Stoichiometry of envelope glycoprotein trimers in the entry of human immunodeficiency virus type 1

The human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins (Envs) function as a trimer, mediating virus entry by promoting the fusion of the viral and target cell membranes. HIV-1 Env trimers induce membrane fusion through a pH-independent pathway driven by the interaction between an Env trimer and its cellular receptors, CD4 and CCR5/CXCR4. We studied viruses with mixed heterotrimers of wild-type and dominant-negative Envs to determine the number (T) of Env trimers required for HIV-1 entry. To our surprise, we found that a single Env trimer is capable of supporting HIV-1 entry; i.e., T = 1. A similar approach was applied to investigate the entry stoichiometry of envelope glycoproteins from amphotropic murine leukemia virus (A-MLV), avian sarcoma/leukosis virus type A (ASLV-A), and influenza A virus. When pseudotyped on HIV-1 virions, the A-MLV and ASLV-A Envs also exhibit a T = 1 entry stoichiometry. In contrast, eight to nine influenza A virus hemagglutinin trimers function cooperatively to achieve membrane fusion and virus entry, using a pH-dependent pathway. The different entry requirements for cooperativity among Env trimers for retroviruses and influenza A virus may influence viral strategies for replication and evasion of the immune system.     

Recombinant extracellular domains of tetraspanin proteins are potent inhibitors of the infection of macrophages by human immunodeficiency virus type 1

Human immunodeficiency virus type 1 (HIV-1) infection of human macrophages can be inhibited by antibodies which bind to the tetraspanin protein CD63, but not by antibodies that bind to other members of the tetraspanin family. This inhibitory response was limited to CCR5 (R5)-tropic virus and was only observed using macrophages, but not T cells. Here, we show that recombinant soluble forms of the large extracellular domain (EC2) of human tetraspanins CD9, CD63, CD81, and CD151 produced as fusion proteins with glutathione S-transferase (GST) can all potently and completely inhibit R5 HIV-1 infection of macrophages with 50% inhibitory concentration values of 0.11 to 1.2 nM. Infection of peripheral blood mononuclear cells could also be partly inhibited, although higher concentrations of EC2 proteins were required. Inhibition was largely coreceptor independent, as macrophage infections by virions pseudotyped with CXCR4 (X4)-tropic HIV-1 or vesicular stomatitis virus (VSV)-G glycoproteins were also inhibited, but was time dependent, since addition prior to or during, but not after, virus inoculation resulted in potent inhibition. Incubation with tetraspanins did not decrease CD4 or HIV-1 coreceptor expression but did block virion uptake. Colocalization of fluorescently labeled tetraspanin EC2 proteins and HIV-1 virions within, and with CD4 and CXCR4 at the cell surfaces of, macrophages could be detected, and internalized tetraspanin EC2 proteins were directed to vesicular compartments that contained internalized dextran and transferrin. Collectively, the data suggest that the mechanism of inhibition of HIV-1 infection by tetraspanins is at the step of virus entry, perhaps via interference with binding and/or the formation of CD4-coreceptor complexes within microdomains that are required for membrane fusion events.     

Wild-type-like viral replication potential of human immunodeficiency virus type 1 envelope mutants lacking palmitoylation signals

Palmitoylation of the cytoplasmic domain of the human immunodeficiency type virus type 1 (HIV-1) envelope (Env) transmembrane protein, gp41, has been implicated in Env targeting to detergent-resistant lipid rafts, Env incorporation into the virus, and viral infectivity. In contrast, we provide evidence here to show that HIV-1 infectivity, Env targeting to lipid rafts, and Env incorporation into the virus are independent of cytoplasmic tail palmitoylation. The T-cell (T)-tropic HXB2-based virus, which utilizes CXCR4 as the entry coreceptor, carrying a Cys-to-Ser mutation at residue 764 or 837 or at both replicated with wild-type (WT) virus replication kinetics in CD4+ T cells. The properties of Env expression, precursor processing, cell surface expression, and Env incorporation of these three mutant viruses were normal compared to those of the WT virus. These three mutant Env proteins all effectively mediated one-cycle virus infection. When the Cys residues were replaced by Ala residues, all single and double mutants still retained the phenotypes of infectivity, Env incorporation, and lipid raft localization of the WT Env. When Cys-to-Ala substitutions were introduced into the macrophage (M)-tropic ConB virus, which utilizes CCR5 as the coreceptor, these mutations did not affect the replication potential, Env phenotypes, lipid raft targeting, or Env assembly into the virus of the WT Env. These T- and M-tropic mutants also productively replicated in human primary CD4+ T cells. Moreover, mutations at both Cys residues significantly reduced the level of palmitoylation of the Env. Our results together support the notion that palmitoylation of the cytoplasmic tail of the HIV-1 Env is not essential for the HIV-1 virus life cycle.     

