Upregulation of survivin by HIV-1 Vpr

The human survivin gene belongs to the family of inhibitor of apoptosis proteins (IAP) and is involved in apoptosis inhibition and regulation of cell division. The survivin gene is the only member of the IAP family whose expression is known to be regulated through the cell cycle. Survivin expression reaches the highest levels during the G₂/M transition and then is rapidly degraded during the G₁ phase. Here we report that the human immunodeficiency virus type 1 (HIV-1) upregulates Survivin expression via survivin promoter transactivation. Vpr, an HIV-1 accessory protein that induces cell cycle arrest in G₂/M, is necessary and sufficient for this effect. Blocking Vpr-induced G₂/M arrest leads to elimination of the survivin promoter transactivation by Vpr. Our results suggest that Survivin may be actively involved in regulating cell viability during HIV-1 infection.     

Human Endogenous Retrovirus K Gag Coassembles with HIV-1 Gag and Reduces the Release Efficiency and Infectivity of HIV-1

Human endogenous retroviruses (HERVs), which are remnants of ancestral retroviruses integrated into the human genome, are defective in viral replication. Because activation of HERV-K and coexpression of this virus with HIV-1 have been observed during HIV-1 infection, it is conceivable that HERV-K could affect HIV-1 replication, either by competition or by cooperation, in cells expressing both viruses. In this study, we found that the release efficiency of HIV-1 Gag was 3-fold reduced upon overexpression of HERV-K(CON) Gag. In addition, we observed that in cells expressing Gag proteins of both viruses, HERV-K(CON) Gag colocalized with HIV-1 Gag at the plasma membrane. Furthermore, HERV-K(CON) Gag was found to coassemble with HIV-1 Gag, as demonstrated by (i) processing of HERV-K(CON) Gag by HIV-1 protease in virions, (ii) coimmunoprecipitation of virion-associated HERV-K(CON) Gag with HIV-1 Gag, and (iii) rescue of a late-domain-defective HERV-K(CON) Gag by wild-type (WT) HIV-1 Gag. Myristylation-deficient HERV-K(CON) Gag localized to nuclei, suggesting cryptic nuclear trafficking of HERV-K Gag. Notably, unlike WT HERV-K(CON) Gag, HIV-1 Gag failed to rescue myristylation-deficient HERV-K(CON) Gag to the plasma membrane. Efficient colocalization and coassembly of HIV-1 Gag and HERV-K Gag also required nucleocapsid (NC). These results provide evidence that HIV-1 Gag heteromultimerizes with HERV-K Gag at the plasma membrane, presumably through NC-RNA interaction. Intriguingly, HERV-K Gag overexpression reduced not only HIV-1 release efficiency but also HIV-1 infectivity in a myristylation- and NC-dependent manner. Altogether, these results indicate that Gag proteins of endogenous retroviruses can coassemble with HIV-1 Gag and modulate the late phase of HIV-1 replication.     

Biochemical analyses of the interactions between human immunodeficiency virus type 1 Vpr and p6(Gag)

The nonstructural human immunodeficiency virus type 1 Vpr protein is packaged into progeny virions at significant levels (approximately 200 copies/virion). Genetic analyses have demonstrated that efficient Vpr packaging is dependent upon a leucine-X-X-leucine-phenylalanine (LXXLF) motif located in the p6(Gag) domain of the structural Gag polyprotein. Recombinant proteins spanning full-length Vpr (Vpr(1-97)) or the amino-terminal 71 amino acids (Vpr(1-71)) formed specific complexes with recombinant p6 proteins in vitro. Complex formation required an intact LXXLF motif and exhibited an intrinsic dissociation constant of approximately 75 microM. Gel filtration and cross-linking analyses further revealed that Vpr(1-71) self-associated in solution. Our experiments demonstrate that Vpr can bind directly and specifically to p6 and suggest that oligomerization of both Vpr and Gag may serve to increase the avidity and longevity of Vpr-Gag complexes, thereby ensuring efficient Vpr packaging.     

