals with the CD4-LTR/beta-gal indicator cells. In a majority of primary isolates (five of eight), including isolates from asymptomatic patients, rare virus-infected cells that can activate the beta-galactosidase gene are present.

Amino Acid Sequence↗