A whole genome screen for HIV restriction factors.

BACKGROUND: Upon 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. RESULTS: To 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. CONCLUSIONS: We 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.

Genetic and biological characterization of recombinant HIV type 1 with Env derived from long-term nonprogressor (LTNP) viruses.

Multiple factors are known to contribute to nonprogressive disease in long-term nonprogressors (LTNP). We previously selected LTNPs, in which broadly neutralizing antibodies against HIV-1 very likely contribute to disease prevention. Here, we characterize those LTNPs further. We analyzed sequences of the viral genes env, nef, vpr, tat, and rev as well as the cellular ccr5, HLA-B*5701, and HLA-B*27 genes derived from eight LTNPs, as mutations in these genes have been associated with the LTNP status in some studies. Furthermore, we compared the replication rates of recombinant reporter viruses carrying envelope proteins from LTNPs to control viruses from patients with similar CD4 count and viral load. Concerning the cellular factors, none of the eight LTNPs showed the 32-base pair deletion in the ccr5 gene, and HLA-B*5701 and HLA-B*27 alleles were detected in only one LTNP, respectively. The reading frames for the regulatory genes nef, vpr, tat, and rev were all open. Although Env sequences from LTNPs differed from those of control patients with respect to the length of variable domains and the number of N-glycosylation sites, these differences were not statistically significant and did not lead to differences in infectivity of recombinant reporter viruses.

Human TRIM5alpha mediated restriction of different HIV-1 subtypes and Lv2 sensitive and insensitive HIV-2 variants.

In order to characterize the antiviral activity of human TRIM5alpha in more detail human derived indicator cell lines over expressing wild type human TRIM5alpha were generated and challenged with HIV-1 and HIV-2 viruses pseudotyped with HIV envelope proteins in comparison to VSV-G pseudotyped particles. HIV envelope protein pseudotyped particles (HIV-1[NL4.3], HIV-1[BaL]) showed a similar restriction to infection (12 fold inhibition) compared to VSV-G pseudotyped viruses after challenging TZM-huTRIM5alpha cells. For HIV-2 a stronger restriction to infection was observed when the homologous envelope protein Env42S was pseudotyped onto these particles compared to VSV-G pseudotyped HIV-2 particles (8.6 fold inhibition versus 3.4 fold inhibition). It has been shown that HIV-2 is restricted by the restriction factor Lv2, acting on capsid like TRIM5alpha. A mutation of amino acid 73 (I73V) of HIV-2 capsid renders this virus Lv2-insensitive. Lv2-insensitive VSV-G pseudotyped HIV-2/I73V particles showed a similar restriction to infection as did HIV-2[VSV-G] particles (4 fold inhibition). HIV-2 envelope protein (Env42S)-pseudotyped HIV-2/I73V particles revealed a 9.3 fold increase in infection in TZM cells but remained restricted in TZM-huTRIM5alpha cells (80.6 fold inhibition) clearly indicating that at least two restriction factors, TRIM5alpha and Lv2, act on incoming HIV-2 particles. Further challenge experiments using primary isolates from different HIV-1 subtypes and from HIV-1 group O showed that wild type human TRIM5alpha restricted infection independent of coreceptor use of the infecting particle but to variable degrees (between 1.2 and 19.6 fold restriction).

Inhibition of HIV-1 group M and O isolates by fusion inhibitors.

We examined the susceptibility of HIV-1 group M and O isolates to the fusion inhibitors T-20 and T-1249. Unexpectedly, HIV-1 O isolates were as sensitive as group M viruses to inhibition by T-20 but were usually less susceptible to T-1249. Our data suggest that T-20 has broad antiretroviral activity and would be effective in individuals with HIV-1 O infection. However, polymorphisms in gp41 might affect the sensitivity of HIV-1 O to second-generation fusion inhibitors.

In vitro inhibition of R5 HIV-1 infectivity by X4 V3-derived synthetic peptides.

