Accessory Gene Products Research Articles

T-lymphocyte epitope identification and their use in vaccine development for HIV-1.

Cellular immune responses mediated by CD8+ cytotoxic T-lymphocytes (CTL) and CD4+ helper T-lymphocytes (HTL) are needed to effectively control and clear many viral pathogens, including HIV-1. Thus, vaccines for HIV-1 capable of inducing CTL and HTL responses are now the focus of multiple academic and industry-based research and development programs. The use of defined, minimal CTL and HTL epitopes in vaccines has several potential advantages. Firstly, it is possible to use epitopes that are conserved thus targeting the majority of viral variants within a given clade or across clades. Secondly, epitopes from multiple viral structural or accessory gene products can be included in vaccines, which supports the induction cellular immune responses with significant breadth. Finally, dominance relationships between epitopes can be altered to increase immune recognition of subdominant epitopes. HTL and CTL epitopes from HIV-1 have recently been identified and characterized in numbers that are large enough to support their use in experimental vaccines. Initial studies with prototype DNA vaccines encoding epitopes indicate the need to include intracellular targeting sequences, to direct the encoded gene products to different cellular compartments, and amino acid spacer sequences between epitopes to optimize the processing, and subsequent presentation, of individual epitopes. Vaccines composed of CTL or HTL epitopes are now being developed for clinical testing.

HIV/host interactions: new lessons from the Red Queen's country.

HIV/host interactions: new lessons from the Red Queen's country.

Low or no antibody responses to human immunodeficiency virus type 1 Nef in infected carriers with subtype E, in contrast to subtype B that showed antibodies preferentially recognizing subtype-specific Nef epitopes

The viral accessory gene product Nef has been shown to play an important role in human immunodeficiency virus type 1 (HIV-1)-induced pathogenesis. Only little information is available regarding the differences in the host immune responses against Nef protein and its function in vivo among different subtypes of HIV-1. In the present study, we showed marked differences in the immune responses to Nef protein between subtypes B and E. The amino acid sequence in subtype E Nef showed 72% homology with that in subtype B. Most murine monoclonal antibodies obtained by immunization with subtype B or E Nef protein showed cross-reactivity with both Nef proteins (80 and 67%, respectively). Next, we focused on the immune responses among infected Japanese and Thai individuals. Subtyping of the individuals into B and E was carried out by enzyme-linked immunosorbent assay (ELISA) using synthetic peptides corresponding to the V3 loop representing the principal neutralizing domain. Most of the sera from these individuals reacted strongly with Gag p24 proteins derived from subtypes B and E at similar levels. However, the immune responses among these individuals to Nef protein were markedly different. Some subtype B-infected Japanese and Thai individuals (40 and 35%, respectively) showed higher levels of anti-Nef antibodies, although these antibodies preferentially recognized epitopes specific to subtype B. On the other hand, most of the subtype E-infected Japanese and Thai individuals showed low or no antibody responses to Nef proteins. Thus, immune responses to Nef were markedly different between subtypes B- and E-infected carriers, suggesting different function(s) for Nef in AIDS pathogenesis. Further, vaccine design must take into account the different subtypes of HIV-1.

Induction of Apoptosis by the Vpr Protein of Human Immunodeficiency Virus Type 1 Occurs Independently of G2 Arrest of the Cell Cycle

