The long cytoplasmic tail of gp41 is required in a cell type-dependent manner for HIV-1 envelope glycoprotein incorporation into virions

Lentiviruses, including HIV-1, have transmembrane envelope (Env) glycoproteins with cytoplasmic tails that are quite long compared with those of other retroviruses. However, mainly because of the lack of biochemical studies performed in cell types that are targets for HIV-1 infection, no clear consensus exists regarding the function of the long lentiviral Env cytoplasmic tail in virus replication. In this report, we characterize the biological and biochemical properties of an HIV-1 mutant lacking the gp41 cytoplasmic tail. We find that the gp41 cytoplasmic tail is necessary for the efficient establishment of a productive, spreading infection in the majority of T cell lines tested, peripheral blood mononuclear cells, and monocyte-derived macrophages. Biochemical studies using a high-level, transient HIV-1 expression system based on pseudotyping with the vesicular stomatitis virus glycoprotein demonstrate that in HeLa and MT-4 cells, mutant Env incorporation into virions is reduced only 3-fold relative to wild type. In contrast, gp120 levels in virions produced from a number of other T cell lines and primary macrophages are reduced more than 10-fold by the gp41 truncation. The Env incorporation defect imposed by the cytoplasmic tail truncation is not the result of increased shedding of gp120 from virions or reduced cell-surface Env expression. These results demonstrate that in the majority of T cell lines, and in primary cell types that serve as natural targets for HIV-1 infection in vivo, the gp41 cytoplasmic tail is essential for efficient Env incorporation into virions.

Stable exposure of the coreceptor-binding site in a CD4-independent HIV-1 envelope protein

We recently derived a CD4-independent virus from HIV-1/IIIB, termed IIIBx, which interacts directly with the chemokine receptor CXCR4 to infect cells. To address the underlying mechanism, a cloned Env from the IIIBx swarm (8x) was used to produce soluble gp120. 8x gp120 bound directly to cells expressing only CXCR4, whereas binding of IIIB gp120 required soluble CD4. Using an optical biosensor, we found that CD4-induced (CD4i) epitopes recognized by mAbs 17b and 48d were more exposed on 8x than on IIIB gp120. The ability of 8x gp120 to bind directly to CXCR4 and to react with mAbs 17b and 48d in the absence of CD4 indicated that this gp120 exists in a partially triggered but stable state in which the conserved coreceptor-binding site in gp120, which overlaps with the 17b epitope, is exposed. Substitution of the 8x V3 loop with that from the R5 virus strain BaL resulted in an Env (8x-V3BaL) that mediated CD4-independent CCR5-dependent virus infection and a gp120 that bound to CCR5 in the absence of CD4. Thus, in a partially triggered Env protein, the V3 loop can change the specificity of coreceptor use but does not alter CD4 independence, indicating that these properties are dissociable. Finally, IIIBx was more sensitive to neutralization by HIV-positive human sera, a variety of anti-IIIB gp120 rabbit sera, and CD4i mAbs than was IIIB. The sensitivity of this virus to neutralization and the stable exposure of a highly conserved region of gp120 suggest new strategies for the development of antibodies and small molecule inhibitors to this functionally important domain.

Influence of follicular dendritic cells on decay of HIV during antiretroviral therapy

Drug treatment of HIV type 1 (HIV-1) infection leads to a rapid initial decay of plasma virus followed by a slower second phase of decay. To investigate the role of HIV-1 retained on follicular dendritic cells (FDCs) in this process, we have developed and analyzed a mathematical model for HIV-1 dynamics in lymphoid tissue (LT) that includes FDCs. Analysis of clinical data using this model indicates that decay of HIV-1 during therapy may be influenced by release of FDC-associated virus. The biphasic character of viral decay can be explained by reversible multivalent binding of HIV-1 to receptors on FDCs, indicating that the second phase of decay is not necessarily caused by long-lived or latently infected cells. Furthermore, viral clearance and death of short-lived productively infected cells may be faster than previously estimated. The model, with reasonable parameter values, is consistent with kinetic measurements of viral RNA in plasma, viral RNA on FDCs, productively infected cells in LT, and CD4+ T cells in LT during therapy.

