Transcriptional regulation of HIV-1 LTR during antigen-dependent activation of primary T cells by dendritic cells.

Numerous factors are known to bind human immunodeficiency virus (HIV) long terminal repeat (LTR) and activate viral transcription, but little is known as to how they function in naturally activated T cells and to what extent their binding is relevant to HIV replication in vivo. To characterize the HIV LTR-binding factors responsible for antigen-dependent activation of HIV, we examined replication of LTR mutant viruses in CD4+ T cells activated by different stimuli. NF-kappaB or Sp1 mutant virus replicated well in CD4+ T cells activated by phorbol ester and calcium ionophore. When they were activated by antigen-pulsed dendritic cells, the replication of the Sp1-deleted virus was severely impaired in CD45RA+, but not in CD45RO+ T cell subsets that dominantly produce interleukin-2 (IL-2). Stimulation via CD3/CD28 induced a high level of IL-2 production in both T cell subsets, but Sp1-deleted virus poorly replicated in CD45RA+ subset. The level of NF-kappaB and Sp1-binding factors did not differ between these subsets. Our results suggest that additional cofactors distinct from IL-2-inducing signaling molecules are important for LTR activation during antigen-dependent T cell activation.

Identification of insertion mutations in HIV-1 reverse transcriptase causing multiple drug resistance to nucleoside analogue reverse transcriptase inhibitors.

OBJECTIVE: A novel 2-amino acid insertion between codons 69 and 70 of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) which confers multiple drug resistance has recently been reported. Independently, we have identified similar insertion mutations in Japanese hemophiliacs and attempted to analyze their emergence in conjunction with therapy regimens and their contribution to drug resistance using recombinant technology. METHODS: The plasma and peripheral blood mononuclear cells (PBMCs) of 348 HIV-1-infected hemophiliacs were screened for HIV-1 RT mutations relevant to nucleoside analogue inhibitors and isolating viruses. Contribution of each insertion to drug resistance was studied by introducing the mutations into a T-cell line-tropic NL4-3 infectious clone and testing the drug susceptibilities of the recovered virus. RESULTS: Insertion of the 2-amino acid residue was found in 4 of the 348 cases and was strongly associated with prolonged chemotherapy with zidovudine (AZT) and didanosine (ddI). The virus isolated from 1 of the 4 cases possessed the same insertion. Characterization of these virus and the recombinant NL4-3 with the insertion strongly suggested that the insertion caused resistance not only to AZT and ddI but also to lamivudine (3TC) and zalcitabine (ddC). CONCLUSION: A 2-amino acid insertion between codons 69 and 70 of RT was detected in 4 of 348 (1.1%) Japanese hemophiliacs and was found to be associated with multiple drug resistance to nucleoside analogue RT inhibitors.

Transactivation is a conserved function among primate lentivirus Vpr proteins but is not shared by Vpx.

OBJECTIVE: To investigate the transactivating activity of Vpr proteins from human immunodeficiency viruses types 1 and 2 (HIV-1 and HIV-2) and simian immunodeficiency viruses (SIVs) on various primate lentivirus long terminal repeats (LTRs), and to determine whether the Vpx proteins shared by HIV-2 and SIV are able to transactivate any HIV or SIV promoter. STUDY DESIGN/METHODS: The vpr and vpx genes of the HIVs and SIVs encode virion-associated proteins, which are implicated in viral replication and pathogenesis. HIV-1 Vpr is involved in the transport of the preintegration complex (PIC) to the nucleus, transactivates the viral LTR, and induces cell cycle arrest. HIV-2 and SIV Vpx proteins share amino acid sequence similarities with Vpr and are involved in PIC translocation into the nucleus but are unable to induce cell cycle arrest. We cloned and expressed the vpr and vpx genes from several primate lentiviruses and tested their transactivating ability on HIV-1, HIV-2, SIVmac and SIVagm LTRs cloned upstream of the CAT reporter gene. RESULTS: All Vpr tested had a transactivating effect on several viral LTRs; however, none of the Vpx proteins showed a detectable transactivating effect. CONCLUSIONS: These results indicate that the transactivating properties of Vpr proteins were conserved throughout evolution in primate lentiviruses, which suggests that they have an important role in virus replication.

Role of the C terminus Gag protein in human immunodeficiency virus type 1 virion assembly and maturation.

Lentiviral Gag polyproteins have a proline-rich protein, p6, at their C terminus. There are conflicting reports about the function of p6 in virus release. In the present work, mutants that affect p6 of human immunodeficiency virus type 1 (HIV-1) Gag polyprotein were constructed and analysed. None of the mutants prevented virus release completely; however, detachment of budding particles was less efficient as evidenced by electron microscopy. Virions of the p6 truncation mutant B2TAA had a significantly reduced number of Pol proteins (p66, p51 and p34) and an increased amount of incompletely processed Gag proteins compared with the parental virus. A mutation that altered the cleavage site between p6 and p1 did not significantly affect virus assembly, virus release or protein processing with the exception of cleavage between p6 and p1. However, virions of this mutant (B2P6C) exhibited irregular-shaped core structures that were distinct from the cone-shaped core structure seen in the parental virion. B2P6C mutant virus was non-infectious in CD4+ T cells. These results suggest that mutations in p6 affect efficient detachment of budding particles from the cell surface. Proper cleavage between p6 and p1 may be critical for the formation of the distinctive cone-shaped core structure of HIV-1 virions.

