Uracil DNA glycosylase specifically interacts with Vpr of both human immunodeficiency virus type 1 and simian immunodeficiency virus of sooty mangabeys, but binding does not correlate with cell cycle arrest.

The Vpr protein encoded by human immunodeficiency virus type 1 (HIV-1) is important for growth of virus in macrophages and prevents infected cells from passing into mitosis (G2 arrest). The cellular target for these functions is not known, but Vpr of HIV-1 and the related Vpr from simian immunodeficiency virus of sooty mangabeys (SIV(SM)) bind the DNA repair enzyme UNG, while the Vpx protein of SIV(SM) does not. Nonetheless, a mutational analysis of Vpr showed that binding to UNG is neither necessary nor sufficient for the effect of Vpr on the cell cycle.     

Functional Murine Leukemia Virus Vectors Pseudotyped with the Visna Virus Envelope Show Expanded Visna Virus Cell Tropism

Pseudotype virus vectors serve as a powerful tool for the study of virus receptor usage and entry. We describe the development of murine leukemia virus (MuLV) particles pseudotyped with the visna virus envelope glycoprotein and encoding a green fluorescent protein reporter as a tool to study the expression of the visna virus receptor. Functional MuLV/visna virus pseudotypes were obtained when the cytoplasmic tail of the visna virus envelope TM protein was truncated to 3, 7, or 11 amino acids in length. MuLV/visna virus particles were used to transduce a panel of cell types from various organisms, including sheep, goat, human, hamster, mouse, monkey, and quail. The majority of the cells examined were susceptible to MuLV/visna pseudotype viruses, supporting the notion that the visna virus cellular receptor is a widely expressed protein found in many species. Of 16 different cell types tested, only mouse embryo fibroblast NIH 3T3 cells, hamster ovary CHO cells, and the human promonocyte cell line U937 cells were not susceptible to transduction by the pseudotyped virus. The production of functional MuLV/visna virus pseudotypes has provided a sensitive, biologically relevant system to study visna virus cell entry and envelope-receptor interactions.     

The cell cycle independence of HIV infections is not determined by known karyophilic viral elements

Human immunodeficiency virus and other lentiviruses infect cells independent of cell cycle progression, but gammaretroviruses, such as the murine leukemia virus (MLV) require passage of cells through mitosis. This property is thought to be important for the ability of HIV to infect resting CD4+ T cells and terminally differentiated macrophages. Multiple and independent redundant nuclear localization signals encoded by HIV have been hypothesized to facilitate migration of viral genomes into the nucleus. The integrase (IN) protein of HIV is one of the HIV elements that targets to the nucleus; however, its role in nuclear entry of virus genomes has been difficult to describe because mutations in IN are pleiotropic. To investigate the importance of the HIV IN protein for infection of non-dividing cells, and to investigate whether or not IN was redundant with other viral signals for cell cycle-independent nuclear entry, we constructed an HIV-based chimeric virus in which the entire IN protein of HIV was replaced by that of MLV. This chimeric virus with a heterologous IN was infectious at a low level, and was able to integrate in an IN-dependent manner. Furthermore, this virus infected non-dividing cells as well as it infected dividing cells. Moreover, we used the chimeric HIV with MLV IN to further eliminate all of the other described nuclear localization signals from an HIV genome--matrix, IN, Viral Protein R, and the central polypurine tract--and show that no combination of the virally encoded NLS is essential for the ability of HIV to infect non-dividing cells.     

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.     

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.     

Cell cycle dependence of foamy retrovirus infection.

In common with oncoviruses but unlike the lentivirus human immunodeficiency virus type 1, foamy (spuma) viruses require host cell proliferation for productive infection. We show that human immunodeficiency virus type 1 replicates in RD-CD4 cells regardless of the growth arrest condition of the cells, while murine leukemia virus is unable to infect growth-arrested RD-CD4 cells or cells progressing through a partial cell cycle that includes S phase but not mitosis. Human foamy virus, like murine leukemia virus, does not productively infect G1/S or G2 growth-arrested cells. Two other foamy viruses, simian foamy virus type 1, isolated from a macaque, and simian foamy virus type 6, isolated from a chimpanzee, also fail to establish productive infection in G1/S-arrested cells.     

Human immunodeficiency virus type 1 Vpr arrests the cell cycle in G2 by inhibiting the activation of p34cdc2-cyclin B.

Human immunodeficiency virus type 1 (HIV-1) vpr inhibits the replication of tumor cell lines and peripheral blood mononuclear cells. Here it is demonstrated that expression of vpr, either in the context of a provirus or from an independent genetic element, induces a discrete cell cycle arrest, with cells containing 4N DNA. Low cyclin B-associated kinase activity, as well as the status of p34cdc2 and cdc25C phosphorylation, indicates that the cascade of reactions which drives the cell into mitosis has not been initiated. The phosphatase inhibitor okadaic acid releases the block, suggesting that Vpr perturbs upstream regulatorsof the G2-M transition. These studies demonstrate that HIV-1 vpr has profound effects on the cellular factors which control entry into mitosis and indicate vpr's potential contribution to the cellular pathology associated with HIV-1 infection.     