Anti-HIV-1 and chemotactic activities of human stromal cell-derived factor 1α (SDF-1α) and SDF-1β are abolished by CD26/dipeptidyl peptidase IV-mediated cleavage

CD26 is a leukocyte-activation antigen that is expressed on T lymphocytes and macrophages and possesses dipeptidyl peptidase IV (DPPIV) activity, whose natural substrates have not been identified yet. CXC chemokines, stromal cell-derived factor 1α (SDF-1α) and 1β (SDF-1β), sharing the receptor CXCR-4, are highly efficacious chemoattractants for resting lymphocytes and CD34+ progenitor cells, and they efficiently block the CXCR-4-mediated entry into cells of T cell line tropic strains of HIV type 1 (HIV-1). Here we show that both the chemotactic and antiviral activities of these chemokines are abrogated by DPPIV-mediated specific removal of the N-terminal dipeptide, not only when the chemokines are produced in transformed mouse L cell line to express human CD26 but also when they were exposed to a human T cell line (H9) physiologically expressing CD26. Mutagenesis of SDF-1α confirmed the critical requirement of the N-terminal dipeptide for its chemotactic and antiviral activities. These data suggest that CD26-mediated cleavage of SDF-1α and SDF-1β likely occurs in human bodies and promotes HIV-1 replication and disease progression. They may also explain why memory function of CD4+ cells is preferentially lost in HIV-1 infection. Furthermore, CD26 would modulate various other biological processes in which SDF-1α and SDF-1β are involved.

The HIV-1 regulatory proteins Tat and Rev are frequently targeted by cytotoxic T lymphocytes derived from HIV-1-infected individuals

The HIV-1 regulatory proteins Rev and Tat are expressed early in the virus life cycle and thus may be important targets for the immune control of HIV-1-infection and for effective vaccines. However, the extent to which these proteins are targeted in natural HIV-1 infection as well as precise epitopes targeted by human cytotoxic T lymphocytes (CTL) remain to be defined. In the present study, 57 HIV-1-infected individuals were screened for responses against Tat and Rev by using overlapping peptides spanning the entire Tat and Rev proteins. CD8+ T cell responses against Tat and Rev were found in up to 19 and 37% of HIV-1-infected individuals, respectively, indicating that these regulatory proteins are important targets for HIV-1-specific CTL. Despite the small size of these proteins, multiple CTL epitopes were identified in each. These data indicate that Tat and Rev are frequently targeted by CTL in natural HIV-1 infection and may be important targets for HIV vaccines.

DC-SIGNR, a DC-SIGN homologue expressed in endothelial cells, binds to human and simian immunodeficiency viruses and activates infection in trans

DC-SIGN, a C-type lectin expressed on the surface of dendritic cells (DCs), efficiently binds and transmits HIVs and simian immunodeficiency viruses to susceptible cells in trans. A DC-SIGN homologue, termed DC-SIGNR, has recently been described. Herein we show that DC-SIGNR, like DC-SIGN, can bind to multiple strains of HIV-1, HIV-2, and simian immunodeficiency virus and transmit these viruses to both T cell lines and human peripheral blood mononuclear cells. Binding of virus to DC-SIGNR was dependent on carbohydrate recognition. Immunostaining with a DC-SIGNR-specific antiserum showed that DC-SIGNR was expressed on sinusoidal endothelial cells in the liver and on endothelial cells in lymph node sinuses and placental villi. The presence of this efficient virus attachment factor on multiple endothelial cell types indicates that DC-SIGNR could play a role in the vertical transmission of primate lentiviruses, in the enabling of HIV to traverse the capillary endothelium in some organs, and in the presentation of virus to CD4-positive cells in multiple locations including lymph nodes.