The HIV-1 accessory gene product Vpr can inhibit cell proliferation via cell cycle arrest at the G2 phase, and it can induce apoptosis after G2 arrest. We found recently that C81, a carboxy-terminally truncated form of Vpr, induced apoptosis via G1 arrest but did not induce G2 arrest of the cell cycle. Thus, it seemed possible that expression of Vpr in cells might cause apoptosis independently of the ability of Vpr to induce G2 arrest. We demonstrate here that Vpr-induced apoptosis occurs by a mechanism that does not necessarily require induction of G2 arrest. First, it was found that the extent of apoptosis reached a maximum even when few cells were arrested at the G2 phase of the cell cycle and was reduced in inverse proportion to the increased induction of G2 arrest. Thus, the extent of induction of G2 arrest was not correlated with the extent of Vpr-induced apoptosis. Furthermore, we replaced the Ile/Leu residues in the leucine zipper-like domain of Vpr with Ala or Pro and used cells that expressed the mutant protein to demonstrate that Vpr caused apoptosis in a manner that was independent of G2 arrest. Finally, replacement of Ile/Leu by Pro at positions 60, 67, 74, and 81 within the leucine zipper-like domain of wild-type Vpr and C81 revealed that the Ile/Leu residues at positions 60, 67, and 74 in the leucine zipper-like domain were indispensable for induction of apoptosis induced by Vpr and by C81 and confirmed, in addition, that both processes might be regulated by the same pathway. C81 appears to be a useful tool for elucidation of the mechanism of apoptosis induced by expression of Vpr protein.

A Carboxy-Terminally Truncated Form of the Vpr Protein of Human Immunodeficiency Virus Type 1 Retards Cell Proliferation Independently of G2 Arrest of the Cell Cycle

Vpr, one of the accessory gene products of HIV-1, is a 96-residue protein with several functions. It is involved in import of the HIV-1 preintegration complex into the nucleus of nondividing cells, in cellular differentiation, inducing cell cycle arrest at the G2/M phase, in immune suppression, and in enhancement of replication of the virus. We found recently that Vpr interferes with the proliferation of mouse NIH3T3 fibroblasts but fails to arrest these cells in the G2 phase. Thus, it seems possible that Vpr might retard cell proliferation via a novel pathway that is distinct from G2 arrest. To elucidate the mechanism by which Vpr induces the retardation of cell growth, we developed a panel of expression vectors that encoded Vpr molecules with deletions of specific putative domains, namely, the first α-helical domain, the second α-helical domain, a leucine zipper-like domain, and an arginine-rich carboxy-terminal domain. These vectors were introduced into HeLa cells since expression of Vpr can induce G2 arrest in such cells. A carboxy-terminally truncated form of Vpr, C81, which failed to induce G2 arrest, led to the G1 arrest and retained the ability to prevent cell proliferation. All the other mutant proteins had completely lost the capacity to induce G2 arrest and to suppress growth. Substitutions of Ile/Leu for Pro at positions 60, 67, 74, and 81 within the leucine zipper-like domain of Vpr or of C81 revealed that Ile60, Leu67, and Ile74 play an important role in the C81-induced suppression of growth, while Ile74 and Ile81 were found to be indispensable for Vpr-induced G2 arrest. Collectively, our results strongly suggest that Vpr can retard cell proliferation independently of G2 arrest of the cell cycle.

Transposition genes of the Bacteroides mobilizable transposon Tn4555: role of a novel targeting gene.

Conjugative transposons have been identified in several bacterial species, most notably the Gram-positive Enterococci and the Gram-negative Bacteroides. In Bacteroides species, these elements encode a complete conjugative machinery, which mediates their own intercellular transfer, and they can mobilize in trans co-resident elements. One such mobilizable element is the antibiotic resistance transposon, Tn4555, which was previously found to integrate into a specific genome target site via a site-specific recombination mechanism. In this work, we demonstrate that three Tn4555 genes were involved in integration of the element. These were int encoding a lambda-type integrase, which was absolutely required for integration of the transposon, and two accessory genes, which increased the frequency of integration. Interestingly, one of these accessory gene products, TnpA, directed the insertion of Tn4555 into the genome target site; in the absence of tnpA, the insertion pattern was essentially random. This is the first example of a site-specific recombinase that uses a specific targeting protein.

HIV-1 Nef mediates lymphocyte chemotaxis and activation by infected macrophages.