Dendritic cells preferentially transfer CXCR4-using human immunodeficiency virus type 1 variants to CD4+ T lymphocytes in trans

Human immunodeficiency virus type 1 (HIV-1) preferentially utilizes the CCR5 coreceptor for target cell entry in the acute phase of infection, while later in disease progression the virus switches to the CXCR4 coreceptor in approximately 50% of patients. In response to HIV-1 the adaptive immune response is triggered, and antibody (Ab) production is elicited to block HIV-1 entry. We recently determined that dendritic cells (DCs) can efficiently capture Ab-neutralized HIV-1, restore infectivity, and transmit infectious virus to target cells. Here, we tested the effect of Abs on trans transmission of CCR5 or CXCR4 HIV-1 variants. We observed that transmission of HIV-1 by immature as well as mature DCs was significantly higher for CXCR4- than CCR5-tropic viral strains. Additionally, neutralizing Abs directed against either the gp41 or gp120 region of the envelope such as 2F5, 4E10, and V3-directed Abs inhibited transmission of CCR5-tropic HIV-1, whereas Ab-treated CXCR4-tropic virus demonstrated unaltered or increased transmission. To further study the effects of coreceptor usage we tested molecularly cloned HIV-1 variants with modifications in the envelope that were based on longitudinal gp120 V1 and V3 variable loop sequences from a patient progressing to AIDS. We observed that DCs preferentially facilitated infection of CD4⁺ T lymphocytes of viral strains with an envelope phenotype found late in disease. Taken together, our results illustrate that DCs transmit CXCR4-tropic HIV-1 much more efficiently than CCR5 strains; we hypothesize that this discrimination could contribute to the in vivo coreceptor switch after seroconversion and could be responsible for the increase in viral load.     

Specific reactions between purified HIV-1 particles and CD4+ cell membrane fragments in a cell-free system of virus fusion or entry

The initial step of human immunodeficiency virus type 1 (HIV-1) infection has been studied by Env-mediated fusion or entry assays with appropriate cells expressing CD4 or CXCR4/CCR5 receptors in cultures, where many factors underlying cellular activities likely regulate the fusion/entry efficiency. Here we attempted to develop a more simplified in vitro cell-free fusion/entry reaction that mimics HIV-1 infection in cultures. Membrane fragments of target cells and intact infectious HIV-1 particles were purified, mixed and incubated. The core p24 protein was released from the purified virions and detected by ELISA without detergents in the supernatant of the reaction mixtures. This release reaction proceeded temperature-dependently and in a dose-dependent manner between the virion and membrane fractions, and was specific for HIV-1 Env and CD4. Env-deleted or VSV-G-pseudotyped HIV-1 released little p24, if any. Pretreatment of the membrane fragments with anti-CD4 antibodies inhibited the p24 induction from both X4-tropic and R5-tropic HIV-1. Furthermore, X4 but not R5 HIV-1 reacted with the membrane prepared from intrinsically CXCR4-positive HeLa-CD4 cells, whereas both viruses reacted with that prepared from CCR5-transduced HeLa-CD4 cells, indicating that this cell-free reaction mimics coreceptor usage of HIV-1 infection. Therefore, a potent entry inhibitor of X4 HIV-1, SDF-1alpha, blocked the release from X4 but not R5 HIV-1. Inversely, a weak entry inhibitor of R5 HIV-1, MIP-1beta, partially affected only the release from R5 HIV-1. These results suggest that this cell-free reaction system provides a useful tool to study biochemical fusion/entry mechanisms of HIV-1 and its inhibitors.     