The aim of the present study was to investigate the inhibitory effect of synthetic peptides derived from the principle neutralizing domain of the V3 loop of the HIV-1 gp120 in the infectivity rates of HIV-1 variants with different tropism. Assessment of the viral infectivity was determined by detection of soluble HIV p24gag antigen in the culture supernatants of PM-1 T cells and primary macrophages after in vitro infection with the R5, Ba-L and X4, NL4.3 variants in the presence or absence of soluble V3-derived synthetic peptides. Our results showed a clear inhibition of Ba-L infectivity in both the PM-1 T cells and primary macrophages. The degree of inhibition was related to the number of basic amino acids in the peptide. The most effective inhibitory peptide, at a concentration of 50 ng/ml, was the one with the highest cationic potential, achieving over 60% inhibition to the PM-1 T cell line and over 90% to primary macrophages. The same peptides did not affect the NL4.3 infectivity. In addition to our previously reported observations on the electrostatic nature of the V3-CCR5 interaction, we show here that V3-like peptides from the more electropositive X4 variants may be useful as effective antagonists and potential infectivity blockers of the R5 variants.

CDC42 Use in Viral Cell Entry Processes by RNA Viruses.

The cellular actin cytoskeleton presents a barrier that must be overcome by many viruses, and it has become increasingly apparent many viral species have developed a diverse repertoire of mechanisms to hijack cellular actin-regulating signalling pathways as part of their cell entry processes. The Rho family GTPase Cdc42 is appreciated as a key moderator of cellular actin dynamics, and the development of specific Cdc42-inhibiting agents has given us an unprecedented ability to investigate its individual role in signalling pathways. However, investigative use of said agents, and the subsequent characterisation of the role Cdc42 plays in viral entry processes has been lacking. Here, we describe the current literature on the role of Cdc42 in human immunodeficiency virus (HIV)-1 cell entry, which represents the most investigated instance of Cdc42 function in viral cell entry processes, and also review evidence of Cdc42 use in other RNA virus cell entries, demonstrating prime areas for more extensive research using similar techniques.

Fitness ranking of individual mutants drives patterns of epistatic interactions in HIV-1.

Fitness interactions between mutations, referred to as epistasis, can strongly impact evolution. For RNA viruses and retroviruses with their high mutation rates, epistasis may be particularly important to overcome fitness losses due to the accumulation of deleterious mutations and thus could influence the frequency of mutants in a viral population. As human immunodeficiency virus type 1 (HIV-1) resistance to azidothymidine (AZT) requires selection of sequential mutations, it is a good system to study the impact of epistasis. Here we present a thorough analysis of a classical AZT-resistance pathway (the 41-215 cluster) of HIV-1 variants by fitness measurements in single round infection assays covering physiological drug concentrations ex vivo. The sign and value of epistasis varied and did not predict the epistatic effect on the mutant frequency. This complex behavior is explained by the fitness ranking of the variants that strongly depends on environmental factors, i.e., the presence and absence of drugs and the host cells used. Although some interactions compensate fitness losses, the observed small effect on the relative mutant frequencies suggests that epistasis might be inefficient as a buffering mechanism for fitness losses in vivo. While the use of epistasis-based hypotheses to make general assumptions on the evolutionary dynamics of viral populations is appealing, our data caution their interpretation without further knowledge on the characteristics of the viral mutant spectrum under different environmental conditions.

Susceptibility to virus-cell fusion at the plasma membrane is reduced through expression of HIV gp41 cytoplasmic domains.