Gene therapy against retroviral diseases.

Eventually, gene therapy may be a valid option for chronic viral infections, including retroviral infections. Human retroviral diseases fit two categories: (1) those that result from a monoclonal outgrowth of a human T-cell leukemia virus type I (HTLV-I)-infected cell, as in the case of adult T cell leukemia (ATL); and (2) those that appear to result directly from virus load rather than monoclonal outgrowth--such as tropical spastic paraparesis/HTLV-I associated myelopathy (TSP/HAM) and human immunodeficiency virus (HIV)-associated acquired immune deficiency syndrome (AIDS). For ATL gene therapy, corrective mechanisms directed at regulatory sequences rather than viral sequences may be most important, though perhaps anti-tax therapy would be useful. For TSP/HAM and AIDS, gene therapy directed to control virus replication may be most useful. For anti-retroviral therapy, one may use dominant negative mutants and a variety of other approaches that direct toxins or compete out viral regulatory gene signal sequences. For maximum benefit, such therapy should be directed to different essential genes (eg gag, pol, env, tat or rev) involved in the virus replication cycle and utilize different toxic approaches. A major impediment to the use of gene therapy for AIDS is our inability to transfect a significant fraction of target cells in vivo. Except for reconstituted mice, retroviral systems of animals have been under-utilized as models for gene therapy. Naturally occurring retroviral diseases of cats, goats, horses, and other species provide models for future development.

Identification of two highly homologous presynaptic proteins distinctly localized at the dendritic and somatic synapses.

Through screening of a murine brain cDNA library, we have isolated two brain specific cDNAs encoding highly homologous proteins, named 921-L and 921-S, comprised of 134 amino acids with 80% identity. Immunohistological study with the mAbs raised against the bacterially expressed 921 proteins showed that 921-L protein is distributed at the dendritic region and 921-S at the neuronal somatic surface. Immuno-electron microscopic study revealed that both 921 proteins are localized at the presynaptic terminal, indicating that the 921 proteins are differentially expressed at the dendritic and somatic presynapses.

An electron-lucent region within the virion distinguishes HIV-1 from HIV-2 and simian immunodeficiency virus.

Ultrastructural comparisons of immature or budding particles of human immunodeficiency virus (HIV) types 1 and 2 and simian immunodeficiency virus of macaques (SIVmac) revealed no significant difference between these genetically distinct, but related, viruses. However, a region encompassing the core of mature HIV-1 virions was found to be more electron lucent than that observed in HIV-2 and SIVmac. This ultrastructural distinction cannot be attributed to HIV-1-specific vpu, HIV-2/SIV-specific vpx, or virion-associated vpr gene products.

Vpx of simian immunodeficiency virus is localized primarily outside the virus core in mature virions.

Human immunodeficiency virus type 2 and the related simian immunodeficiency virus (SIV) contain a unique regulatory gene, vpx. The Vpx protein is packaged in mature virions and is required for efficient viral replication in peripheral blood lymphocytes and macrophages. To study the localization of Vpx in mature virions, conical and bar-shaped core structures of SIV from macaques (SIVmac) were purified. The SIVmac core has a density of approximately 1.25 g/cm3, compared with 1.16 g/cm3 for an intact virion. The relative proportions of major capsid protein (p27) and reverse transcriptase activity were similar for intact virions and core structures. The majority of matrix protein (p14) was removed from the purified core structure, suggesting its association with the viral membrane. Similarly, most of the Vpx protein was absent from the purified core structure. This result suggests that as with the matrix protein, the majority of Vpx proteins are localized outside the virus core. The localization of Vpx suggests that it may be involved in virus entry such as penetration or uncoating.

A virion-specific inhibitory molecule with therapeutic potential for human immunodeficiency virus type 1.

A potential new approach for gene therapy against human immunodeficiency virus type 1 (HIV-1) infection is the design of a nonstructural gene-based virion-specific inhibitory molecule that is packaged with virus to destroy its infectivity. We tested this approach for HIV-1 by using Vpx, a virion-associated protein of HIV-2 and simian immunodeficiency virus. Vpx was incorporated into HIV-1 virions and the resulting cell-free virus lost infectivity in CD4+ human T cells. This demonstrates the therapeutic potential of an accessory gene-based virion-specific inhibitory molecule. Vpx and its derivatives can be regarded as a new class of anti-HIV-1 molecule.