Cofactor requirement for human immunodeficiency virus type 1 entry into a CD4-expressing human cell line.

Expression of the human immunodeficiency virus type 1 (HIV-1) receptor CD4 on many nonhuman and some human cell lines is not sufficient to permit HIV-1 infection. We describe a human glioblastoma cell line (U373-MG) which remains resistant to HIV-1 despite the added expression of an authentic CD4 molecule. The block to HIV-1 infection of these cells is strain independent and appears to be at viral entry. Heterokaryons of CD4-expressing U373-MG (U373-CD4) cells fused to HeLa cells allow HIV-1 entry. A U373-CD4/HeLa hybrid clone allows efficient HIV-1 replication. These results suggest that HeLa cells express a factor(s) that can complement the viral entry defect of U373-CD4 cells and is necessary for efficient CD4-mediated HIV-1 infection.     

Accumulation and activation of natural killer cells in local intraperitoneal HIV-1/MuLV infection results in early control of virus infected cells

Natural killer (NK) cells are important effectors in resistance to viral infections. The role of NK cells in the acute response to human immunodeficiency virus 1 (HIV-1) infected cells was investigated in a mouse model based on a HIV-1/murine leukemia virus (MuLV) pseudovirus. Splenocytes infected with HIV-1/MuLV were injected intraperitoneally and local immunologic responses and persistence of infected cells were investigated. In vivo depletion with an anti-NK1.1 antibody showed that NK cells are important in resistance to virus infected cells. Moreover, NK cell frequency in the peritoneal cavity increased in response to infected cells and these NK cells had a more mature phenotype, as determined by CD27 and Mac-1 expression. Interestingly, after injection of HIV-1/MuLV infected cells, but not MuLV infected cells, peritoneal NK cells had an increased cytotoxic activity. In conclusion, NK cells play a role in the early control of HIV-1/MuLV infected cells in vivo.     

Cdk1 Activity Is Required for Mitotic Activation of Aurora A during G2/M Transition of Human Cells

In mammalian cells entry into and progression through mitosis are regulated by multiple mitotic kinases. How mitotic kinases interact with each other and coordinately regulate mitosis remains to be fully understood. Here we employed a chemical biology approach using selective small molecule kinase inhibitors to dissect the relationship between Cdk1 and Aurora A kinases during G₂/M transition. We find that activation of Aurora A first occurs at centrosomes at late G₂ and is required for centrosome separation independently of Cdk1 activity. Upon entry into mitosis, Aurora A then becomes fully activated downstream of Cdk1 activation. Inactivation of Aurora A or Plk1 individually during a synchronized cell cycle shows no significant effect on Cdk1 activation and entry into mitosis. However, simultaneous inactivation of both Aurora A and Plk1 markedly delays Cdk1 activation and entry into mitosis, suggesting that Aurora A and Plk1 have redundant functions in the feedback activation of Cdk1. Together, our data suggest that Cdk1, Aurora A, and Plk1 mitotic kinases participate in a feedback activation loop and that activation of Cdk1 initiates the feedback loop activity, leading to rapid and timely entry into mitosis in human cells. In addition, live cell imaging reveals that the nuclear cycle of cells becomes uncoupled from cytokinesis upon inactivation of both Aurora A and Aurora B kinases and continues to oscillate in a Cdk1-dependent manner in the absence of cytokinesis, resulting in multinucleated, polyploidy cells.     

Inhibiting MAP kinase activity prevents calcium transients and mitosis entry in early sea urchin embryos

A transient calcium increase triggers nuclear envelope breakdown (mitosis entry) in sea urchin embryos. Cdk1/cyclin B kinase activation is also known to be required for mitosis entry. More recently, MAP kinase activity has also been shown to increase during mitosis. In sea urchin embryos, both kinases show a similar activation profile, peaking at the time of mitosis entry. We tested whether the activity of both kinases is required for mitosis entry and whether either kinase controls mitotic calcium signals. We found that reducing the activity of either mitotic kinase prevents nuclear envelope breakdown, despite the presence of a calcium transient, when cdk1/cyclin B kinase activity is alone inhibited. When MAP kinase activity alone was inhibited, the calcium signal was absent, suggesting that MAP kinase activity is required to generate the calcium transient that triggers nuclear envelope breakdown. However, increasing intracellular free calcium by microinjection of calcium buffers or InsP(3) while MAP kinase was inhibited did not itself induce nuclear envelope breakdown, indicating that additional MAP kinase-regulated events are necessary. After MAP kinase inhibition early in the cell cycle, the early events of the cell cycle (pronuclear migration/fusion and DNA synthesis) were unaffected, but chromosome condensation and spindle assembly are prevented. These data indicate that in sea urchin embryos, MAP kinase activity is part of a signaling complex alongside two components previously shown to be essential for entry into mitosis: the calcium transient and the increase in cdk1/cyclinB kinase activity.     

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.     