The cytoplasmic tail of the HIV transmembrane protein plays an important role in viral infection. In this study we analyzed the role of retroviral cytoplasmic tails in modulating the cytoskeleton and interfering with virus-cell fusion. HeLaP4 cells expressing different HIV cytoplasmic tail constructs showed reduced acetylated tubulin levels whereas the cytoplasmic tail of MLV did not alter microtubule stability indicating a unique function for the lentiviral cytoplasmic tail. The effect on tubulin is mediated through the membrane proximal region of the HIV cytoplasmic tail and was independent of membrane localization. Site-directed mutagenesis identified three motifs in the HIV-2 cytoplasmic tail required to effect the reduction in acetylated tubulin. Both the YxxPhi domain and amino acids 21 to 45 of the HIV-2 cytoplasmic tail need to be present to change the level of acetylated tubulin in transfected cells. T-cells stably expressing one HIV-2 cytoplasmic tail derived construct showed also a reduction in acetylated tubulin thus confirming the importance of this effect not only for HeLaP4 and 293T cells. Challenge experiments using transiently transfected HeLaP4 cells and T cells stably expressing an HIV cytoplasmic tail construct revealed both reduced virus-cell fusion and replication of HIV-1(NL4.3) compared to control cells. In the virus-cell fusion assay only virions pseudotyped with either HIV or MLV envelopes showed reduced fusion efficiency, whereas VSV-G pseudotyped virions where not affected by the expression of HIV derived cytoplasmic tail constructs, indicating that fusion at the plasma but not endosomal membrane is affected. Overexpression of human histone-deacetylase 6 (HDAC6) and constitutively active RhoA resulted in a reduction of acetylated tubulin and reduced virus-cell fusion as significant as that observed following expression of HIV cytoplasmic tail constructs. Inhibition of HDAC6 showed a strong increase in acetylated tubulin and increase of virus-cell fusion confirming the correlation between post-translational modification of tubulin and virus-cell fusion. These results thus identify tubulin and its post-translational modification as a new cellular target for interference with HIV-cell fusion.

Mimotopes selected with antibodies from HIV-1-neutralizing long-term non-progressor plasma.

A promising approach to identify HIV-1 vaccine candidates is to dissect the natural immune response against the virus in persons controlling the infection over decades without any antiviral therapy. Here we focus on a group of such persons, eight long-term non-progressors (LTNP), in which we proved the presence of broadly neutralizing antibodies against HIV-1 in the plasma as very likely cause for their LTNP status. The aim of this study was to identify the epitopes for these neutralizing antibodies, as these should represent immunogens potentially able to elicit neutralizing antibodies upon vaccination. We screened random peptide phage libraries with plasma antibodies from eight LTNP. After several rounds of positive and negative selection, about 700 HIV-specific mimotopes were sequenced. The mimotope sequences were analyzed for homology to HIV-1 Env, in particular for their capacity to represent conformational epitopes on the surface of the gp120 structure using our software 3DEX. Related phage groups were analyzed for crossreactivity with the LTNP plasma by ELISA as well as for their capacity to induce HIV-1-neutralizing antibodies in mice. Based on this study interesting mimotopes can now be selected for further immunization studies.

Involvement of clathrin-mediated endocytosis in human immunodeficiency virus type 1 entry.

Productive entry of human immunodeficiency virus (HIV) is believed to occur by direct fusion at the plasma membrane. Endocytic uptake of HIV particles has been observed in several studies but is considered to be nonproductive, leading to virus degradation in the lysosome. We show here that endocytosis contributes significantly to productive HIV entry in HeLa cells by using trans dominant-negative mutants of dynamin and Eps15. Inducible expression of a dominant-negative mutant of dynamin in a CD4-positive HeLa cell line reduced HIV infection by 40 to 80%. This effect was independent of the infectious dose and was observed for three different isolates. Analysis of reverse transcription products by real-time PCR and of virus entry by delivery of a virion-associated Vpr-beta-lactamase fusion protein revealed a similar reduction, indicating that the block occurred at the entry stage. A strong reduction of HIV entry was also observed upon transient transfection of a different trans dominant-negative variant of dynamin, and this reduction correlated with the relative inhibition of transferrin endocytosis. Expression of a dominant-negative variant of Eps15, which is specific for clathrin-dependent endocytosis, reduced HIV entry in HeLa cells by ca 95%, confirming the role of endocytosis for productive infection. In contrast, no effect was observed for a dominant-negative variant of caveolin. We conclude that dynamin-dependent, clathrin-mediated endocytosis can lead to productive entry of HIV in HeLa cells, suggesting this pathway as an alternative route of virus entry.

Role of HIV-2 envelope in Lv2-mediated restriction.

