Biologic features of HIV-1 that correlate with virulence in the host.

Individuals infected with the human immunodeficiency virus type 1 (HIV-1) may be asymptomatic or have AIDS-related complex or the acquired immuno deficiency syndrome (AIDS). Little is known about the factors that influence progression of infection to AIDS. In this study of isolates of HIV-1 obtained at intervals during the infection of four individuals, the development of disease was found to be correlated with the emergence of HIV-1 variants that were more cytopathic in vitro as the disease progressed and that replicated more efficiently in a wide variety of different human cells. The biologic properties of HIV-1 in vitro thus appear to reflect its virulence in the host. Further studies of such sequentially isolated viruses may lead to the identification of viral genes that govern pathogenesis.

Differential effects of nef on HIV replication: implications for viral pathogenesis in the host.

Stable lymphoid cell lines expressing the human immunodeficiency virus type 1 (HIV-1) nef gene product, p27, were established. The presence of p27 in the lymphoid cells suppressed replication of some strains of both HIV-1 and HIV-2. This observation indicates that nef could be important in the establishment of HIV latency. In contrast, fast replicating and highly cytopathic HIV-1 isolates recovered from patients with advanced disease states were not affected by the negative effect of nef present in these lymphoid cell lines. This lack of response to nef appears to constitute another viral feature that correlates with disease progression. Thus, manipulating expression of the nef gene in vivo might influence pathogenesis in the host.

AIDS retrovirus (ARV-2) clone replicates in transfected human and animal fibroblasts.

A molecular clone of the AIDS-associated retrovirus (ARV-2) was transfected into human T lymphocyte and monocyte cell lines as well as mouse, mink, monkey, and human fibroblast lines. A replicating virus with cytopathic and biologic properties of ARV-2 was recovered from all the cell lines. The animal and human fibroblast cells are resistant to direct infection by ARV, and in these experiments virus production in the fibroblast lines, especially mouse, was reduced compared to human lymphocytes. However, human fibroblasts were more permissive to virus expression than mouse cells. These results show that, whereas the primary block to ARV infection in certain cells may occur at the cell surface, intracellular mechanisms can also participate in controlling virus replication. The results have relevance to vaccine development and encourage further work with modified molecular clones to examine regions of the ARV genome necessary for cytopathology and replication.

Distinct pathogenic sequela in rhesus macaques infected with CCR5 or CXCR4 utilizing SHIVs.

Infection of macaques with chimeric simian-human immunodeficiency virus (SHIV) provides an excellent in vivo model for examining the influence of envelope on HIV-1 pathogenesis. Infection with a pathogenic CCR5 (R5)-specific enveloped virus, SHIVSF162P, was compared with infection with the CXCR4 (X4)-specific SHIVSF33A.2. Despite comparable levels of viral replication, animals infected with the R5 and X4 SHIV had distinct pathogenic outcomes. SHIVSF162P caused a dramatic loss of CD4+ intestinal T cells followed by a gradual depletion in peripheral CD4+ T cells, whereas infection with SHIVSF33A.2 caused a profound loss in peripheral T cells that was not paralleled in the intestine. These results suggest a critical role of co-receptor utilization in viral pathogenesis and provide a reliable in vivo model for preclinical examination of HIV-1 vaccines and therapeutic agents in the context of the HIV-1 envelope protein.

Characterization of a noncytopathic HIV-2 strain with unusual effects on CD4 expression.

A new isolate of the human immunodeficiency virus type 2, designated HIV-2UC1, was recovered from an Ivory Coast patient with normal lymphocyte numbers who died with neurologic symptoms. Like some HIV-1 isolates, HIV-2UC1 grows rapidly to high titers in human peripheral blood lymphocytes and macrophages and has a differential ability to productively infect established human cell lines of lymphocytic and monocytic origin. Moreover, infection with this isolate also appears to involve the CD4 antigen. However, unlike other HIV isolates, HIV-2UC1 does not cause cytopathic effects in susceptible T cells nor does it lead to loss of CD4 antigen expression on the cell surface. These results indicate that HIV-2 may be found in individuals with neurologic symptoms and that the biological characteristics of this heterogeneous subgroup can differ from those typical of HIV-1.

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.

Human T cell leukemia virus type 1 Tax associates with a molecular chaperone complex containing hTid-1 and Hsp70.