PUL21a-Cyclin A2 Interaction is Required to Protect Human Cytomegalovirus-Infected Cells from the Deleterious Consequences of Mitotic Entry

Entry into mitosis is accompanied by dramatic changes in cellular architecture, metabolism and gene expression. Many viruses have evolved cell cycle arrest strategies to prevent mitotic entry, presumably to ensure sustained, uninterrupted viral replication. Here we show for human cytomegalovirus (HCMV) what happens if the viral cell cycle arrest mechanism is disabled and cells engaged in viral replication enter into unscheduled mitosis. We made use of an HCMV mutant that, due to a defective Cyclin A2 binding motif in its UL21a gene product (pUL21a), has lost its ability to down-regulate Cyclin A2 and, therefore, to arrest cells at the G1/S transition. Cyclin A2 up-regulation in infected cells not only triggered the onset of cellular DNA synthesis, but also promoted the accumulation and nuclear translocation of Cyclin B1-CDK1, premature chromatin condensation and mitotic entry. The infected cells were able to enter metaphase as shown by nuclear lamina disassembly and, often irregular, metaphase spindle formation. However, anaphase onset was blocked by the still intact anaphase promoting complex/cyclosome (APC/C) inhibitory function of pUL21a. Remarkably, the essential viral IE2, but not the related chromosome-associated IE1 protein, disappeared upon mitotic entry, suggesting an inherent instability of IE2 under mitotic conditions. Viral DNA synthesis was impaired in mitosis, as demonstrated by the abnormal morphology and strongly reduced BrdU incorporation rates of viral replication compartments. The prolonged metaphase arrest in infected cells coincided with precocious sister chromatid separation and progressive fragmentation of the chromosomal material. We conclude that the Cyclin A2-binding function of pUL21a contributes to the maintenance of a cell cycle state conducive for the completion of the HCMV replication cycle. Unscheduled mitotic entry during the course of the HCMV replication has fatal consequences, leading to abortive infection and cell death.     

Actin-Dependent Receptor Colocalization Required for Human Immunodeficiency Virus Entry into Host Cells

Human immunodeficiency virus (HIV) envelope binds CD4 and a chemokine receptor in sequence, releasing hydrophobic viral gp41 residues into the target membrane. HIV entry required actin-dependent concentration of coreceptors, which could be disrupted by cytochalasin D (CytoD) without an effect on cell viability or mitosis. Pretreatment of peripheral blood mononuclear cells, but not virus, inhibited entry and infection. Immunofluorescent confocal microscopy of activated cells revealed CD4 and CXCR4 in nonoverlapping patterns. Addition of gp120 caused polarized cocapping of both molecules with subsequent pseudopod formation, while CytoD pretreatment blocked these membrane changes completely.     

Species-specific, postentry barriers to primate immunodeficiency virus infection

By using replication-defective vectors derived from human immunodeficiency virus type 1 (HIV-1), simian immunodeficiency virus (SIV(mac)), and murine leukemia virus (MuLV), all of which were pseudotyped with the vesicular stomatitis virus (VSV) G glycoprotein, the efficiency of postentry, early infection events was examined in target cells of several mammalian species. Titers of HIV-1 vectors were significantly lower than those of SIV(mac) and MuLV vectors in most cell lines and primary cells from Old World monkeys. By contrast, most New World monkey cells exhibited much lower titers for the SIV(mac) vector compared with those of the HIV-1 vector. Prosimian cells were resistant to both HIV-1 and SIV(mac) vectors, although the MuLV vector was able to infect these cells. Cells from other mammalian species were roughly equivalent in susceptibility to the three vectors, with the exception of rabbit cells, which were specifically resistant to the HIV-1 vector. The level of HIV-1 vector expression was very low in transduced cells of rodent, rabbit, cow, and pig origin. Early postentry restriction of primate immunodeficiency virus infection exhibits patterns largely coincident with species borders and applies to diverse cell types within an individual host, suggesting the involvement of species-specific, widely expressed cellular factors.     

Requirements of cyclin a for mitosis are independent of its subcellular localization

Cyclin A (CycA), the only essential mitotic cyclin in Drosophila, is cytoplasmic during interphase and accumulates in the nucleus during prophase. We show that interphase localization is mediated by Leptomycin B (LMB)-sensitive nuclear export. This is a feature shared with human CyclinB1, and it is assumed that nuclear accumulation is necessary for mitotic entry. Here, we tested if the unique mitotic function of CycA requires nuclear accumulation. We fused subcellular localization signals to CycA and tested their mitotic capability. Surprisingly, nuclear accumulation was not required, and even a membrane-tethered form of CycA was able to induce mitosis. We noted that Cyclin B (CycB) protein disappears prematurely in CycA mutants, reminiscent of rca1 mutants. Rca1 is an inhibitor of Fizzy-related-APC/C activity, and in rca1 mutants, mitotic cyclins are degraded in G2 of the 16(th) embryonic cell cycle. Overexpression of Rca1 can restore mitosis in CycA mutants, indicating that the mitotic failure of CycA mutants is caused by premature activation of the APC/C. The essential mitotic function of CycA is therefore not the activation of numerous mitotic substrates by Cdk1-dependent phosphorylation. Rather, CycA-dependent kinase activity is required to inhibit one inhibitor of mitosis, the Fzr protein.