We have characterized envelope protein pseudotyped HIV-2 particles derived from two HIV-2 isolates termed prCBL23 and CBL23 in order to define the role of the envelope protein for the Lv2-mediated restriction to infection. Previously, it has been described that the primary isolate prCBL23 is restricted to infection of several human cell types, whereas the T cell line adapted isolate CBL23 is not restricted in these cell types. Molecular cloning of the two isolates revealed that the env and the gag gene are responsible for the observed phenotype and that this restriction is mediated by Lv2, which is distinct from Ref1/Lv1 (Schmitz, C., Marchant, D., Neil, S.J., Aubin, K., Reuter, S., Dittmar, M.T., McKnight, A., Kizhatil, K., Albritton, L.M., 2004. Lv2, a novel postentry restriction, is mediated by both capsid and envelope. J. Virol. 78 (4), 2006-2016). We generated pseudotyped viruses consisting of HIV-2 (ROD-ADeltaenv-GFP, ROD-ADeltaenv-RFP, or ROD-ADeltaenv-REN) and the prCBL23 or CBL23 envelope proteins as well as chimeric proteins between these envelopes. We demonstrate that a single amino acid exchange at position 74 in the surface unit of CBL23-Env confers restriction to infection. This single point mutation causes tighter CD4 binding, resulting in a less efficient fusion into the cytosol of the restricted cell line. Prevention of endosome formation and prevention of endosome acidification enhance infectivity of the restricted particles for GHOST/X4 cells indicating a degradative lysosomal pathway as a cause for the reduced cytosolic entry. The described restriction to infection of the primary isolate prCBL23 is therefore largely caused by an entry defect. A remaining restriction to infection (19-fold) is preserved when endosomal acidification is prevented. This restriction to infection is also dependent on the presence of the point mutation at position 74 (G74E).

T20-insensitive HIV-1 from naive patients exhibits high viral fitness in a novel dual-color competition assay on primary cells.

The relationship between sensitivity to antiviral drugs and viral fitness is of paramount importance in understanding the long-term implications of clinical resistance. Here we report the development of a novel recombinant virus assay to study entry inhibitor-resistant HIV variants using a biologically relevant cell type, primary CD4 T-cells. We have modified the replication-competent molecular clone HIV(NL4-3) to express a reporter protein (Renilla luciferase), Green Fluorescent Protein (EGFP), or Red Fluorescent Protein (DsRed2) upon infection, thus allowing quantification of replication. Luciferase-expressing virus was used to evaluate drug sensitivity, while co-infection with viruses carrying the green and red fluorescent proteins was employed in the competitive fitness assay. Using envelope proteins from three T20 insensitive variants, lower levels of resistance were observed in primary CD4 T-cells than had been previously reported for cell lines. Importantly, dual-color competition assays demonstrated comparable or higher fitness for these variants despite their reduced T20 sensitivity. We conclude that reduced sensitivity to T20 is compatible with high viral fitness in the absence of selection pressure. Thus, simultaneously measuring both resistance and viral fitness using this newly described dual-color competition assay will likely provide important information about resistant viral variants that emerge during therapy with entry inhibitors.

Determinants of human immunodeficiency virus type 1 resistance to membrane-anchored gp41-derived peptides.

The expression of a membrane-anchored gp41-derived peptide (M87) has been shown to confer protection from infection through human immunodeficiency virus type 1 (HIV-1) (Hildinger et al., J. Virol. 75:3038-3042, 2001). In an effort to characterize the mechanism of action of this membrane-anchored peptide in comparison to the soluble peptide T-20, we selected resistant variants of HIV-1(NL4-3) and HIV-1(BaL) by serial virus passage using PM1 cells stably expressing peptide M87. Sequence analysis of the resistant isolates showed different patterns of selected point mutations in heptad repeat regions 1 and 2 (HR1 and HR2, respectively) for the two viruses analyzed. For HIV-1(NL4-3) a single amino acid change at position 33 in HR1 (L33S) was selected, whereas for HIV-1(BaL) the majority of the sequences obtained showed two amino acid changes, one in HR1 and one in HR2 (I48V/N126K). In both selections the most important contiguous 3-amino-acid sequence, GIV, within HR1, associated with resistance to soluble T-20, was not changed. Site-directed mutagenesis studies confirmed the importance of the characterized point mutations to confer resistance to M87 as well as to soluble T-20 and T-649. Replication capacity and dual-color competition assays revealed that the double mutation I48V/N126K in HIV-1(BaL) results in a strong reduction of viral fitness, whereas the L33S mutation in HIV-1(NL4-3) did enhance viral fitness compared to the respective parental viruses. However, the selected point mutations did not confer resistance to the more recently described optimized membrane-anchored fusion inhibitor M87o (Egelhofer et al., J. Virol. 78:568-575, 2004), strengthening the importance of this novel antiviral concept for gene therapy approaches.

