Michael P. Sherman

Nucleocytoplasmic Shuttling by Human Immunodeficiency Virus Type 1 Vpr

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) is capable of infecting nondividing cells such as macrophages because the viral preintegration complex is able to actively traverse the limiting nuclear pore due to the redundant and possibly overlapping nuclear import signals present in Vpr, matrix, and integrase. We have previously recognized the presence of at least two distinct and novel nuclear import signals residing within Vpr that, unlike matrix and integrase, bypass the classical importin α/β-dependent signals and do not require energy or a RanGTP gradient. We now report that the carboxy-terminal region of Vpr (amino acids 73 to 96) contains a bipartite nuclear localization signal (NLS) composed of multiple arginine residues. Surprisingly, when the leucine-rich Vpr(1–71) fragment, previously shown to harbor an NLS, or full-length Vpr is fused to the C terminus of a green fluorescent protein-pyruvate kinase (GFP-PK) chimera, the resultant protein is almost exclusively detected in the cytoplasm. However, the addition of leptomycin B (LMB), a potent inhibitor of CRM1-dependent nuclear export, produces a shift from a cytoplasmic localization to a nuclear pattern, suggesting that these Vpr fusion proteins shuttle into and out of the nucleus. Studies of nuclear import with GFP-PK–Vpr fusion proteins in the presence of LMB reveals that both of the leucine-rich α-helices are required for effective nuclear uptake and thus define a unique NLS. Using a modified heterokaryon analysis, we have localized the Vpr nuclear export signal to the second leucine-rich helix, overlapping a portion of the amino-terminal nuclear import signal. These studies thus define HIV-1 Vpr as a nucleocytoplasmic shuttling protein.

Functional and Structural Characterization of Synthetic HIV-1 Vpr That Transduces Cells, Localizes to the Nucleus, and Induces G2 Cell Cycle Arrest

Human immunodeficiency virus (HIV) Vpr contributes to nuclear import of the viral pre-integration complex and induces G2 cell cycle arrest. We describe the production of synthetic Vpr that permitted the first studies on the structure and folding of the full-length protein. Vpr is unstructured at neutral pH, whereas under acidic conditions or upon addition of trifluorethanol it adopts α-helical structures. Vpr forms dimers in aqueous trifluorethanol, whereas oligomers exist in pure water.1H NMR spectroscopy allows the signal assignment of N- and C-terminal amino acid residues; however, the central section of the molecule is obscured by self-association. These findings suggest that the in vivo folding of Vpr may require structure-stabilizing interacting factors such as previously described interacting cellular and viral proteins or nucleic acids. In biological studies we found that Vpr is efficiently taken up from the extracellular medium by cells in a process that occurs independent of other HIV-1 proteins and appears to be independent of cellular receptors. Following cellular uptake, Vpr is efficiently imported into the nucleus of transduced cells. Extracellular addition of Vpr induces G2 cell cycle arrest in dividing cells. Together, these findings raise the possibility that circulating forms of Vpr observed in HIV-infected patients may exert biological effects on a broad range of host target cells.

Cyclophilin A interacts with HIV-1 Vpr and is required for its functional expression

Viral protein R (Vpr) of human immunodeficiency virus, type 1 (HIV-1) is the major virion-associated accessory protein that affects a number of biological functions in the retroviral life cycle, including promotion of the transport of the preintegration complex into the nucleus and the induction of G2 host cell cycle arrest. Our recent investigation of the conformational heterogeneity of the proline residues in the N terminus of Vpr suggested a functional interaction between Vpr and a host peptidylprolyl cis/trans isomerase (PPIase) that might regulate the cis/trans interconversion of the imidic bond within the conserved proline residues of Vpr in vivo. Using surface plasmon resonance spectroscopy, Far Western blot, and pulldown experiments a physical interaction of Vpr with the major host PPIase cyclophilin A (CypA) is now demonstrated. The interaction domain involves the N-terminal region of Vpr including an essential role for proline in position 35. The CypA inhibitor cyclosporin A and non-immunosuppressive PPIase inhibitors such as NIM811 and sanglifehrin A block expression of Vpr without affecting pre- or post-translational events such as transcription, intracellular transport, or virus incorporation of Vpr. Similarly to CypA inhibition, Vpr expression is also reduced in HIV-1 infected CypA–/– knock-out T cells. This study thus shows that in addition to the interaction between CypA and HIV-1 capsid occurring during early steps in virus replication, CypA is also important for the de novo synthesis of Vpr and that in the absence of CypA activity, the Vpr-mediated cell cycle arrest is completely lost in HIV-1-infected T cells.