The matrix protein of human immunodeficiency virus type 1 is required for incorporation of viral envelope protein into mature virions.

Accumulating evidence suggests that the matrix (MA) protein of retroviruses plays a key role in virus assembly by directing the intracellular transport and membrane association of the Gag polyprotein. In this report, we show that the MA protein of human immunodeficiency virus type 1 is also critical for the incorporation of viral Env proteins into mature virions. Several deletions introduced in the MA domain (p17) of human immunodeficiency virus type 1 Gag polyprotein did not greatly affect the synthesis and processing of the Gag polyprotein or the formation of virions. Analysis of the viral proteins revealed normal levels of Gag and Pol proteins in these mutant virions, but the Env proteins, gp120 and gp41, were hardly detectable in the mutant virions. Our data suggest that an interaction between the viral Env protein and the MA domain of the Gag polyprotein is required for the selective incorporation of Env proteins during virus assembly. Such an interaction appears to be very sensitive to conformational changes in the MA domain, as five small deletions in two separate regions of p17 equally inhibited viral Env protein incorporation. Mutant viruses were not infectious in T cells. When mutant and wild-type DNAs were cotransfected into T cells, the replication of wild-type virus was also hindered. These results suggest that the incorporation of viral Env protein is a critical step for replication of retroviruses and can be a target for the design of antiviral strategies.

Human immunodeficiency virus vpr gene encodes a virion-associated protein.

The vpr gene of human immunodeficiency virus type 1 (HIV-1) is one of the seven accessory genes that are believed to have roles in the virus replication cycle. We report here the detection of a 13 kD vpr protein in sucrose gradient-purified HIV-1. This protein was not detected in cells infected with a virus having a truncated vpr gene that lacks the potential to encode for 26 C-terminal amino acid residues. These findings raise the possibility that virion-associated vpr proteins may be involved in the early life cycle of HIV-1 replication and suggest that the C-terminal region of the vpr gene is essential for its expression.

The arcB gene of Escherichia coli encodes a sensor-regulator protein for anaerobic repression of the arc modulon.

The arcA (dye) and arcB genes of Escherichia coli are responsible for anaerobic repression of target operons and regulons of aerobic function (the arc modulon). The amino acid sequence of ArcA (Dye) indicated that it is the regulator protein of a two-component control system. Here we show that ArcB is a membrane sensor protein on the basis of its deduced amino acid sequence (778 residues), hydropathicity profile, and cellular distribution. On the carboxyl end of the ArcB sequence there is an additional domain showing homology with conserved regions of regulator proteins. Deletion into this domain destroyed ArcB function. ArcB conserved a histidine residue for autophosphorylation of the sensor proteins, and aspartic residues important for the regulator proteins.

Human immunodeficiency virus type 1 has an additional coding sequence in the central region of the genome.

Eight coding regions designated gag, pol, env, sor, R, tat, art/trs, and 3' orf have been identified in the genome of the human immunodeficiency virus type 1 (HIV-1). Several other open reading frames have the potential to encode additional viral proteins. In this study, we show that HIV-1 has another coding sequence whose product is expressed during natural infection. Unlike antibody to other HIV-1 proteins, the prevalence of antibody to the product encoded by this region is elevated in patients with acquired immune deficiency syndrome (AIDS). Because no analogous coding region has been identified in HIV-2, the antibody to the product of this coding region may serve as a marker to distinguish infection with HIV-1 from infection with HIV-2.

Poly (ADP-Ribose) synthetase. Separation and identification of three proteolytic fragments as the substrate-binding domain, the DNA-binding domain, and the automodification domain.

Poly(ADP-ribose) synthetase of Mr = 120,000 is cleaved by limited proteolysis with alpha-chymotrypsin into two fragments of Mr = 54,000 (54K) and Mr = 66,000 (66K). When the native enzyme is modified with 3-(bromoacetyl)pyridine, both portions of the enzyme are alkylated; however, alkylation of the 54K portions of the enzyme is protected by the addition of the substrate, NAD, or its analog, nicotinamide, suggesting that the substrate-binding site is localized in the 54K fragment. When the enzyme previously automodified with a low concentration of [adenine-U-14C] NAD is digested with alpha-chymotrypsin, the radioactivity is detected exclusively in the 66K fragment. The 66K fragment thus labeled is further cleaved with papain into two fragments of Mr = 46,000 and Mr = 22,000. With these two fragments, the label is detected only in the 22K fragment, but not in the 46K fragment. The 46K fragment binds to a DNA-cellulose column with the same affinity as that of the native enzyme, while the 22K fragment and the 54K fragment have little affinity for the DNA ligand. These results indicate that poly (ADP-ribose) synthetase contains three separable domains, the first possessing the site for binding of the substrate, NAD, the second containing the site for binding of DNA, and the third acting as the site(s) for accepting poly(ADP-ribose).