Trimeric membrane-anchored gp41 inhibits HIV membrane fusion.

The HIV-1 envelope glycoprotein is composed of a receptor binding subunit, gp120 that is non-covalently linked to the membrane-anchored fusion protein, gp41. Triggered by cellular receptor binding, the trimeric envelope complex mediates the fusion of viral and cellular membranes through the rearrangement of the fusion protein subunit into a six-helical bundle core structure. Here we describe the biophysical and functional properties of a membrane-anchored fragment of gp41 (gp41ctm) that includes the complete C-terminal heptad repeat region 2, the connecting part, and the transmembrane region. We show that the transmembrane domain of the envelope glycoprotein is sufficient for trimerization in vitro, contributing most of the alpha-helical content of gp41ctm. Trimeric gp41ctm is protease-resistant and recognizes neutralizing antibodies 2F5 and 4E10. However, gp41ctm and gp41ctm proteoliposomes elicit no clear neutralizing immune responses in preliminary mouse studies. We further show that gp41ctm and surprisingly also gp41ctm proteoliposomes have potent anti-viral activity. Our data suggest that liposome-anchored gp41ctm exerts its inhibitory action outside of the initial fusion contact site, and its implications for the fusion reaction are discussed.

The N-linked glycan g15 within the V3 loop of the HIV-1 external glycoprotein gp120 affects coreceptor usage, cellular tropism, and neutralization.

We have studied infectivity and neutralization of X4, R5, and R5X4 tropic HIV-1 mutants, which are lacking N-linked glycosylation sites for glycans g13, g14, g15, and g17 in the V3 loop region of gp120. X4-tropic NL4-3 mutants lacking combinations of g14/15 or g15/17 showed markedly higher infectivity in CXCR4-specific infection. The role of g15 in CCR5-specific infection was investigated using viruses with high (NL-918, R5-monotropic), medium (NL-991, R5-monotropic), and low (NL-952, R5X4-dualtropic) CCR5-specific infectivity. For NL-991, a reduction of infectivity on GHOST-CCR5 cells was observed for a mutant lacking g15. For NL-952 mutants all lacking g15, a complete loss of CCR5-specificity was observed and NL-952 was shifted from R5X4 to X4 tropism. For all mutants of NL4-3, NL-991, and NL-952, where the lack of g15 markedly influenced infectivity or coreceptor usage, neutralization was enhanced. In contrast, NL-918 mutants with or without g15 showed no difference in neutralization and no difference in GHOST-CCR5 infection rates. Thus, for viruses with a low or medium CCR5-specificity the role of g15 for changing CCR5-usage and sensitivity to neutralization was more significant than for viruses with high infection rates on GHOST-CCR5 cells. Our data demonstrate that V3 glycans play an important role in the usage of CXCR4 and CCR5. The lack of g15 was relevant for a more efficient use of CXCR4, whereas interaction with CCR5 was facilitated in the presence of g15. This study also demonstrates that glycan g15 is involved in blocking of neutralizing antibodies and shifting HIV tropism from R5X4 to X4.

HIV: epidemiology and strategies for therapy and vaccination.