Human Immunodeficiency Virus Type 1 Vpr Induces DNA Replication Stress In Vitro and In Vivo

ABSTRACT The human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) causes cell cycle arrest in G2. Vpr-expressing cells display the hallmarks of certain forms of DNA damage, specifically activation of the ataxia telangiectasia mutated and Rad3-related kinase, ATR. However, evidence that Vpr function is relevant in vivo or in the context of viral infection is still lacking. In the present study, we demonstrate that HIV-1 infection of primary, human CD4+ lymphocytes causes G2 arrest in a Vpr-dependent manner and that this response requires ATR, as shown by RNA interference. The event leading to ATR activation in CD4+ lymphocytes is the accumulation of replication protein A in nuclear foci, an indication that Vpr likely induces stalling of replication forks. Primary macrophages are refractory to ATR activation by Vpr, a finding that is consistent with the lack of detectable ATR, Rad17, and Chk1 protein expression in these nondividing cells. These observations begin to explain the remarkable resilience of macrophages to HIV-1-induced cytopathicity. To study the in vivo consequences of Vpr function, we isolated CD4+ lymphocytes from HIV-1-infected individuals and interrogated the cell cycle status of anti-p24Gag-immunoreactive cells. We report that infected cells in vivo display an aberrant cell cycle profile whereby a majority of cells have a 4N DNA content, consistent with the onset of G2 arrest.

Human Immunodeficiency Virus Type 1 Vpr Contains Two Leucine-Rich Helices That Mediate Glucocorticoid Receptor Coactivation Independently of Its Effects on G2 Cell Cycle Arrest

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) Vpr participates in nuclear targeting of the viral preintegration complex in nondividing cells and induces G2 cell cycle arrest in proliferating cells, which creates an intracellular milieu favorable for viral replication. Vpr also activates the transcription of several promoters and enhancers by a poorly understood mechanism. Vpr enhances glucocorticoid receptor (GR) signaling and may mediate the effects of steroids on HIV replication. More specifically, recombinant Vpr can potentiate virion production from U937 cells, downregulate NF-κB induction, and enhance programmed cell death, all effects also mediated by glucocorticoids. Vpr has been proposed to act as a GR coactivator, although other studies suggest that these enhancing effects are merely a consequence of G2 cell cycle arrest. We now demonstrate that Vpr functions as a GR coactivator and that this activity is independent of cell cycle arrest. In addition, we show that the Vpr-induced coactivation requires an intact glucocorticoid response element, that it is dependent on the presence of hormone and the corresponding receptor, and that it is mediated by the two highly conserved leucine-rich domains within Vpr that resemble the GR coactivator signature motif.

PROTOTYPICAL HTLV-I/II INFECTION IS RARE IN LGL LEUKEMIA

The etiology of LGL leukemia is not known; however, we recently detected HTLV-II in a patient with LGL leukemia. In this study, we found that sera from 6 of 28 patients with LGL leukemia were positive for HTLV-I/II using a whole virus ELISA; moreover, the ELISA-negative sera were near the positive cut-off value. Therefore, we performed additional studies on these sera using commercially available assays which can confirm and distinguish HTLV-I from HTLV-II infection. Serum from only one patient was confirmed positive using conventional criteria (HTLV-II+). Sera from 25 patients (89%) had indeterminate reactivity on Western blot assays. Of these, sera from 21 (84%) reacted to gag protein p24; 12 (48%) reacted with recombinant env protein p21e, and 10 (40%) reacted with both. We could not detect HTLV-I/II pol or pX gene sequences in these patients using polymerase chain reaction analyses, with the exception of the HTLV-II-infected patient described previously. These data show that most patients with LGL leukemia are not infected with prototypical HTLV-I or HTLV-II. The frequent reactivity of patient sera to HTLV-I/II gag protein p24 and to env protein p21e, however, suggests that a deleted or variant form of HTLV-I/II may be associated with LGL leukemia.