Tax, an oncogenic viral protein encoded by human T cell leukemia virus type 1 (HTLV-1), induces cellular transformation of T lymphocytes by modulating a variety of cellular gene expressions [1]. Identifying cellular partners that interact with Tax constitutes the first step toward elucidating the molecular basis of Tax-induced transformation. Here, we report a novel Tax-interacting protein, hTid-1. hTid-1, a human homolog of the Drosophila tumor suppressor protein Tid56, was initially characterized based on its interaction with the HPV-16 E7 oncoprotein [2]. hTid-1 and Tid56 are members of the DnaJ family [2,3], which contains a highly conserved signature J domain that regulates the activities of heat shock protein 70 (Hsp70) by serving as cochaperone [4-6]. In this context, the molecular chaperone complex is involved in cellular signaling pathways linked to apoptosis, protein folding, and membrane translocation and in modulation of the activities of tumor suppressor proteins, including retinoblastoma, p53, and WT1[7-12]. We find that expression of hTid-1 inhibits the transformation phenotype of two human lung adenocarcinoma cell lines. We show that Tax interacts with hTid-1 via a central cysteine-rich domain of hTid-1 while a signature J domain of hTid-1 mediates its binding to Hsp70 in HEK cells. Importantly, Tax associates with the molecular chaperone complex containing both hTid-1 and Hsp70 and alters the cellular localization of hTid-1 and Hsp70. In the absence of Tax, expression of the hTid-1/Hsp70 molecular complex is targeted to perinuclear mitochondrial clusters. In the presence of Tax, hTid-1 and its associated Hsp70 are sequestered within a cytoplasmic "hot spot" structure, a subcellular distribution that is characteristic of Tax in HEK cells.

HIV-1 variation: consequences for disease progression and vaccine strategies.

HIV-1 displays a high degree of biological heterogeneity in vitro, differing in tropism, kinetics of replication, cytopathogenicity, resistance to antiviral drugs and susceptibility to serum neutralization. Some of these properties can be linked to pathogenesis in the host. HIV variation is a major challenge to the development of effective vaccine or antiviral therapies.

Comparison of in vivo plasma and peripheral blood mononuclear cell HIV-1 quasi-species to short-term tissue culture isolates: an analysis of tat and C2-V3 env regions.

OBJECTIVE: To determine whether the HIV-1 genomes that grow out in vitro from peripheral blood mononuclear cells (PBMC) better represent the in vivo quasi-species present in plasma or PBMC. RESULTS: For one patient (9606), PBMC culture represented more accurately the plasma rather than the in vivo PBMC quasi-species distribution, because a large number of tat-defective proviruses present in PBMC in vivo were not detected in plasma nor in the PBMC cultures. For a second patient (9605), PBMC culture was representative of both in vivo PBMC and plasma tat sequences, but selection of C2-V3 env sequences was observed in PBMC cultures compared with sequences present in both plasma and PBMC in vivo. This selection consisted of the absence in vitro of genomes with certain amino-acid substitutions at or near conserved glycosylation sites of the C2 region at positions 276 and 289. Site 276 has been reported to be important for viral infectivity, and these substitutions may therefore have affected infectivity. In the third patient (10095), selection of both tat and C2-V3 sequences was observed in culture as compared to plasma and PBMC in vivo. In contrast to the first two patients, this third patient contained V3 sequences in vivo that were predicted to impart syncytium induction and enhanced replication capacity. It was these sequences that grew out preferentially in vitro. CONCLUSION: This study suggests that short-term PBMC culture is representative of HIV-1 genomes present in PBMC and plasma in vivo to the degree that they are infectious.

HIV heterogeneity and viral pathogenesis.

HIV research in the past year has elucidated many questions relevant to strategies for treatment and control. For instance, there is a greater understanding of the diversity of HIV isolates as well as the wide range of potential cells sensitive to infection. The search for a safe, effective vaccine now calls for more caution in the light of the discovery of neutralization-resistant variants and antibody-mediated enhancement of infection. Efforts to control HIV must take into account the various mechanisms of virus entry into host cells, and the processes involved in cytopathic effects. Moreover, the role of cells of the mononuclear phagocyte system as reservoirs for HIV particles should be recognized. Together with new information about cytokine induction of HIV, the concept of latent infection of monocytes and macrophages has profound implications for virus persistence and dissemination, especially in the seronegative individual. While many factors about HIV have been uncovered in the past year, several questions remain unanswered and new ones have arisen. For instance, in how many ways does the virus kill in host cell? What causes latency and why does it occur in some but not all hosts? How can virus-filled macrophage vesicles be reached by therapeutic agents or prevented from releasing HIV? What surveillance mechanisms allow some productively infected hosts (cells as well as individuals) to survive beyond expectation? These and other questions should provoke future research on this presently complex and challenging pathogen.