In the decades since its discovery, the acquired immunodeficiency syndrome AIDS has developed into one of the leading causes of death worldwide, with a dramatic situation in many countries of subsaharan Africa. A large body of knowledge has been acquired regarding the molecular biology and replication properties of the causative agent of AIDS, the human immunodeficiency virus (HIV). However, despite these advances the global spread of HIV and especially its spread in developing countries continues almost unabated. During the last decade, drugs inhibiting HIV replication have been introduced into clinical use and highly active antiretroviral therapy (HAART) using a combination of several drugs has been proven to slow or halt the course of the disease in most patients. However, HAART is currently only available in countries of the developed world and the emergence of resistant variants may rapidly lead to a partial loss of this therapeutic arsenal. Thus, the development of a prophylactic vaccine against HIV must be the ultimate goal to control the global pandemic of AIDS. This minireview summarises the state of the epidemic and discusses current treatment strategies as well as future developments. Finally, strategies towards the development of a vaccine against HIV are presented and recent studies using prime-boost regimens to achieve protection from disease in animal models while not preventing infection with the challenge virus are discussed.

Construction and characterization of a fluorescently labeled infectious human immunodeficiency virus type 1 derivative.

The introduction of a label which can be detected in living cells opens new possibilities for the direct analysis of dynamic processes in virus replication, such as the transport and assembly of structural proteins. Our aim was to generate a tool for the analysis of the trafficking of the main structural protein of human immunodeficiency virus type 1 (HIV-1), Gag, as well as for the analysis of virus-host cell interactions in an authentic setting. We describe here the construction and characterization of infectious HIV derivatives carrying a label within the Gag polyprotein. Based on our initial finding that a short epitope tag could be inserted near the C terminus of the matrix domain of Gag without affecting viral replication, we constructed HIV derivatives carrying the egfp gene at the analogous position, resulting in the expression of a Gag-EGFP fusion protein in the authentic viral context. Particles displaying normal viral protein compositions were released from transfected cells, and Gag-EGFP was efficiently processed by the viral protease, yielding the expected products. Furthermore, particles with mature morphology were observed by thin-section electron microscopy. The modified virus was even found to be infectious, albeit with reduced relative infectivity. By preparing mixed particles containing equimolar amounts of Gag-EGFP and Gag, we were able to obtain highly fluorescently labeled virion preparations which displayed normal morphology and full wild-type infectivity, demonstrating that the process of HIV particle assembly displays a remarkable flexibility. The fluorescent virus derivative is a useful tool for investigating the interaction of HIV with live cells.

Lv2, a novel postentry restriction, is mediated by both capsid and envelope.

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.

Biological analysis of human immunodeficiency virus type 1 R5 envelopes amplified from brain and lymph node tissues of AIDS patients with neuropathology reveals two distinct tropism phenotypes and identifies envelopes in the brain that confer an enhanced tropism and fusigenicity for macrophages.

Complete envelope genes were amplified from autopsy brain tissue of five individuals who had died of AIDS and had neurological complications. Lymph node samples were included for two of the patients. Nineteen different envelope clones from the five patients had distinct V1V2 sequences. Thirteen of the envelopes were functional and conferred fusigenicity and infectivity for CD4(+) CCR5(+) cells. Infectivity and cell-cell fusion assays showed that most envelopes used both CCR5 and CCR3. One brain-derived envelope used a broad range of coreceptors, while three other brain envelopes from one individual were restricted to CCR5. However, there was no correlation between tissue of origin and coreceptor use. Envelopes showed two very distinct phenotypes depending on their capacity to infect macrophages and to exploit low levels of CD4 and/or CCR5 for infection. Envelopes that were highly fusigenic and tropic for macrophages were identified in brain tissue from four of the five patients. The enhanced macrophage tropism correlated with reduced sensitivity to inhibition by Q4120, a CD4-specific antibody, but not with sensitivity to the CCR5 inhibitor, TAK779. The highly macrophage-tropic envelopes were able to infect cells expressing low levels of CD4 and/or CCR5. Comparison with several well-characterized macrophage-tropic envelopes showed that the four identified patient envelopes were at the top limit of macrophage tropism. In contrast, all four lymph node-derived envelopes exhibited a non-macrophage-tropic phenotype and required high levels of CD4 for infection. Our data support the presence of envelopes that are highly fusigenic and tropic for macrophages in the brains of patients with neurological complications. These envelopes are able to infect cells that express low levels of CD4 and/or CCR5 and may have adapted for replication in brain macrophages and microglia, which are known to express limited amounts of CD4.