A Functional Interaction between gp41 and gp120 Is Observed for Monomeric but Not Oligomeric,Uncleaved HIV-1 Env gp140

The envelope glycoprotein (Env) is the sole antigenic feature on the surface of HIV and the target for the humoral immune system. Soluble, uncleaved gp140 Env constructs truncated at the transmembrane domain are being investigated intensively as potential vaccine immunogens by many groups, and understanding their structural properties is essential. We used hydrogen/deuterium-exchange mass spectrometry and small-angle X-ray scattering to probe structural order in a panel of commonly used gp140 constructs and matched gp120 monomers. We observed that oligomeric forms of uncleaved gp140, generally presumed to be trimeric, contain a protease-resistant form of gp41 akin to the postfusion, helical bundle conformation and appear to lack specific interactions between gp120 and gp41. In contrast, the monomeric form of gp140 shows significant stabilization of the gp120 inner domain imparted by the gp41 region, demonstrating excellent agreement with past mutagenesis studies. Moreover, the gp140 monomers respond to CD4 binding in manner that is consistent with the initial stages of Env activation: CD4 binding induces structural ordering throughout gp120 while loosening its association with gp41. The results indicate that uncleaved gp140 oligomers do not represent an authentic prefusion form of Env, whereas gp140 monomers isolated from the same glycoprotein preparations in many ways exhibit function and internal structural order that are consistent with expectations for certain aspects of native Env. gp140 monomers may thus be a useful reagent for advancing structural and functional studies.     

Effects of HIV-1 Vpr on neuroinvasion and neuropathogenesis

Human immunodeficiency virus type I (HIV-1) infection leads to penetration of the central nervous system (CNS) in virtually all infected individuals and HIV-1-induced encephalopathy in a significant number of untreated patients. The molecular mechanisms by which HIV-1 enters the CNS and yields CNS dysfunction are still unclear. Our laboratories and others have begun to explore the direct effects of prioritized HIV-1-specific proteins on diverse human CNS cell types. One of these proteins, the accessory HIV-1 protein Vpr, is a critical moiety in these studies, and will be discussed in this article.     

Roles of the interactions between Env and Gag proteins in the HIV-1 replication cycle

The Env and Gag proteins of HIV-1 are the two major structural proteins of this retrovirus. The interactions between Env and Gag proteins and their regulation in HIV-1 are required for several steps of the replication cycle, involving not only virus assembly, specifically Env incorporation, but also entry steps after virus maturation. A large number of host factors and certain membrane microdomains appear to engage both in transport/trafficking of Env and/or Gag proteins, and in the interactions of these two proteins. The present review briefly summarizes our current knowledge regarding the roles of the interactions between Env and Gag proteins in the virus replication cycle.     

Glutamate metabolism in HIV-1 infected macrophages: Role of HIV-1 Vpr

HIV-1 infected macrophages play a significant role in the neuropathogenesis of AIDS. HIV-1 viral protein R (Vpr) not only facilitates HIV-1 infection but also contribute to long-lived persistence in macrophages. Our previous studies using SILAC-based proteomic analysis showed that the expression of critical metabolic enzymes in the glycolytic pathway and tricarboxylic acid (TCA) cycle were altered in response to Vpr expression in macrophages. We hypothesized that Vpr-induced modulation of glycolysis and TCA cycle regulates glutamate metabolism and release in HIV-1 infected macrophages.

We assessed the amount of specific metabolites induced by Vpr and HIV-1 in macrophages at the intracellular and extracellular level in a time-dependent manner utilizing multiple reaction monitoring (MRM) targeted metabolomics. In addition, stable isotope-labeled glucose and an MRM targeted metabolomics assay were used to evaluate the de novo synthesis and release of glutamate in Vpr overexpressing macrophages and HIV-1 infected macrophages, throughout the metabolic flux of glycolytic pathway and TCA cycle activation.

The metabolic flux studies demonstrated an increase in glucose uptake, glutamate release and accumulation of α-ketoglutarate (α-KG) and glutamine in the extracellular milieu in Vpr expressing and HIV-1 infected macrophages. Interestingly, glutamate pools and other intracellular intermediates (glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), citrate, malate, α-KG, and glutamine) showed a decreased trend except for fumarate, in contrast to the glutamine accumulation observed in the extracellular space in Vpr overexpressing macrophages.

Our studies demonstrate that dysregulation of mitochondrial glutamate metabolism induced by Vpr in HIV-1 infected macrophages commonly seen, may contribute to neurodegeneration via excitotoxic mechanisms in the context of NeuroAIDS.     