Effect of major deletions in the V1 and V2 loops of a macrophage-tropic HIV type 1 isolate on viral envelope structure, cell entry, and replication.

Two HIV-1 envelope mutant proteins were generated by introducing deletions in the first and second hypervariable gp120 regions (V1 and V2 loops, respectively) of a macrophage-tropic primary HIV-1 isolate, SF162, to study the effect of the deleted sequences on envelope structure, viral entry, and replication potentials. The first mutant lacked 17 amino acids of the V1 loop and the latter 30 amino acids of the V2 loop. A comparison of the immunochemical structure of the wild-type and mutant monomeric and virion-associated gp120 molecules revealed that the V1 and V2 loop deletions differentially altered the structure of the V3 loop, the CD4-binding site, and epitopes within conserved regions of gp120. Regardless of differences in structure, both mutated envelope proteins supported viral replication into peripheral blood mononuclear cells to levels comparable to those of the wild-type SF162 virus. However, they decreased the viral replication potential in macrophages, even though they did not alter the coreceptor usage of the viruses. These studies support and extend previous observations that a complex structural interaction between the V1, V2, and V3 loops and elements of the CD4-binding site of gp120 controls entry of virus into cells. The present studies, however, suggest that the effect of the V1 and V2 loops in viral entry is cell dependent.

Generation and structural analysis of soluble oligomeric gp140 envelope proteins derived from neutralization-resistant and neutralization-susceptible primary HIV type 1 isolates.

We generated DNA constructs expressing soluble truncated forms of the envelope of SF162, a neutralization-resistant primary human immunodeficiency virus type 1 isolate, and SF162AV2, a neutralization-susceptible virus derived from SF162 after the deletion of 30 amino acids from the V2 loop. The constructs express the entire gp120 subunit and the extracellular region of the gp41 subunit, with either the presence ("cleaved" forms, designated gp140C) or the absence ("fused" forms, designated gp140F) of the gp120-gp41 cleavage site. Both gp140 forms derived from SF162 and SF162deltaV2 are secreted in the cell medium and are recognized by the oligomer-specific anti-gp41 MAb T4. As is the case for the corresponding virion-associated envelope molecules, the CD4-binding region is occluded within both gp140F and gp140C forms. However, structural differences exist between these two forms. The gp140F proteins are less efficiently recognized than the gp140C proteins by antibodies present in the sera of HIV-infected patients with neutralizing activities against SF162 and SF162AV2. Also, the V3 loop is more exposed on gp140F than gp140C. As is the case for intact virions, on CD4 binding both the gp140F and gp140C proteins undergo conformational changes that result in the exposure of the epitope recognized by MAb 17b, which has been implicated in coreceptor binding. In contrast, during these structural changes the exposure of specific V3 loop epitopes is not increased on either gp140C or gp140F. Taken together, our data indicate that although these gp140 forms differ structurally from the native envelope, their similarities, in particular that of gp140C, outweigh their differences.

An individual with a high prevalence of a tat-defective provirus in peripheral blood.

The first exon of tat was sequenced from 23 provirus genomes randomly amplified directly from an HIV-1-infected individual's peripheral blood. Twelve of the 23 sequences constituted a distinct subset of the quasi-species detected. This subset had in common two inactivating mutations in the tat gene. In addition, two of these defective genomes each had a unique mutation. This is the second instance of a defective early gene being present in a high percentage of the proviruses present in the PBMCs of an HIV-1-infected individual, (the first reported by Martins LP et al.: J Virol 1991;65:4502-4507), and suggests that genomes defective in an early gene can participate in the infectious spread of HIV-1 in vivo.

Detection of human immunodeficiency virus (HIV) in serum and body fluids by sequential competition ELISA.