Infection of macrophage lineage cells is a feature of primate lentivirus replication, and several properties of primate lentiviruses seem to have evolved to promote the infection of macrophages. Here we demonstrate that the accessory gene product Nef induces the production of two CC-chemokines, macrophage inflammatory proteins 1alpha and 1beta, by HIV-1-infected macrophages. Adenovirus-mediated expression of Nef in primary macrophages was sufficient for chemokine induction. Supernatants from Nef-expressing macrophages induced both the chemotaxis and activation of resting T lymphocytes, permitting productive HIV-1 infection. These results indicate a role for Nef in lymphocyte recruitment and activation at sites of virus replication.

Inhibition of Vpr-Induced Cell Cycle Abnormality by Quercetin: A Novel Strategy for Searching Compounds Targeting Vpr

Vpr, an accessory gene product of HIV-1 which induces cell cycle abnormality leading to the increased HIV replication, is supposed to be a possible target for anti-AIDS drugs. We recently established a cell line (MIT-23) in which Vpr-induced cell cycle perturbation could be manipulated by a tetracycline promoter. Here, we screened anti-Vpr activity in 27 kinds of herb drugs using MIT-23 cells. One of the extracts prepared from Houttuyniae herba showed an inhibitory activity. Quercetin (QCT), a compound of this crude drug, efficiently inhibited Vpr function without affecting its expression. Furthermore, data suggested that Vpr-induced transcription from HIV-LTR was considerably abrogated by QCT. These data indicate that QCT, a flavonoid previously reported to inhibit HIV replication, also targets Vpr, implicating that MIT-23 cell provides a novel strategy for screening compounds possessing anti-Vpr activity which would be in turn utilized for clarifying the mechanism of Vpr function.

Expression of a Novel Nef Epitope on the Surface of HIV Type 1-Infected Cells

We compared antibody responses to various structural and accessory gene products of HIV-1 between long-term nonprogressors and patients who have progressed to AIDS. On the basis of our results, we performed epitope mapping of the Nef protein and identified a novel epitope. This novel epitope of the Nef protein was found to be exposed on the surface of HIV-1-infected cells. The antibody response against it correlated with CD4+ cell counts among HIV-1-infected patients (r = 0.457, p < 0.001). Although further research is necessary, we suspect that antibody response against the epitope may be protective against disease progression.

Heterologous expression of correctly assembled methylamine dehydrogenase in Rhodobacter sphaeroides.

The biosynthesis of methylamine dehydrogenase (MADH) from Paracoccus denitrificans requires four genes in addition to those that encode the two structural protein subunits, mauB and mauA. The accessory gene products appear to be required for proper export of the protein to the periplasm, synthesis of the tryptophan tryptophylquinone (TTQ) prosthetic group, and formation of several structural disulfide bonds. To accomplish the heterologous expression of correctly assembled MADH, eight genes from the methylamine utilization gene cluster of P. denitrificans, mauFBEDACJG, were placed under the regulatory control of the coxII promoter of Rhodobacter sphaeroides and introduced into R. sphaeroides by using a broad-host-range vector. The heterologous expression of MADH was constitutive with respect to carbon source, whereas the native mau promoter allows induction only when cells are grown in the presence of methylamine as a sole carbon source and is repressed by other carbon sources. The recombinant MADH was localized exclusively in the periplasm, and its physical, spectroscopic, kinetic and redox properties were indistinguishable from those of the enzyme isolated from P. denitrificans. These results indicate that mauM and mauN are not required for MADH or TTQ biosynthesis and that mauFBEDACJG are sufficient for TTQ biosynthesis, since R. sphaeroides cannot synthesize TTQ. A similar construct introduced into Escherichia coli did not produce detectable MADH activity or accumulation of the mauB and mauA gene products but did lead to synthesizes of amicyanin, the mauC gene product. This finding suggests that active recombinant MADH is not expressed in E. coli because one of the accessory gene products is not functionally expressed. This study illustrates the potential utility of R. sphaeroides and the coxII promoter for heterologous expression of complex enzymes such as MADH which cannot be expressed in E. coli. These results also provide the foundation for future studies on the molecular mechanisms of MADH and TTQ biosynthesis, as well as a system for performing site-directed mutagenesis of the MADH gene and other mau genes.

