Áine McKnight

CD4-independent infection by HIV-2 is mediated by Fusin/CXCR4

Several members of the chemokine receptor family have been shown to function in association with CD4 to permit HIV-1 entry and infection. However, the mechanism by which these molecules serve as CD4-associated cofactors is unclear. In the present report, we show that one member of this family, termed Fusin/ CXCR4, is able to function as an alternative receptor for some isolates of HIV-2 in the absence of CD4. This conclusion is supported by the finding that (1) CD4-independent infection by these viruses is inhibited by an anti-Fusin monoclonal antibody, (2) Fusin expression renders human and nonhuman CD4-negative cell lines sensitive to HIV-2-induced syncytium induction and/or infection, and (3) Fusin is selectively down-regulated from the cell surface following HIV-2 infection. The finding that one chemokine receptor can function as a primary viral receptor strongly suggests that the HIV envelope glycoprotein contains a binding site for these proteins and that differences in the affinity and/or the availability of this site can extend the host range of these viruses to include a number of CD4-negative cell types.

Analysis of memory B cell responses and isolation of novel monoclonal antibodies with neutralizing breadth from HIV-1-infected individuals.

Background The isolation of human monoclonal antibodies (mAbs) that neutralize a broad spectrum of primary HIV-1 isolates and the characterization of the human neutralizing antibody B cell response to HIV-1 infection are important goals that are central to the design of an effective antibody-based vaccine. Methods and Findings We immortalized IgG+ memory B cells from individuals infected with diverse clades of HIV-1 and selected on the basis of plasma neutralization profiles that were cross-clade and relatively potent. Culture supernatants were screened using various recombinant forms of the envelope glycoproteins (Env) in multiple parallel assays. We isolated 58 mAbs that were mapped to different Env surfaces, most of which showed neutralizing activity. One mAb in particular (HJ16) specific for a novel epitope proximal to the CD4 binding site on gp120 selectively neutralized a multi-clade panel of Tier-2 HIV-1 pseudoviruses, and demonstrated reactivity that was comparable in breadth, but distinct in neutralization specificity, to that of the other CD4 binding site-specific neutralizing mAb b12. A second mAb (HGN194) bound a conserved epitope in the V3 crown and neutralized all Tier-1 and a proportion of Tier-2 pseudoviruses tested, irrespective of clade. A third mAb (HK20) with broad neutralizing activity, particularly as a Fab fragment, recognized a highly conserved epitope in the HR-1 region of gp41, but showed striking assay-dependent selectivity in its activity. Conclusions This study reveals that by using appropriate screening methods, a large proportion of memory B cells can be isolated that produce mAbs with HIV-1 neutralizing activity. Three of these mAbs show unusual breadth of neutralization and therefore add to the current panel of HIV-1 neutralizing antibodies with potential for passive protection and template-based vaccine design.

Cell surface receptors, virus entry and tropism of primate lentiviruses

Human immunodeficiency virus (HIV) exploits cell surface receptors to attach to and gain entry into cells. The HIV envelope spike glycoprotein on the surface of virus particles binds both CD4 and a seven-transmembrane coreceptor. These interactions trigger conformational changes in the envelope spike that induce fusion of viral and cellular membranes and entry of the viral core into the cell cytoplasm. Other cell surface receptors also interact with gp120 and aid attachment of virus particles. This review describes these receptors, their roles in HIV entry and their influence on cell tropism.

Llama Antibody Fragments with Cross-Subtype Human Immunodeficiency Virus Type 1 (HIV-1)-Neutralizing Properties and High Affinity for HIV-1 gp120

ABSTRACT Members of the Camelidae family produce immunoglobulins devoid of light chains. We have characterized variable domains of these heavy chain antibodies, the VHH, from llamas immunized with human immunodeficiency virus type 1 (HIV-1) envelope protein gp120 in order to identify VHH that can inhibit HIV-1 infection. To increase the chances of isolating neutralizing VHH, we employed a functional selection approach, involving panning of phage libraries expressing the VHH repertoire on recombinant gp120, followed by a competitive elution with soluble CD4. By immunizing with gp120 derived from an HIV-1 subtype B′/C primary isolate, followed by panning on gp120 from HIV-1 isolates of subtypes A, B, and C, we could select for VHH with cross-subtype neutralizing activity. Three VHH able to neutralize HIV-1 primary isolates of subtypes B and C were characterized. These bound to recombinant gp120 with affinities close to the suggested affinity ceiling for in vivo-maturated antibodies and competed with soluble CD4 for this binding, indicating that their mechanism of neutralization involves interacting with the functional envelope spike prior to binding to CD4. The most potent VHH in terms of low 50% inhibitory concentration (IC50) and IC90 values and cross-subtype reactivity was A12. These results indicate that camelid VHH can be potent HIV-1 entry inhibitors. Since VHH are stable and can be produced at a relatively low cost, they may be considered for applications such as HIV-1 microbicide development. Antienvelope VHH might also prove useful in defining neutralizing and nonneutralizing epitopes on HIV-1 envelope proteins, with implications for HIV-1 vaccine design.