A micro-ELISA based on competition with the biotin-labeled 25 kDa gag (p25gag) recombinant protein of the human immunodeficiency virus (HIV) was compared to commercial antigen capture ELISAs for the detection of viral antigens in a variety of body fluids including serum, cerebro-spinal fluid (CSF), sputum, saliva, milk, semen, vaginal and bronchial fluids, as well as earwash fluid. Two-thirds (24/30) of these specimens contained IgG and/or IgA antibodies to HIV. The results were correlated with the recovery of infectious HIV in culture. The competition ELISA detected the presence of HIV antigen in 4 out of 8 sera, 5 out of 6 CSF and 6 out of 15 other body fluids that were found to contain infectious virus. Comparatively, 5 of the 8 sera, 3 of the 6 CSF, and 2 of the 15 body fluids tested positive for HIV antigen by capture ELISA. The data suggest that the competition test is more effective than the capture method in detecting antigen in CSF and body secretions, which might be due to the presence of immune complexes. However, both ELISA methods showed similar susceptibility to antibody interference in spiked specimens. The results confirm that antigenemia status can be of value in assessing HIV infection when used in combination with other clinical and laboratory data.

A method of video-assisted thoracoscopic surgery for collection of thymic biopsies in rhesus monkeys (Macaca mulatta).

To support an infectious disease study, we developed a surgical procedure to collect serial thymic biopsies in rhesus monkeys (Macaca mulatta). In many instances in which thymic tissue is required from living animals, open surgical approaches (thoracotomies) are used, which result in greater postoperative pain and longer recovery periods than those associated with thoracoscopic procedures. Our intent was to develop a surgical procedure that allowed serial biopsy of the thymus with minimal surgical morbidity. We modified a previously published experimental method of thoracoscopic total thymectomy in the dog to collect thymic biopsies in M. mulatta. Of the 15 animals evaluated, 8 underwent two biopsy procedures separated by a 5- to 6-week interoperative interval. The other seven animals underwent a single biopsy procedure. Thymic tissue was collected successfully during all procedures, with an average surgical time of 15 min. No significant intra- or postoperative complications were noted, and the animals recoveries from the surgical procedures were uneventful. Minimally invasive thoracoscopic surgical techniques can be used successfully to collect thymic tissue from adult and juvenile rhesus monkeys with minimal surgical morbidity.

Distinct biological and serological properties of human immunodeficiency viruses from the brain.

Human immunodeficiency viruses from the brain can be distinguished from peripheral blood isolates by their ability to infect established human cell lines and their sensitivity to serum neutralization. Isolates from the brain and lymph nodes obtained from the same person displayed similar host range tropism and susceptibility to serum neutralization; however, the brain isolate infected macrophages more efficiently. These data suggest that brain isolates may represent a distinct subtype of the human immunodeficiency virus.

An envelope modification that renders a primary, neutralization-resistant clade B human immunodeficiency virus type 1 isolate highly susceptible to neutralization by sera from other clades.

SF162 is a primary (PR), non-syncytium-inducing, macrophagetropic human immunodeficiency virus type 1 (HIV-1) clade B isolate which is resistant to antibody-mediated neutralization. Deletion of the first or second hypervariable envelope gp120 region (V1 or V2 loop, respectively) of this virus does not abrogate its ability to replicate in peripheral blood mononuclear cells and primary macrophages, nor does it alter its coreceptor usage profile. The mutant virus with the V1 loop deletion, SF162DeltaV1, remains as resistant to antibody-mediated neutralization as the wild-type virus SF162. In contrast, the mutant virus with the V2 loop deletion, SF162DeltaV2, exhibits enhanced susceptibility to neutralization by certain monoclonal antibodies whose epitopes are located within the CD4-binding site and conserved regions of gp120. More importantly, SF162DeltaV2 is now up to 170-fold more susceptible to neutralization than SF162 by sera collected from patients infected with clade B HIV-1 isolates. In addition, it becomes susceptible to neutralization by sera collected from patients infected with clade A, C, D, E, and F HIV-1 isolates. These findings suggest that the V2, but not the V1, loop of SF162 shields an as yet unidentified region of the HIV envelope rich in neutralization epitopes and that the overall structure of this region appears to be conserved among clade B, C, D, E, and F HIV-1 PR isolates.