Solution structure of peptides from HIV-1 Vpr protein that cause membrane permeabilization and growth arrest

Vpr, one of the accessory gene products encoded by HIV-1, is a 96-residue protein with a number of functions, including targeting of the viral pre-integration complex to the nucleus and inducing growth arrest of dividing cells. We have characterized by 2D NMR the solution conformations of bioactive synthetic peptide fragments of Vpr encompassing a pair of H(F/S)RIG sequence motifs (residues 71-75 and 78-82 of HIV-1 Vpr) that cause cell membrane permeabilization and death in yeast and mammalian cells. Due to limited solubility of the peptides in water, their structures were studied in aqueous trifluoroethanol. Peptide Vpr59-86 (residues 59-86 of Vpr) formed an alpha-helix encompassing residues 60-77, with a kink in the vicinity of residue 62. The first of the repeated sequence motifs (HFRIG) participated in the well-defined alpha-helical domain whereas the second (HSRIG) lay outside the helical domain and formed a reverse turn followed by a less ordered region. On the other hand, peptides Vpr71-82 and Vpr71-96, in which the sequence motifs were located at the N-terminus, were largely unstructured under similar conditions, as judged by their C(alpha)H chemical shifts. Thus, the HFRIG and HSRIG motifs adopt alpha-helical and turn structures, respectively, when preceded by a helical structure, but are largely unstructured in isolation. The implications of these findings for interpretation of the structure-function relationships of synthetic peptides containing these motifs are discussed.

Solution structure of peptides from HIV-1 Vpr protein that cause membrane permeabilization and growth arrest.

Vpr, one of the accessory gene products encoded by HIV-1, is a 96-residue protein with a number of functions, including targeting of the viral pre-integration complex to the nucleus and inducing growth arrest of dividing cells. We have characterized by 2D NMR the solution conformations of bioactive synthetic peptide fragments of Vpr encompassing a pair of H(F/S)RIG sequence motifs (residues 71-75 and 78-82 of HIV-1 Vpr) that cause cell membrane permeabilization and death in yeast and mammalian cells. Due to limited solubility of the peptides in water, their structures were studied in aqueous trifluoroethanol. Peptide Vpr59-86 (residues 59-86 of Vpr) formed an alpha-helix encompassing residues 60-77, with a kink in the vicinity of residue 62. The first of the repeated sequence motifs (HFRIG) participated in the well-defined alpha-helical domain whereas the second (HSRIG) lay outside the helical domain and formed a reverse turn followed by a less ordered region. On the other hand, peptides Vpr71-82 and Vpr71-96, in which the sequence motifs were located at the N-terminus, were largely unstructured under similar conditions, as judged by their C(alpha)H chemical shifts. Thus, the HFRIG and HSRIG motifs adopt alpha-helical and turn structures, respectively, when preceded by a helical structure, but are largely unstructured in isolation. The implications of these findings for interpretation of the structure-function relationships of synthetic peptides containing these motifs are discussed.

Functional roles of HIV accessory proteins for viral replication (review).

Numerous lentiviruses, including human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2) of causative agents of human AIDS as representative, have been recently isolated from various species of primates. The fundamental and most prominent feature of the viruses is the presence of a number of accessory genes in their genomes. Extensive biological and biochemical studies have demonstrated that the accessory gene products are not essential for viral replication at least in certain types of cells. Quite surprisingly, some of these accessory proteins are absolutely non-essential in any types of cells so far examined. In this brief review, our systematic genetic studies on the importance of the accessory proteins of HIV-1 and HIV-2 for viral replication are described and discussed.