Poly(ADP-ribose) polymerase-1 is required for efficient HIV-1 integration

Poly(ADP-ribose) polymerase-1 (PARP-1; EC 2.4.2.30) is an abundant nuclear enzyme, activated by DNA strand breaks to attach up to 200 ADP-ribose groups to nuclear proteins. As retroviral infection requires integrase-catalyzed DNA strand breaks, we examined infection of pseudotyped HIV type I in fibroblasts from mice with a targeted deletion of PARP-1. Viral infection is almost totally abolished in PARP-1 knockout fibroblasts. This protection from infection reflects prevention of viral integration into the host genome. These findings suggest a potential for PARP inhibitors in therapy of HIV type I infection.

Live attenuated HIV vaccines: Predicting the tradeoff between efficacy and safety

The utility of live attenuated vaccines for controlling HIV epidemics is being debated. Live attenuated HIV vaccines (LAHVs) could be extremely effective in protecting against infection with wild-type strains, but may not be completely safe as the attenuated strain could cause AIDS in some vaccinated individuals. We present a theoretical framework for evaluating the consequences of the tradeoff between vaccine efficacy (in terms of preventing new infections with wild-type strains) and safety (in terms of vaccine-induced AIDS deaths). We use our framework to predict, for Zimbabwe and Thailand, the epidemiological impact of 1,000 different (specified by efficacy and safety characteristics) LAHVs. We predict that paradoxically: (i) in Zimbabwe (where transmission is high) LAHVs would significantly decrease the AIDS death rate, but (ii) in Thailand (where transmission is low) exactly the same vaccines (in terms of efficacy and safety characteristics) would increase the AIDS death rate. Our results imply that a threshold transmission rate exists that determines whether any given LAHV has a beneficial or a detrimental impact. We also determine the vaccine perversity point, which is defined in terms of the fraction of vaccinated individuals who progress to AIDS as a result of the vaccine strain. Vaccination with any LAHV that causes more than 5% of vaccinated individuals to progress to AIDS in 25 years would, even 50 years later, lead to perversity (i.e., increase the annual AIDS death rate) in Thailand; these same vaccines would lead to decreases in the annual AIDS death rate in Zimbabwe.

Control of HIV-1 viremia and protection from AIDS are associated with HLA-Bw4 homozygosity

Certain HLA-B antigens have been associated with lack of progression to AIDS. HLA-B alleles can be divided into two mutually exclusive groups based on the expression of the molecular epitopes HLA-Bw4 and HLA-Bw6. Notably, in addition to its role in presenting viral peptides for immune recognition, the HLA-Bw4, but not HLA-Bw6, motif functions as a ligand for a natural killer cell inhibitory receptor (KIR). Here, we show that profound suppression of HIV-1 viremia is significantly associated with homozygosity for HLA-B alleles that share the HLA-Bw4 epitope. Furthermore, homozygosity for HLA-Bw4 alleles was also significantly associated with the ability to remain AIDS free and to maintain a normal CD4 T cell count in a second cohort of HIV-1-infected individuals with well defined dates of seroconversion. This association was independent of the presence of a mutation in CC chemokine receptor 5 (CCR5) associated with resistance to HIV-1 infection, and it was independent of the presence of HLA alleles that could potentially confound the results. We conclude that homozygosity for HLA-Bw4-bearing B alleles is associated with a significant advantage and that the HLA-Bw4 motif is important in AIDS pathogenesis.

Global survey of genetic variation in CCR5, RANTES, and MIP-1α: Impact on the epidemiology of the HIV-1 pandemic