Development of the antibody response in acute HIV-1 infection.

Background: Cytotoxic T lymphocytes have been shown to reduce viraemia during acute HIV-1 infection; however the role of neutralizing antibodies in this process is unclear. One confounding factor may be artefacts introduced by viral culture. Objective: To assess the development of autologous neutralizing and non-neutralizing antibodies following acute HIV-1 infection using recombinant viruses with envelopes amplified directly from patient peripheral blood mononuclear cells, thereby avoiding in vitro selection. Methods: Disease progression in four homosexual men was monitored from acute infection for up to 2.5 years, in the absence of antiretroviral therapy. Antibodies to viral envelope protein were quantified by enzyme-linked immunosorbent assay. Development of neutralizing antibodies was monitored using a quantitative infectivity reduction assay, sequential serum, recombinant viruses and target cells with defined receptor expression. Results: The time to development of neutralizing antibodies after onset of symptoms was 3, 5, 7 and 16 months in the four patients. There was no correlation between development of neutralizing antibodies and the resolution of viraemia in any of the patients. However, antibodies to the envelope were detectable as early as 2 weeks after onset of symptoms. Conclusions: Neutralizing antibodies do not contribute to the control of viraemia in acute HIV-1 infection. However, antibodies to the envelope could be detected at the time of reduction in plasma viraemia and so other effector functions of antibodies may play a role in viral clearance.

A whole genome screen for HIV restriction factors.

BackgroundUpon cellular entry retroviruses must avoid innate restriction factors produced by the host cell. For human immunodeficiency virus (HIV) human restriction factors, APOBEC3 (apolipoprotein-B-mRNA-editing-enzyme), p21 and tetherin are well characterised.ResultsTo identify intrinsic resistance factors to HIV-1 replication we screened 19,121 human genes and identified 114 factors with significant inhibition of infection. Those with a known function are involved in a broad spectrum of cellular processes including receptor signalling, vesicle trafficking, transcription, apoptosis, cross-nuclear membrane transport, meiosis, DNA damage repair, ubiquitination and RNA processing. We focused on the PAF1 complex which has been previously implicated in gene transcription, cell cycle control and mRNA surveillance. Knockdown of all members of the PAF1 family of proteins enhanced HIV-1 reverse transcription and integration of provirus. Over-expression of PAF1 in host cells renders them refractory to HIV-1. Simian Immunodeficiency Viruses and HIV-2 are also restricted in PAF1 expressing cells. PAF1 is expressed in primary monocytes, macrophages and T-lymphocytes and we demonstrate strong activity in MonoMac1, a monocyte cell line.ConclusionsWe propose that the PAF1c establishes an anti-viral state to prevent infection by incoming retroviruses. This previously unrecognised mechanism of restriction could have implications for invasion of cells by any pathogen.

Detection of Antibody-Dependent Complement-Mediated Inactivation of both Autologous and Heterologous Virus in Primary Human Immunodeficiency Virus Type 1 Infection

ABSTRACT Specific CD8 T-cell responses to human immunodeficiency virus type 1 (HIV-1) are induced in primary infection and make an important contribution to the control of early viral replication. The importance of neutralizing antibodies in containing primary viremia is questioned because they usually arise much later. Nevertheless antienvelope antibodies develop simultaneously with, or even before, peak viremia. We determined whether such antibodies might control viremia by complement-mediated inactivation (CMI). In each of seven patients studied, antibodies capable of CMI appeared at or shortly after the peak in viremia, concomitantly with detection of virus-specific T-cell responses. The CMI was effective on both autologous and heterologous HIV-1 isolates. Activation of the classical pathway and direct viral lysis were at least partly responsible. Since immunoglobulin G (IgG)-antibodies triggered the CMI, specific memory B cells could also be induced by vaccination. Thus, consideration should be given to vaccination strategies that induce IgG antibodies capable of CMI.