Design and Assay of Inhibitors of HIV-1 Vpr Cell Killing and Growth Arrest Activity Using Microbial Assay Systems

Viral protein R (Vpr), one of the accessory gene products encoded by the human immunodeficiency virus type 1 (HIV-1) genome, has a number of functions, including causing a growth arrest of HIV-1-infected cells and possibly the death of uninfected bystander cells. In microbial assay systems, the C-terminal portion of Vpr can cause cell death when added externally, and when expressed in yeast it causes growth arrest. In this study we have sought to obtain inhibitors of the Vpr functions that affect the microbial systems. Our first approach employed peptide display, which identified a number of sequences, including a heptapeptide sequence, GETRAPL, involved in binding to the C-terminus of Vpr. To determine whether GETRAPL could block the extracellular cytocidal activity of Vpr, the heptapeptide was synthesized and found to have some blocking activity in microbial assays. A second approach led to the finding that melittin inhibitors had activity against Vpr extracellular activities. In a third approach, compounds were tested against the Vpr-induced growth arrest. A number of compounds were found to abrogate the growth arrest, and some also inhibited Vpr's extracellular activity.

HIV-1 Vpr interacts with a human 34-kDa mov34 homologue, a cellular factor linked to the G2/M phase transition of the mammalian cell cycle.

Several important and possibly interrelated functions have been identified for the HIV-1 accessory gene product Vpr. These include import of the HIV reverse transcription complex into the nucleus of nondividing cells, cellular differentiation including cell cycle arrest at the G2/M phase border, immune suppression, and enhancement of virus replication. We have cloned a candidate Vpr ligand, termed human Vpr interacting protein (hVIP/MOV34), by using a yeast two-hybrid assay. This gene is homologous to a simultaneously identified 34-kDa human mov34 homologue. The MOV34 family includes proteins that function as transcriptional and proteolytic regulators of cell growth and differentiation. We demonstrate direct interactions between the putative ligand hVIP/MOV34 and Vpr in vitro and in vivo. hVIP/MOV34 localizes to the nucleus and appears to function as a component of the cell cycle cascade. We observe an association between the induction of cell cycle arrest at the G2/M phase border by Vpr and a change in the subcellular localization of hVIP/MOV34 from a nuclear to a perinuclear localization. This was further associated with the inhibition of maturation promoting factor-associated histone H1 kinase activity. We conclude that hVIP/MOV34 is involved in the regulation of the cell cycle and a likely cellular cofactor for HIV-1 Vpr.

Structural studies of synthetic peptide fragments derived from the HIV-1 Vpr protein.

Vpr, one of the accessory gene products of the human immunodeficiency virus-1 (HIV-1) genome, exhibits diverse biological characteristics. Vpr functions as a transcriptional activator of HIV and heterologous promoters. It is capable of arresting cells in cell cycle progression and plays a crucial role in the infection of macrophages. Despite the wealth of information available on the biological aspects of Vpr, the structure of Vpr remains poorly understood. To gain insight into the structure-function relationship of Vpr, peptides corresponding to putative helical regions of Vpr were synthesized and their structures determined by circular dichroism (CD) spectroscopy. The CD studies confirmed the predicted helical structures of these peptides. Based on the data, a hypothetical model for the structure of Vpr was proposed which displays an anti-parallel alpha-helix core structure reminiscent of a helix-loop-helix motif. These findings are consistent with the results from mutational studies of Vpr and provide a plausible structural basis to further investigate the multiple functions of Vpr as a viral protein.

Genetic and biochemical analyses of Actinobacillus pleuropneumoniae urease.

The urease gene cluster from the virulent Actinobacillus pleuropneumoniae serotype 1 strain CM5 was cloned and sequenced. The urease activity was associated with a 6.3-kbp region which contains eight long open reading frames (ORFs). The structural genes, ureABC, are separated from the accessory genes, ureEFGD, by a 615-bp ORF of unknown function, ureX. Homologies were found with the structural and accessory urease gene products of Haemophilus influenzae and, to a lesser extent, with those of other organisms. The urease enzyme subunits had predicted molecular masses of 61.0, 11.3, and 11.0 kDa, and the size of the holoenzyme was estimated to be 337 +/- 13 kDa by gel filtration chromatography. Urease activity was maximal but unstable at 65 degrees C. In cell lysates, the A. pleuropneumoniae urease was stable over a broad pH range (5.0 to 10.6) and the optimal pH for activity was 7.7. The Km was 1.5 +/- 0.1 mM urea when it was assayed at pH 7.7. The low Km suggests that this enzyme would be active in the respiratory tract environment, where urea levels should be similar to those normally found in pig serum (2 to 7 mM).