Conformation of the HIV-1 Gag protein in solution

A single multi-domain viral protein, termed Gag, is sufficient for assembly of retrovirus-like particles in mammalian cells. We have purified the human immunodeficiency virus type 1 (HIV-1) Gag protein (lacking myristate at its N terminus and the p6 domain at its C terminus) from bacteria. This protein is capable of assembly into virus-like particles in a defined in vitro system. We have reported that it is in monomer-dimer equilibrium in solution, and have described a mutant Gag protein that remains monomeric at high concentrations in solution. We report that the mutant protein retains several properties of wild-type Gag. This mutant enabled us to analyze solutions of monomeric protein. Hydrodynamic studies on the mutant protein showed that it is highly asymmetric, with a frictional ratio of 1.66. Small-angle neutron scattering (SANS) experiments confirmed its asymmetry and yielded an R(g) value of 34 A. Atomic-level structures of individual domains within Gag have previously been determined, but these domains are connected in Gag by flexible linkers. We constructed a series of models of the mutant Gag protein based on these domain structures, and tested each model computationally for its agreement with the experimental hydrodynamic and SANS data. The only models consistent with the data were those in which Gag was folded over, with its N-terminal matrix domain near its C-terminal nucleocapsid domain in three-dimensional space. Since Gag is a rod-shaped molecule in the assembled immature virion, these findings imply that Gag undergoes a major conformational change upon virus assembly.     

The interaction between HIV-1 Gag and human lysyl-tRNA synthetase during viral assembly

Human lysyl-tRNA synthetase (LysRS) is a tRNA-binding protein that is selectively packaged into HIV-1 along with its cognate tRNALys isoacceptors. Evidence exists that Gag alone is sufficient for the incorporation of LysRS into virions. Herein, using both in vitro and in vivo methods, we begin to map regions in Gag and LysRS that are required for this interaction. In vitro reactions between wild-type and truncated HIV-1 Gag and human LysRS were monitored using GST-tagged molecules and glutathione-agarose chromatography. Gag/LysRS interaction in vivo was detected in 293FT cells cotransfected with plasmids coding for wild-type or mutant HIV-1 Gag and LysRS, either by monitoring Gag.LysRS complexes immunoprecipitated from cell lysate with anti-LysRS or by measuring the ability of LysRS to be packaged into budded Gag viral-like particles. Based on these studies, we conclude that the Gag/LysRS interaction depends upon Gag sequences within the C-terminal domain of capsid (the last 54 amino acids) and amino acids 208-259 of LysRS. The latter domain includes the class II aminoacyl-tRNA synthetase consensus sequence known as motif 1. Both regions have been implicated in homodimerization of capsid and LysRS, respectively. Sequences falling outside these amino acid stretches can be deleted from either molecule without affecting the Gag/LysRS interaction, further supporting the observation that LysRS is incorporated into Gag viral-like particles independent of its ability to bind tRNALys.     

Discrimination between Functional and Non-functional Cellular Gag Complexes involved in HIV-1 Assembly

HIV-1 Gag and Gag-Pol are responsible for viral assembly and maturation and represent a major paradigm for enveloped virus assembly. Numerous intracellular Gag-containing complexes (GCCs) have been identified in cellular lysates using sucrose gradient ultracentrifugation. While these complexes are universally present in Gag-expressing cells, their roles in virus assembly are not well understood. Here we demonstrate that most GCC species are predominantly comprised of monomeric or dimeric Gag molecules bound to ribosomal complexes, and as such, are not on-pathway intermediates in HIV assembly. Rather, these GCCs represent a population of Gag that is not yet functionally committed for incorporation into a viable virion precursor. We hypothesize that these complexes act as a reservoir of monomeric Gag that can incorporate into assembling viruses, and serve to mitigate non-specific intracellular Gag oligomerization. We have identified a subset of large GCC complexes, comprising more than 20 Gag molecules, that may be equivalent to membrane-associated puncta previously shown to be bona fide assembling-virus intermediates. This work provides a clear rationale for the existence of diverse GCCs, and serves as the foundation for characterizing on-pathway intermediates early in virus assembly.     

Effect of HIV-1 Vpr on cell cycle regulators

Cell cycle is one of the most complex processes in the life of a dividing cell. It involves numerous regulatory proteins, which direct the cell through a specific sequence of events for the production of two daughter cells. Cyclin-dependent kinases (cdks), which complex with the cyclin proteins, are the main players in the cell cycle. They can regulate the progression of the cells through different stages regulated by several proteins including p53, p21(WAF1), p19, p16, and cdc25. Downstream targets of cyclin-cdk complexes include pRB and E2F. A cell cycle can be altered to the advantage of many viral agents, most notably polyomaviruses, papillomaviruses, adenoviruses, and retroviruses. In addition, viral protein R (Vpr) is a protein encoded by the human immunodeficiency virus type 1 (HIV-1). HIV-1, the causative agent of acquired immunodeficiency syndrome (AIDS), is a member of the lentivirus class of retroviruses. This accessory protein plays an important role in the regulation of the cell cycle by causing G(2) arrest and affecting cell cycle regulators. Vpr prevents infected cells from proliferating, and collaborates with the matrix protein (MA) to enable HIV-1 to enter the nucleus of nondividing cells. Studies from different labs including ours showed that Vpr affects the functions of cell cycle proteins, including p53 and p21(WAF1). Thus, the replication of HIV-1, and ultimately its pathogenesis, are intrinsically tied to cell-cycle control.     