Nuclear Export of Vpr Is Required for Efficient Replication of Human Immunodeficiency Virus Type 1 in Tissue Macrophages

ABSTRACT Retroviruses must gain access to the host cell nucleus for subsequent replication and viral propagation. Human immunodeficiency virus type 1 (HIV-1) and other primate lentiviruses are distinguished from the gammaretroviruses by their ability to infect nondividing cells such as macrophages, an important viral reservoir in vivo. Rather than requiring nuclear membrane breakdown during cell division, the HIV-1 preintegration complex (PIC) enters the nucleus by traversing the central aqueous channel of the limiting nuclear pore complex. The HIV-1 PIC contains three nucleophilic proteins, matrix, integrase, and Vpr, all of which have been implicated in nuclear targeting. The mechanism by which Vpr can display such nucleophilic properties and yet also be available for incorporation into virions assembling at the plasma membrane is unresolved. We recently characterized Vpr as a nucleocytoplasmic shuttling protein that contains two novel nuclear import signals and an exportin-1-dependent nuclear export signal (NES). We now demonstrate that mutation of this NES impairs the incorporation of Vpr into newly formed virions. Furthermore, we find that the Vpr NES is required for efficient HIV replication in tissue macrophages present in human spleens and tonsils. These findings underscore how the nucleocytoplasmic shuttling of Vpr not only contributes to nuclear import of the HIV-1 PIC but also enables Vpr to be present in the cytoplasm for incorporation into virions, leading to enhancement of viral spread within nondividing tissue macrophages.

Insights into the Biology of HIV-1 Viral Protein R

HIV-1 viral protein R (Vpr) is a small, highly conserved accessory protein encoded by the HIV genome that serves many functions in the viral life cycle. Vpr induces G2 cell cycle arrest, which is thought to indirectly enhance viral replication by increasing transcription from the LTR. Vpr has also been implicated in facilitating infection of nondividing cells, most notably macrophages. Because Vpr is a nucleo-cytoplasmic shuttling protein, its role in enhancing viral replication in macrophages may be mediated through enhanced entry of the HIV preintegration complex through the limiting nuclear pore. Free Vpr is detectable in the serum of patients, and in vitro studies implicate extracellular forms of Vpr as an effector of cellular responses mediated through its ability to transduce through intact cytoplasmic membranes. We review the biologic properties of Vpr, focusing on its mechanism of action, role in HIV replication, and significance for host pathogenesis.

DNA sequence analysis of the gene encoding the HTLV-I p21e transmembrane protein reveals inter- and intraisolate genetic heterogeneity.

DNA sequence analysis was performed on a 235-bp region of the p21 e transmembrane protein gene of the human T-cell lymphoma/leukemia virus type I (HTLV-I) which encompassess the putative immunosuppressive peptide. Polymerase chain reaction-based amplification was used to generate multiple molecular clones from isolates derived from fresh or cultured cells from 19 individuals. A dendrogram was constructed using the p21e DNA sequence information to compare the sequences among isolates in the current study and other previously published HTLV-I isolates including strains from Africa and Papua New Guinea. Examination of multiple clones from individual isolates revealed the presence of multiple genotypes in patients with tropical spastic paraparesis/HTLV-I-associated myelopathy and adult T-cell leukemia/lymphoma. These findings suggest that HTLV-I, like HIV, may be present as a quasispecies in vivo. Our studies, however, failed to identify specific sequence motifs that segregated exclusively with the lymphoproliferative or neurological forms of the disease.

Human T-cell lymphoma/leukemia retroviruses and malignancy

The primate T-cell lymphoma/leukemia viruses (PTLVs) are a group of plus-sense, single-stranded, RNA-containing particles that reverse transcribe their genomes into DNA and subsequently integrate into the host chromosome as proviruses. The PTLVs are a subgroup of the Oncovirinae (oncornaviruses), which includes human T-cell lymphoma/leukemia viruses types I and II (HTLV-I and HTLV-II) and simian T-cell lymphoma virus type I (STLV-I). These retroviruses are linked antigenically and by sequence homology. In particular, they all possess not only the viral structural protein coding genes gag, for core proteins, pol for reverse transcriptase, integrase, and protease, and env for membrane proteins, but also tax and rex genes within a unique region known as the pX gene, which, through a series of mRNA splicing events, code for a transactivator protein (Tax) and a repressor of expression protein (Rex) (figure 5-1). The Tax and Rex proteins work in trans to control viral expression by acting upon regulatory regions within the long terminal repeats (LTRs) of the pro virus. The fact that these proteins control not only virus expression but also alter cellular gene expression has made them a subject of intense research and a model for carcinogenesis.