Expression of CC chemokine receptor 5 (CCR5), the major coreceptor for HIV-1 cell entry, and its ligands (e.g., RANTES and MIP-1α) is widely regarded as central to the pathogenesis of HIV-1 infection. By surveying nearly 3,000 HIV+ and HIV− individuals from worldwide populations for polymorphisms in the genes encoding RANTES, MIP-1α, and CCR5, we show that the evolutionary histories of human populations have had a significant impact on the distribution of variation in these genes, and that this may be responsible, in part, for the heterogeneous nature of the epidemiology of the HIV-1 pandemic. The varied distribution of RANTES haplotypes (AC, GC, and AG) associated with population-specific HIV-1 transmission- and disease-modifying effects is a striking example. Homozygosity for the AC haplotype was associated with an increased risk of acquiring HIV-1 as well as accelerated disease progression in European Americans, but not in African Americans. Yet, the prevalence of the ancestral AC haplotype is high in individuals of African origin, but substantially lower in non-Africans. In a Japanese cohort, AG-containing RANTES haplotype pairs were associated with a delay in disease progression; however, we now show that their contribution to HIV-1 pathogenesis and epidemiology in other parts of the world is negligible because the AG haplotype is infrequent in non-Far East Asians. Thus, the varied distribution of RANTES, MIP-1α, and CCR5 haplotype pairs and their population-specific phenotypic effects on HIV-1 susceptibility and disease progression results in a complex pattern of biological determinants of HIV-1 epidemiology. These findings have important implications for the design, assessment, and implementation of effective HIV-1 intervention and prevention strategies.

A point mutation in the HIV-1 Tat responsive element is associated with postintegration latency.

Study of the mechanism of HIV-1 postintegration latency in the ACH2 cell line demonstrates that these cells failed to increase HIV-1 production following treatment with exogenous Tat. Reasoning that the defect in ACH2 cells involves the Tat response, we analyzed the sequence of tat cDNA and Tat responsive element (TAR) from the virus integrated in ACH2. Tat cDNA sequence is closely related to that of HIV LAI, and the encoded protein is fully functional in terms of long terminal repeat (LTR) transactivation. Cloning of a region corresponding to the 5'-LTR from ACH2, however, identified a point mutation (C37 -> T) in TAR. This mutation impaired Tat responsiveness of the LTR in transient transfection assays, and the measured defect was complemented in cells that had been treated with tetradecanoyl phorbol acetate or tumor necrosis factor type alpha (TNF-alpha). A compensatory mutation in TAR (G28 -> A), designed to reestablish base pairing in the TAR hairpin, restored wild-type Tat responsiveness. When the (C37 -> T) mutation was introduced in an infectious clone of HIV-1, no viral production was measured in the absence of TNF-alpha, whereas full complementation was observed when the infection was conducted in the presence of TNF-alpha or when a compensatory mutation (G28 -> A) was introduced into TAR. These experiments identify a novel mutation associated with HIV-1 latency and suggest that alterations in the Tat-TAR axis can be a crucial determinant of the latent phenotype in infected individuals.

Pattern of gp120 sequence divergence linked to a lack of clinical progression in human immunodeficiency virus type 1 infection.

Differential rates of AIDS development and/or T4 lymphocyte depletion in HIV-1-infected individuals remain unexplained. The hypothesis that qualitative differences in selection pressure in vivo may account for different rates of disease progression was addressed in nine eligible study participants from a cohort of 315 homosexual men who have been followed since 1985. Disproportionately fewer changes in variable regions and more in C3 of gp12O were found to be significantly associated with slower disease progression. Our finding provides the first example to demonstrate that differential selection pressure related to the emergence of HIV-1 variants is associated with long term nonprogression. Candidate vaccines that elicit strong selection pressure on C3 of gp120 are likely to provide better protection than those targeting variable regions.

Human immunodeficiency virus type 1 infection despite prior immunization with a recombinant envelope vaccine regimen.

With efforts underway to develop a preventive human immunodeficiency virus type 1 (HIV-1) vaccine, it remains unclear which immune responses are sufficient to protect against infection and whether prior HIV-1 immunity can alter the subsequent course of HIV-1 infection. We investigated these issues in the context of a volunteer who received six HIV-1LAI envelope immunizations and 10 weeks thereafter acquired HIV-1 infection through a high-risk sexual exposure. In contrast to nonvaccinated acutely infected individuals, anamnestic HIV-1-specific B- and T-cell responses appeared within 3 weeks in this individual, and neutralizing antibody preceded CD8+ cytotoxic responses. Despite an asymptomatic course and an initial low level of detectable infectious virus, a progressive CD4+ cell decline and dysfunction occurred within 2 years. Although vaccination elicited immunity to HIV-1 envelope, which was recalled upon HIV-1 exposure, it was insufficient to prevent infection and subsequent immunodeficiency.