The Promiscuous CC Chemokine Receptor D6 Is a Functional Coreceptor for Primary Isolates of Human Immunodeficiency Virus Type 1 (HIV-1) and HIV-2 on Astrocytes

ABSTRACT The role of coreceptors other than CCR5 and CXCR4 in the pathogenesis of human immunodeficiency virus (HIV) disease is controversial. Here we show that a promiscuous CC chemokine receptor, D6, can function as a coreceptor for various primary dual-tropic isolates of HIV type 1 (HIV-1) and HIV-2. Furthermore, D6 usage is common among chimeric HIV-1 constructs bearing the gp120 proteins of isolates from early seroconverting patients. D6 mRNA and immunoreactivity were demonstrated to be expressed in HIV-1 target cells such as macrophages, peripheral blood mononuclear cells, and primary astrocytes. In primary astrocytes, an RNA interference-mediated knockdown of D6 expression inhibited D6-tropic isolate infection. D6 usage may account for some previous observations of alternative receptor tropism for primary human cells. Thus, D6 may be an important receptor for HIV pathogenesis in the brain and for the early dissemination of virus in the host.

Identification of a subset of human immunodeficiency virus type 1 (HIV-1), HIV-2, and simian immunodeficiency virus strains able to exploit an alternative coreceptor on untransformed human brain and lymphoid cells

ABSTRACT The chemokine receptors CCR5 and CXCR4 are the major coreceptors for human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV). At least 12 other chemokine receptors or close relatives support infection by particular HIV and SIV strains on CD4+ transformed indicator cell lines in vitro. However, the role of these alternative coreceptors in vivo is presently thought to be insignificant. Infection of cell lines expressing high levels of recombinant CD4 and coreceptors thus does not provide a true indication of coreceptor use in vivo. We therefore tested primary untransformed cell cultures that lack CCR5 and CXCR4, including astrocytes and brain microvascular endothelial cells (BMVECs), for naturally expressed alternative coreceptors functional for HIV and SIV infection. An adenovirus vector (Ad-CD4) was used to express CD4 in CD4− astrocytes and thus confer efficient infection if a functional coreceptor is present. Using a large panel of viruses with well-defined coreceptor usage, we identified a subset of HIV and SIV strains able to infect two astrocyte cultures derived from adult brain tissue. Astrocyte infection was partially inhibited by several chemokines, indicating a role for the chemokine receptor family in the observed infection. BMVECs were weakly positive for CD4 but negative for CCR5 and CXCR4 and were susceptible to infection by the same subset of isolates that infected astrocytes. BMVEC infection was efficiently inhibited by the chemokine vMIP-I, implicating one of its receptors as an alternative coreceptor for HIV and SIV infection. Furthermore, we tested whether the HIV type 1 and type 2 strains identified were able to infect peripheral blood mononuclear cells (PBMCs) via an alternative coreceptor. Several strains replicated in Δ32/Δ32 CCR5 PBMCs with CXCR4 blocked by AMD3100. This AMD3100-resistant replication was also sensitive to vMIP-I inhibition. The nature and potential role of this alternative coreceptor(s) in HIV infection in vivo is discussed.

Lv2, a Novel Postentry Restriction, Is Mediated by both Capsid and Envelope

ABSTRACT The characterization of restrictions to lentivirus replication in cells identifies critical steps in the viral life cycle and potential therapeutic targets. We previously reported that a human immunodeficiency virus type 2 (HIV-2) isolate was restricted to infection in some human cells, which led us to identify a step in the life cycle of HIV-2 detected after reverse transcription but prior to nuclear entry. The block is bypassed with a vesicular stomatitis virus glycoprotein G (VSV-G) envelope (A. McKnight et al., J. Virol. 75:6914-6922, 2001). We hypothesized that, although the restriction is apparent at a post-reverse transcription step, the lack of progress results from a failure of the virus to reach a cellular compartment with access to the nucleus. Here we analyzed molecular clones of the restricted virus, MCR, and an unrestricted virus, MCN. Using sequence analysis and gene swapping, we mapped the viral determinants to gag and env. Site-directed mutagenesis identified a single amino acid at position 207 in CA to be responsible for the gag restriction. Pseudotype experiments indicate that this step is also important for the infection of cells by HIV-1. The HIV-1 NL4.3 core is restricted if supplied with a restricted MCR envelope but not with VSV-G. Also the NL4.3 envelope rescues the restricted core of HIV-2 MCR. Abrogation experiments with MLV demonstrate that the restriction is distinct from Fv1/Ref1/Lv1. We propose that this represents a new lentiviral restriction, Lv2. Thus, the envelope and capsid of HIV act to ensure that the virus is delivered into an appropriate cellular compartment that allows postentry events in viral replication to proceed efficiently.