Infection of specific dendritic cells by CCR5-tropic human immunodeficiency virus type 1 promotes cell-mediated transmission of virus resistant to broadly neutralizing antibodies

The tropism of human immunodeficiency virus type 1 for chemokine receptors plays an important role in the transmission of AIDS. Although CXCR4-tropic virus is more cytopathic for T cells, CCR5-tropic strains are transmitted more frequently in humans for reasons that are not understood. Phenotypically immature myeloid dendritic cells (mDCs) are preferentially infected by CCR5-tropic virus, in contrast to mature mDCs, which are not susceptible to infection but instead internalize virus into a protected intracellular compartment and enhance the infection of T cells. Here, we define a mechanism to explain preferential transmission of CCR5-tropic viruses based on their interaction with mDCs and sensitivity to neutralizing antibodies. Infected immature mDCs differentiated normally and were found to enhance CCR5-tropic but not CXCR4-tropic virus infection of T cells even in the continuous presence of neutralizing antibodies. Infectious synapses also formed normally in the presence of such antibodies. Infection of immature mDCs by CCR5-tropic virus can therefore establish a pool of infected cells that can efficiently transfer virus at the same time that they protect virus from antibody neutralization. This property of DCs may enhance infection, contribute to immune evasion, and could provide a selective advantage for CCR5-tropic virus transmission.     

Apoptosis of bystander T cells induced by human immunodeficiency virus type 1 with increased envelope/receptor affinity and coreceptor binding site exposure

Apoptosis of uninfected bystander CD4(+) T cells contributes to T-cell depletion during human immunodeficiency virus type 1 (HIV-1) pathogenesis. The viral and host mechanisms that lead to bystander apoptosis are not well understood. To investigate properties of the viral envelope glycoproteins (Env proteins) that influence the ability of HIV-1 to induce bystander apoptosis, we used molecularly cloned viruses that differ only in specific amino acids in Env. The ability of these strains to induce bystander apoptosis was tested in herpesvirus saimiri-immortalized primary CD4(+) T cells (CD4/HVS), which resemble activated primary T cells. Changes in Env that increase affinity for CD4 or CCR5 or increase coreceptor binding site exposure enhanced the capacity of HIV-1 to induce bystander apoptosis following viral infection or exposure to nonreplicating virions. Apoptosis induced by HIV-1 virions was inhibited by CD4, CXCR4, and CCR5 antibodies or by the CXCR4 inhibitor AMD3100, but not the fusion inhibitor T20. HIV-1 virions with mutant Envs that bind CXCR4 but are defective for CD4 binding or membrane fusion induced apoptosis, whereas CXCR4 binding-defective mutants did not. These results demonstrate that HIV-1 virions induce apoptosis through a CXCR4- or CCR5-dependent pathway that does not require Env/CD4 signaling or membrane fusion and suggest that HIV-1 variants with increased envelope/receptor affinity or coreceptor binding site exposure may promote T-cell depletion in vivo by accelerating bystander cell death.     

Dendritic cells are less susceptible to human immunodeficiency virus type 2 (HIV-2) infection than to HIV-1 infection

Human immunodeficiency virus type 1 (HIV-1) infection of dendritic cells (DCs) has been documented in vivo and may be an important contributor to HIV-1 transmission and pathogenesis. HIV-1-specific CD4⁺ T cells respond to HIV antigens presented by HIV-1-infected DCs and in this process become infected, thereby providing a mechanism through which HIV-1-specific CD4⁺ T cells could become preferentially infected in vivo. HIV-2 disease is attenuated with respect to HIV-1 disease, and host immune responses are thought to be contributory. Here we investigated the susceptibility of primary myeloid DCs (mDCs) and plasmacytoid DCs (pDCs) to infection by HIV-2. We found that neither CCR5-tropic primary HIV-2 isolates nor a lab-adapted CXCR4-tropic HIV-2 strain could efficiently infect mDCs or pDCs, though these viruses could infect primary CD4⁺ T cells in vitro. HIV-2-exposed mDCs were also incapable of transferring virus to autologous CD4⁺ T cells. Despite this, we found that HIV-2-specific CD4⁺ T cells contained more viral DNA than memory CD4⁺ T cells of other specificities in vivo. These data suggest that either infection of DCs is not an important contributor to infection of HIV-2-specific CD4⁺ T cells in vivo or that infection of DCs by HIV-2 occurs at a level that is undetectable in vitro. The frequent carriage of HIV-2 DNA within HIV-2-specific CD4⁺ T cells, however, does not appear to be incompatible with preserved numbers and functionality of HIV-2-specific CD4⁺ T cells in vivo, suggesting that additional mechanisms contribute to maintenance of HIV-2-specific CD4⁺ T-cell help in vivo.     