Synthesis and targeted delivery of an azidothymidine homodinucleotide conferring protection to macrophages against retroviral infection.

The infectivity and replication of human (HIV-1), feline (FIV), and murine (LP-BM5) immunodeficiency viruses are all inhibited by several nucleoside analogues after intracellular conversion to their triphosphorylated derivatives. At the cellular level, the main problems in the use of these drugs concern their limited phosphorylation in some cells (e.g., macrophages) and the cytotoxic side effects of nucleoside analogue triphosphates. To overcome these limitations a new nucleoside analogue homodinucleotide, di(thymidine-3'-azido-2',3'-dideoxy-D-riboside)-5'-5'-p1-p2-pyrophosphat e (AZTp2AZT), was designed and synthesized. AZTp2AZT was a poor in vitro inhibitor of HIV reverse transcriptase, although it showed antiviral and cytotoxic activities comparable to those of the parent AZT when added to cultures of a HTLV-1 transformed cell line. AZTp2AZT encapsulated into erythrocytes was remarkably stable. Induction of erythrocyte-membrane protein clusterization and subsequent phagocytosis of AZTp2AZT-loaded cells allowed the targeted delivery of this impermeant drug to macrophages where its metabolic activation occurs. The addition of AZTp2AZT-loaded erythrocytes to human, feline, and murine macrophages afforded almost complete in vitro protection of these cells from infection by HIVBa-L, FIV, and LP-BM5, respectively. Therefore, AZTp2AZT, unlike the membrane-diffusing azidothymidine, acts as a very efficient antiretroviral prodrug following selective targeting to macrophages by means of loaded erythrocytes.

Selective pressure of a quinoxaline nonnucleoside inhibitor of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) on HIV-1 replication results in the emergence of nucleoside RT-inhibitor-specific (RT Leu-74-->Val or Ile and Val-75-->Leu or Ile) HIV-1 mutants.

The quinoxaline nonnucleoside RT inhibitor (NNRTI) (S)-4-isopropoxycarbonyl-6-methoxy-3-(methylthiomethyl)-3,4- dihydroquinoxaline-2(1H)-thione (HBY 097) was used to select for drug-resistant HIV-1 variants in vitro. The viruses first developed mutations affecting the NNRTI-binding pocket, and five of six strains displayed the RT G190-->E substitution, which is characteristic for HIV-1 resistance against quinoxalines. In one variant, a new mutant (G190-->Q) most likely evolved from preexisting G190-->E mutants. The negative charge introduced by the G190-->E substitution was maintained at that site of the pocket by simultaneous selection for V179-->D together with G190-->Q. After continued exposure to the drug, mutations at positions so far known to be specific for resistance against nucleoside RT inhibitors (NRTIs) (L74-->V/I and V75-->L/I) were consistently detected in all cultures. The inhibitory activities of the cellular conversion product of 2',3'-dideoxyinosine (ddI, didanosine), 2',3'-dideoxyadenosine (ddA) and of 2',3'-didehydro-3'-deoxythymidine (d4T, stavudine) against these late-passage viruses were shown to be enhanced with the L74-->V/I RT mutant virus as compared with the wild-type (wt) HIV-1MN isolate. Clonal analysis proved linkage of the codon 74 and codon 75 mutations to the NNRTI-specific mutations in all RT gene fragments. The nonnucleoside- and nucleoside-resistance mutation sites are separated by approximately 35 A. We propose that the two sites "communicate" through the template-primer which is situated in the DNA-binding cleft between these two sites. Quinoxalines cause high selective pressure on HIV-1 replication in vitro; however, the implication of these findings for the treatment of HIV-1 infection has yet to be determined.