HIV-1 Vpr suppresses immune activation and apoptosis through regulation of nuclear factor kappa B.

The HIV-1 accessory gene product Vpr can influence viral pathogenesis by affecting viral replication as well as host cell transcription and proliferation. We have investigated the effects of Vpr on host cell activation and confirm that it influences cellular proliferation. However, we have also found that Vpr modulates T-cell receptor (TCR)-triggered apoptosis in a manner similar to that of glucocorticoids. In the absence of TCR-mediated activation, Vpr induces apoptosis whereas in its presence, Vpr interrupts the expected induction of apoptosis. This regulation of apoptosis is linked to Vpr suppression of NF-kappa B activity via the induction of I kappa B, an inhibitor of NF-kappa B. Further, Vpr suppresses expression of IL-2, IL-10, IL-12, TNF alpha and IL-4, all of which are NF-kappa B-dependent. The effects of Vpr could be reversed by RU486. Our finding that Vpr can regulate NF-kappa B supports the hypothesis that some aspects of viral pathogenesis are the consequence of cell dysregulation by Vpr.

Molecular Determinants of Nef Function.

The human immunodeficiency virus (HIV), the causative agent of the acquired immune deficiency syndrome (AIDS), in addition to encoding for the gag, pol and env structural genes common to all retroviruses also encodes six accessory genes: tat, rev, nef, vpr, vpu and vif. These accessory genes are responsible for the regulation of HIV replication. Recent advances in our understanding of the function(s) of these genes have illustrated the complex interplay between HIV, the infected cell and the host. In addition, identification of cellular proteins interacting with accessory gene products have provided new tools to study cellular processes. The topic of this review, nef, has been shown in vitro to induce the cell surface downregulation of CD4, the receptor for HIV, to enhance the infectivity of HIV particles and to associate with at least one cellular serine/threonine kinase. In vivo, Nef is essential for the efficient virus replication responsible for disease progression. In this review, several prominent aspects of Nef function are discussed including its effect on CD4 trafficking, on signaling pathways and on virus infectivity enhancement. Copyright 1997 S. Karger AG, Basel

Activation of PAK by HIV and SIV Nef: importance for AIDS in rhesus macaques

Background The primate lentiviruses, human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2) and simian immunodeficiency virus (SIV), encode a conserved accessory gene product, Nef. In vivo, Nef is important for the maintenance of high virus loads and progression to AIDS in SIV-infected adult rhesus macaques. In tissue culture cells expressing Nef, this viral protein interacts with a cellular serine kinase, designated Nef-associated kinase.Results This study identifies the Nef-associated kinase as a member of the p21-activated kinase (PAK) family of kinases and investigates the role of this Nef-associated kinase in vivo. Mutants of Nef that do not associate with the cellular kinase are unable to activate the PAK-related kinase in infected cells. To determine the role of cellular kinase association in viral pathogenesis, macaques were infected with SIV containing point-mutations in Nef that block PAK activation. Virus recovered at early time points after inoculation with mutant virus was found to have reverted to prototype Nef function and sequence. Reversion of the kinase-negative mutant to a kinase-positive genotype in macaques infected with the mutant virus preceded the induction of high virus loads and disease progression.Conclusions Nef associates with and activates a PAK-related kinase in lymphocytes infected in vitro. Moreover, the Nef-mediated activation of a PAK-related kinase correlates with the induction of high virus loads and the development of AIDS in the infected host. These findings reveal that there is a strong selective pressure in vivo for the interaction between Nef and the PAK-related kinase.