Role of CCR5 in infection of primary macrophages and lymphocytes by macrophage-tropic strains of human immunodeficiency virus: resistance to patient-derived and prototype isolates resulting from the delta ccr5 mutation.

The alpha-chemokine receptor fusin (CXCR-4) and beta-chemokine receptor CCR5 serve as entry cofactors for T-cell (T)-tropic and macrophage (M)-tropic human immunodeficiency virus type 1 (HIV-1) strains, respectively, when expressed with CD4 in otherwise nonpermissive cells. Some M-tropic and dual-tropic strains can also utilize other beta-chemokine receptors, such as CCR2b and CCR3. A mutation of CCR5 (delta ccr5) was recently found to be common in certain populations and appears to confer protection against HIV-1 in vivo. Here, we show that this mutation results in a protein that is expressed intracellularly but not on the cell surface. Primary CD4 T cells from delta ccr5 homozygous individuals were highly resistant to infection with prototype M-tropic HIV-1 strains, including an isolate (YU-2) that uses CCR5 and CCR3, but were permissive for both a T-tropic strain (3B) and a dual-tropic variant (89.6) that uses CXCR-4, CCR5, CCR3, or CCR2b. These cells were also resistant to M-tropic patient isolates but were readily infected by T-tropic patient isolates. Primary macrophages from delta ccr5 homozygous individuals were also resistant to infection with M-tropic strains, including YU-2, but the dual-tropic strain 89.6 was able to replicate in them even though macrophages are highly resistant to CXCR-4-dependent T-tropic isolates. These data show that CCR5 is the essential cofactor for infection of both primary macrophages and T lymphocytes by most M-tropic strains of HIV-1. They also suggest that CCR3 does not function for HIV-1 entry in primary lymphocytes or macrophages, but that a molecule(s) other than CCR5 can support entry into macrophages by certain virus isolates. These studies further define the cellular basis for the resistance to HIV-1 infection of individuals lacking functional CCR5.     

HIV-1 selectively infects a subset of nonmaturing BDCA1-positive dendritic cells in human blood

The infection of cultured monocyte-derived dendritic cells (DCs) with HIV-1 involves CD4 and CCR5 receptors, while transmission to T cells is enhanced at least in part by the lectin DC-SIGN/CD209. In the present study, we studied BDCA-1+ myeloid DCs isolated directly from human blood. These cells express CD4 and low levels of CCR5 and CXCR4 coreceptors, but not DC-SIGN. The myeloid DCs replicate two R5 viruses, BaL and YU2, and transfer infection to activated T cells. The virus productively infects a small fraction of the blood DCs that fail to mature in culture, as indicated by the maturation markers CD83 and DC-LAMP/CD208, and the expression of high CD86 and MHC class II, in contrast to many noninfected DCs. A greater proportion of BDCA-1+ DCs are infected when the virus is pseudotyped with the vesicular stomatitis envelope VSV-G (5-15%), as compared with the R5 virus (0.3-3.5%), indicating that HIV-1 coreceptors may limit the susceptibility of DCs to become infected, or the endocytic route of viral entry used by HIV/vesicular stomatitis virus enhances infectivity. When infected and noninfected cells are purified by cell sorting, the former uniformly express HIV p24 gag and are virtually inactive as stimulators of the allogeneic MLR, in contrast to potent stimulation by noninfected DCs from the same cultures. These results point to two roles for a small fraction of blood DCs in HIV-1 pathogenesis: to support productive infection and to evade the direct induction of T cell-mediated immunity.     