Identification of a CArG Box-Dependent Enhancer within the Cysteine-Rich Protein 1 Gene That Directs Expression in Arterial but Not Venous or Visceral Smooth Muscle Cells

Smooth muscle cells (SMCs) are heterogeneous with respect to their contractile, synthetic, and proliferative properties, though the regulatory factors responsible for their phenotypic diversity remain largely unknown. To further our understanding of smooth muscle gene regulation, we characterized the cis-regulatory elements of the murine cysteine-rich protein 1 gene (CRP1/Csrp1). CRP1 is expressed in all muscle cell types during embryogenesis and predominates in vascular and visceral SMCs in the adult. We identified a 5-kb enhancer within the CRP1 gene that is sufficient to drive expression in arterial but not venous or visceral SMCs in transgenic mice. This enhancer also exhibits region-specific activity in the outflow tract of the heart and the somites. Within the 5-kb CRP1 enhancer, we found a single CArG box that binds serum response factor (SRF), and by mutational analysis, demonstrate that the activity of the enhancer is dependent on this CArG element. Our findings provide further evidence for the existence of distinct regulatory programs within SMCs and suggest a role for SRF in the activation of the CRP1 gene.     

Interactions of HIV-1 Gag with assembly cofactors

HIV-1 Gag is the only protein required for retroviral particle assembly. There is evidence suggesting that phosphatidylinositol phosphate and nucleic acid are essential for viruslike particle assembly. To elucidate structural foundations of interactions of HIV-1 Gag with the assembly cofactors PI(4,5)P2 and RNA, we employed mass spectrometric protein footprinting. In particular, the NHS-biotin modification approach was used to identify the lysine residues that are exposed to the solvent in free Gag and are protected from biotinylation by direct protein-ligand or protein-protein contacts in Gag complexes with PI(4,5)P2 and/or RNA. Of 21 surface lysines readily modified in free Gag, only K30 and K32, located in the matrix domain, were strongly protected in the Gag-PI(4,5)P2 complex. Nucleic acid also protected these lysines, but only at significantly higher concentrations. In contrast, nucleic acids and not PI(4,5)P2 exhibited strong protection of two nucleocapsid domain residues: K391 and K424. In addition, K314, located in the capsid domain, was specifically protected only in the presence of both PI(4,5)P2 and nucleic acid. We suggest that concerted binding of PI(4,5)P2 and nucleic acid to the matrix and nucleocapsid domains, respectively, promotes protein-protein interactions involving capsid domains. These protein-protein interactions must be involved in virus particle assembly.     

Role of myristylation in HIV-1 Gag assembly

Assembly of the human immunodeficiency virus type 1 (HIV-1) first occurs on the plasma membrane of host cells where binding is driven by strong electrostatic interactions between the N-terminal matrix (MA) domain of the structural precursor polyprotein, Gag, and the membrane. MA is also myristylated, but the exact role this modification plays is not clear. In this study, we compared the protein oligomerization and membrane binding properties of Myr(+) and Myr(-) Gag(MA) expressed in COS-1 cells. Sedimentation studies in solution showed that both the myristylated Gag precursor and the mature MA product were detected in larger complexes than their unmyristylated counterparts, and the myristylated MA protein bound liposomes with approximately 3-fold greater affinity than unmyristylated MA. Aromatic residues near the N-terminal region of the MA protein were more accessible to chymotrypsin in the unmyristylated form and, consistent with this, an epitope in the N-terminal region was more exposed. Moreover, the cyclophilin binding site in the CA domain downstream of MA was more accessible in the unmyristylated Gag protein, while the Tsg101 binding site in the C-terminal region was equally available in the unmyristylated and myristylated Gag proteins. Taken together, our results suggest that myristylation promotes assembly by inducing conformational changes and facilitating MA multimerization. This observation offers a novel role for myristylation.