Human immunodeficiency virus type 1 infection and replication in normal human oral keratinocytes

Recent epidemiologic studies show increasing human immunodeficiency virus type 1 (HIV-1) transmission through oral-genital contact. This paper examines the possibility that normal human oral keratinocytes (NHOKs) might be directly infected by HIV or might convey infectious HIV virions to adjacent leukocytes. PCR analysis of proviral DNA constructs showed that NHOKs can be infected by CXCR4-tropic (NL4-3 and ELI) and dualtropic (89.6) strains of HIV-1 to generate a weak but productive infection. CCR5-tropic strain Ba-L sustained minimal viral replication. Antibody inhibition studies showed that infection by CXCR4-tropic viral strains is mediated by the galactosylceramide receptor and the CXCR4 chemokine coreceptor. Coculture studies showed that infectious HIV-1 virions can also be conveyed from NHOKs to activated peripheral blood lymphocytes, suggesting a potential role of oral epithelial cells in the transmission of HIV infection.     

Phenotypic studies on recombinant human immunodeficiency virus type 1 (HIV-1) containing CRF01_AE env gene derived from HIV-1-infected patient,residing in central Thailand

Human immunodeficiency virus type 1 (HIV-1) env genes were cloned from blood samples of HIV-1-infected Thai patients, and 35 infectious CRF01_AE envelope glycoprotein (Env)-recombinant viruses were established. In this report, we examined the neutralization susceptibility of these viruses to human monoclonal antibodies, 2G12, IgG1 b12, 2F5 and 4E10, pooled patient plasma, coreceptor antagonists and fusion inhibitor, T-20. The neutralization susceptibility of CRF01_AE Env-recombinant viruses to 2F5, 4E10, patient plasma, coreceptor antagonists and T-20 varied, while most viruses showed low susceptibility to 2G12 and IgG1 b12. Several dual-tropic viruses showed lower susceptibility to 2F5 and 4E10 than CXCR4- or CCR5-tropic viruses. Neutralization susceptibility of the CRF01_AE Env-recombinant virus to pooled patient plasma was negatively correlated with the length of the V1/V2 region or the number of potential N-linked glycosylation sites in conserved regions of gp120. No correlation was found between the coreceptor usage and neutralization susceptibility of the virus to T-20, whereas several dual-tropic viruses showed higher susceptibility to coreceptor antagonists than CXCR4- or CCR5-tropic viruses. We propose that these CRF01_AE Env-recombinant viruses are useful to further study the molecular mechanism of the susceptibility of CRF01_AE Env to neutralizing antibodies and viral entry inhibitors.     

Nef can enhance the infectivity of receptor-pseudotyped human immunodeficiency virus type 1 particles

Nef is an accessory protein of human immunodeficiency virus type 1 (HIV-1) that enhances the infectivity of progeny virions when expressed in virus-producing cells. The requirement for Nef for optimal infectivity is, at least in part, determined by the envelope (Env) glycoprotein, because it can be eliminated by pseudotyping HIV-1 particles with pH-dependent Env proteins. To investigate the role of Env in the function of Nef, we have examined the effect of Nef on the infectivity of Env-deficient HIV-1 particles pseudotyped with viral receptors for cells expressing cognate Env proteins. We found that Nef significantly enhances the infectivity of CD4-chemokine receptor pseudotypes for cells expressing HIV-1 Env. Nef also increased the infectivity of HIV-1 particles pseudotyped with Tva, the receptor for subgroup A Rous sarcoma virus (RSV-A), even though Nef had no effect if the pH-dependent Env protein of RSV-A was used for pseudotyping. However, Nef does not always enhance viral infectivity if the normal orientation of the Env-receptor interaction is reversed, because the entry of Env-deficient HIV-1 into cells expressing the vesicular stomatitis virus G protein was unaffected by Nef. Together, our results demonstrate that the presence of a viral Env protein during virus production is not required for the ability of Nef to increase viral infectivity. Furthermore, since the infectivity of Tva pseudotypes was blocked by inhibitors of endosomal acidification, we conclude that low-pH-dependent entry does not always bypass the requirement for Nef.     

Primary intestinal epithelial cells selectively transfer R5 HIV-1 to CCR5+ cells

The upper gastrointestinal tract is a principal route of HIV-1 entry in vertical transmission and after oral-genital contact. The phenotype of the newly acquired virus is predominantly R5 (CCR5-tropic) and not X4 (CXCR4-tropic), although both R5 and X4 viruses are frequently inoculated onto the mucosa. Here we show that primary intestinal (jejunal) epithelial cells express galactosylceramide, an alternative primary receptor for HIV-1, and CCR5 but not CXCR4. Moreover, we show that intestinal epithelial cells transfer R5, but not X4, viruses to CCR5+ indicator cells, which can efficiently replicate and amplify virus expression. Transfer was remarkably efficient and was not inhibited by the fusion blocker T-20, but was substantially reduced by colchicine and low (4 degrees C) temperature, suggesting endocytotic uptake and microtubule-dependent transcytosis of HIV-1. Our finding that CCR5+ intestinal epithelial cells select and transfer exclusively R5 viruses indicates a mechanism for the selective transmission of R5 HIV-1 in primary infection acquired through the upper gastrointestinal tract.     

Nef stimulates human immunodeficiency virus type 1 replication in primary T cells by enhancing virion-associated gp120 levels: coreceptor-dependent requirement for Nef in viral replication

The Nef protein enhances human immunodeficiency virus type 1 (HIV-1) replication through an unknown mechanism. We and others have previously reported that efficient HIV-1 replication in activated primary CD4(+) T cells depends on the ability of Nef to downregulate CD4 from the cell surface. Here we demonstrate that Nef greatly enhances the infectivity of HIV-1 particles produced in primary T cells. Nef-defective HIV-1 particles contained significantly reduced quantities of gp120 on their surface; however, Nef did not affect the levels of virion-associated gp41, indicating that Nef indirectly stabilizes the association of gp120 with gp41. Surprisingly, Nef was not required for efficient replication of viruses that use CCR5 for entry, nor did Nef influence the infectivity or gp120 content of these virions. Nef also inhibited the incorporation of CD4 into HIV-1 particles released from primary T cells. We propose that Nef, by downregulating cell surface CD4, enhances HIV-1 replication by inhibiting CD4-induced dissociation of gp120 from gp41. The preferential requirement for Nef in the replication of X4-tropic HIV-1 suggests that the ability of Nef to downregulate CD4 may be most important at later stages of disease when X4-tropic viruses emerge.     

Low pH is required for avian sarcoma and leukosis virus Env-dependent viral penetration into the cytosol and not for viral uncoating

A novel entry mechanism has been proposed for the avian sarcoma and leukosis virus (ASLV), whereby interaction with specific cell surface receptors activates or primes the viral envelope glycoprotein (Env), rendering it sensitive to subsequent low-pH-dependent fusion triggering in acidic intracellular organelles. However, ASLV fusion seems to proceed to a lipid mixing stage at neutral pH, leading to the suggestion that low pH might instead be required for a later stage of viral entry such as uncoating (L. J. Earp, S. E. Delos, R. C. Netter, P. Bates, and J. M. White. J. Virol. 77:3058-3066, 2003). To address this possibility, hybrid virus particles were generated with the core of human immunodeficiency virus type 1 (HIV-1), a known pH-independent virus, and with subgroups A or B ASLV Env proteins. Infection of cells by these pseudotyped virions was blocked by lysosomotropic agents, as judged by inhibition of HIV-1 DNA synthesis. Furthermore, by using HIV-1 cores that contain a Vpr-beta-lactamase fusion protein (Vpr-BlaM) to monitor viral penetration into the cytosol, we demonstrated that virions bearing ASLV Env, but not HIV-1 Env, enter the cytosol in a low-pH-dependent manner. This effect was independent of the presence of the cytoplasmic tail of ASLV Env. These studies provide strong support for the model, indicating that low pH is required for ASLV Env-dependent viral penetration into the cytosol and not for viral uncoating.     

Preferential infection of dendritic cells during human immunodeficiency virus type 1 infection of blood leukocytes

Human immunodeficiency virus type 1 (HIV-1) transmission by the parenteral route is similar to mucosal transmission in the predominance of virus using the CCR5 coreceptor (R5 virus), but it is unclear whether blood dendritic cells (DCs), monocytes, or T cells are the cells initially infected. We used ex vivo HIV-1 infection of sorted blood mononuclear cells to model the in vivo infection of blood leukocytes. Using quantitative real-time PCR to detect full-length HIV-1 DNA, both sorted CD11c⁺ myeloid and CD11c⁻ plasmacytoid DCs were more frequently infected than other blood mononuclear cells, including CD16⁺ or CD14⁺ monocytes or resting CD4⁺ T cells. There was a strong correlation between CCR5 coreceptor use and preferential DC infection across a range of HIV-1 isolates. After infection of unsorted blood mononuclear cells, HIV-1 was initially detected in the CD11c⁺ DCs and later in other leukocytes, including clustering DCs and activated T cells. DC infection with R5 virus was productive, as shown by efficient transmission to CD4⁺ T cells in coculture. Blood DCs infected with HIV-1 in vitro and cultured alone expressed only low levels of multiply spliced HIV-1 RNA unless cocultured with CD4⁺ T cells. Early selective infection of immature blood DCs by R5 virus and upregulation of viral expression during DC-T-cell interaction and transmission provide a potential pathway for R5 selection following parenteral transmission.     

Estimating the threshold surface density of Gp120-CCR5 complexes necessary for HIV-1 envelope-mediated cell-cell fusion

Reduced expression of CCR5 on target CD4(+) cells lowers their susceptibility to infection by R5-tropic HIV-1, potentially preventing transmission of infection and delaying disease progression. Binding of the HIV-1 envelope (Env) protein gp120 with CCR5 is essential for the entry of R5 viruses into target cells. The threshold surface density of gp120-CCR5 complexes that enables HIV-1 entry remains poorly estimated. We constructed a mathematical model that mimics Env-mediated cell-cell fusion assays, where target CD4(+)CCR5(+) cells are exposed to effector cells expressing Env in the presence of a coreceptor antagonist and the fraction of target cells fused with effector cells is measured. Our model employs a reaction network-based approach to describe protein interactions that precede viral entry coupled with the ternary complex model to quantify the allosteric interactions of the coreceptor antagonist and predicts the fraction of target cells fused. By fitting model predictions to published data of cell-cell fusion in the presence of the CCR5 antagonist vicriviroc, we estimated the threshold surface density of gp120-CCR5 complexes for cell-cell fusion as ～20 μm(-2). Model predictions with this threshold captured data from independent cell-cell fusion assays in the presence of vicriviroc and rapamycin, a drug that modulates CCR5 expression, as well as assays in the presence of maraviroc, another CCR5 antagonist, using sixteen different Env clones derived from transmitted or early founder viruses. Our estimate of the threshold surface density of gp120-CCR5 complexes necessary for HIV-1 entry thus appears robust and may have implications for optimizing treatment with coreceptor antagonists, understanding the non-pathogenic infection of non-human primates, and designing vaccines that suppress the availability of target CD4(+)CCR5(+) cells.     

Cleavage of the Murine Leukemia Virus Transmembrane Env Protein by Human Immunodeficiency Virus Type 1 Protease: Transdominant Inhibition by Matrix Mutations

We have identified mutations in the human immunodeficiency virus type 1 (HIV-1) matrix protein (MA) which block infectivity of virions pseudotyped with murine leukemia virus (MuLV) envelope (Env) glycoproteins without affecting infectivity conferred by HIV-1 Env or vesicular stomatitis virus G glycoproteins. This inhibition is very potent and displays a strong transdominant effect; infectivity is reduced more than 100-fold when wild-type and mutant molecular clones are cotransfected at a 1:1 ratio. This phenomenon is observed with both ecotropic and amphotropic MuLV Env. The MA mutations do not affect the incorporation of MuLV Env into virions. We demonstrate that in HIV-1 virions pseudotyped with MuLV Env, the HIV-1 protease (PR) efficiently catalyzes the cleavage of the p15(E) transmembrane (TM) protein to p12(E). Immunoprecipitation analysis of pseudotyped virions reveals that the mutant MA blocks this HIV-1 PR-mediated cleavage of MuLV TM. Furthermore, the transdominant inhibition exerted by the mutant MA on wild-type infectivity correlates with the relative level of p15(E) cleavage. Consistent with the hypothesis that abrogation of infectivity imposed by the mutant MA is due to inhibition of p15(E) cleavage, mutant virions are significantly more infectious when pseudotyped with a truncated p12(E) form of MuLV Env. These results indicate that HIV-1 Gag sequences can influence the viral PR-mediated processing of the MuLV TM Env protein p15(E). These findings have implications for the development of HIV-1-based retroviral vectors pseudotyped with MuLV Env, since p15(E) cleavage is essential for activating membrane fusion and virus infectivity.