Isolation and characterization of an infectious molecular clone of the MN strain of HIV-1

Infectious molecular clones of the human immunodeficiency virus (HIV) have been very important tools for the analysis of regulatory gene functions and the study of differential cell tropism. We have cloned and characterized a proviral sequence of HIVmn from mn strain infected H9 cells. This clone, called KP1, was found to be infectious for different cell lines and human peripheral blood lymphocytes (PBL). KP1 proviral DNA was detected in HUT-78 cells and human PBL by polymerase chain reaction (PCR) analysis after infection of these cells with cell-free supernatants from KP1 transfected human rhabdomyosarcoma (RD) cells. To the best of our knowledge, this is the first report of an infectious molecular clone of HIVmn which is a representative of one of the most prevalent strains of HIV-1 in North America and Europe. Biologically active clones of a broadly antigenic strain such as HIVmn will be extremely useful in therapeutic approaches for AIDS.     

Insertion of primary syncytium-inducing (SI) and non-SI envelope V3 loops in human immunodeficiency virus type 1 (HIV-1) LAI reduces neutralization sensitivity to autologous, but not heterologous, HIV-1 antibodies.

The aim of the study was to investigate the influence of V3 loops from naturally occurring viruses on the neutralization sensitivity of a molecularly cloned virus. A selection of well-defined syncytium-inducing (SI) and non-SI V3 loops of a single human immunodeficiency virus type 1-infected individual (H594) and the V3 regions of two SI laboratory strains were inserted in an infectious molecular clone of human immunodeficiency type 1 LAI. Neutralization was performed with a heterologous serum pool and autologous patient serum, using the virus reduction neutralization assay and peripheral blood lymphocytes as target cells. High sensitivity of the chimeric viruses containing the laboratory strain V3 regions to neutralization by H594 sequential sera as well as the heterologous serum pool was found. A statistically significant correlation between the sensitivities of these viruses was seen. In contrast, insertion of the primary isolate NSI and SI envelope V3 loops significantly reduced the neutralization by autologous serum but not by the heterologous serum pool. No correlation was found between the neutralization of the viruses with laboratory strain-derived V3 regions and the viruses with primary isolate V3 domains. We conclude that heterologous antibodies are able to neutralize infectious molecular clones with V3 loops of both SI and NSI viruses, regardless of whether they originated from laboratory strains or primary isolates. However, serum of patient H594 discriminated between the two types of viruses and showed reduced neutralization of the viruses with the autologous NSI and SI primary isolate V3 loops. These results indicated that the neutralization sensitivity of the viruses depended on the capacity of the V3 region to influence the conformation of the virus envelope. These V3-dependent conformational changes partially explain the neutralization sensitivity of laboratory strains and the relative neutralization resistance of primary isolates.     

Molecular architectures of trimeric SIV and HIV-1 envelope glycoproteins on intact viruses: strain-dependent variation in quaternary structure

The initial step in target cell infection by human, and the closely related simian immunodeficiency viruses (HIV and SIV, respectively) occurs with the binding of trimeric envelope glycoproteins (Env), composed of heterodimers of the viral transmembrane glycoprotein (gp41) and surface glycoprotein (gp120) to target T-cells. Knowledge of the molecular structure of trimeric Env on intact viruses is important both for understanding the molecular mechanisms underlying virus-cell interactions and for the design of effective immunogen-based vaccines to combat HIV/AIDS. Previous analyses of intact HIV-1 BaL virions have already resulted in structures of trimeric Env in unliganded and CD4-liganded states at ∼20 Å resolution. Here, we show that the molecular architectures of trimeric Env from SIVmneE11S, SIVmac239 and HIV-1 R3A strains are closely comparable to that previously determined for HIV-1 BaL, with the V1 and V2 variable loops located at the apex of the spike, close to the contact zone between virus and cell. The location of the V1/V2 loops in trimeric Env was definitively confirmed by structural analysis of HIV-1 R3A virions engineered to express Env with deletion of these loops. Strikingly, in SIV CP-MAC, a CD4-independent strain, trimeric Env is in a constitutively “open” conformation with gp120 trimers splayed out in a conformation similar to that seen for HIV-1 BaL Env when it is complexed with sCD4 and the CD4i antibody 17b. Our findings suggest a structural explanation for the molecular mechanism of CD4-independent viral entry and further establish that cryo-electron tomography can be used to discover distinct, functionally relevant quaternary structures of Env displayed on intact viruses.     

Biological phenotype of human immunodeficiency virus type 1 clones at different stages of infection: progression of disease is associated with a shift from monocytotropic to T-cell-tropic virus population.

The composition of human immunodeficiency virus type 1 (HIV-1) clonal populations at different stages of infection and in different compartments was analyzed. Biological HIV-1 clones were obtained by primary isolation from patient peripheral blood mononuclear cells under limiting dilution conditions, with either blood donor peripheral blood lymphocytes or monocyte-derived macrophages (MDM) as target cells, and the biological phenotype of the clones was analyzed. In asymptomatic individuals, low frequencies of HIV-1 clones were observed. These clones were non-syncytium inducing and preferentially monocytotropic. In individuals progressing to disease, a 100-fold increase in frequencies of productively HIV-1-infected cells was observed as a result of a selective expansion of nonmonocytotropic clones. In a person progressing to AIDS within 19 months after infection, only syncytium-inducing clones were detected, shifting from MDM-tropic to non-MDM-tropic over time. From his virus donor, a patient with wasting syndrome, only syncytium-inducing clones, mostly non-MDM-tropic, were recovered. Parallel clonal analysis of HIV-1 populations in cells present in bronchoalveolar lavage fluid and peripheral blood from an AIDS patient revealed a qualitatively and quantitatively more monocytotropic virus population in the lung compartment than in peripheral blood at the same time point. These findings indicate that monocytotropic HIV-1 clones, probably generated in the tissues, are responsible for the persistence of HIV-1 infection and that progression of HIV-1 infection is associated with a selective increase of T-cell-tropic, nonmonocytotropic HIV-1 variants in peripheral blood.     

Inhibition of early and late events of the HIV-1 replication cycle by cytoplasmic Fab intrabodies against the matrix protein, p17.

BACKGROUND: The HIV-1 matrix (MA) protein, p17, contains two subcellular localization signals that facilitate both nuclear import of the viral preintegration complex early during infection and virus particle assembly late in infection. The dual role of MA in both the afferent and efferent arms of the HIV-1 life cycle makes it an important target for intracellular immunization-based gene therapy strategies. MATERIALS AND METHODS: Here we report, using a new bicistronic vector, that an intracellular Fab antibody, or Fab intrabody, directed against a carboxy-terminal epitope of MA from the Clade B HIV-1 genotype, can inhibit HIV-1 infection when expressed in the cytoplasm of actively dividing CD4+ T cells. RESULTS: Marked inhibition of proviral gene expression occurred when single-round HIV-1 CAT virus was used for infections. In challenge experiments using both laboratory strains and syncytium-inducing primary isolates of HIV-1, a substantial reduction in the infectivity of virions released from the cells was also observed. CONCLUSIONS: This novel strategy of simultaneously blocking early and late events of the HIV-1 life cycle may prove useful in clinical gene therapy approaches for the treatment of HIV-1 infection and AIDS, particularly when combined with genetic or pharmacologic-based strategies that inhibit other HIV-1 target molecules simultaneously.     

Characterization of the functional properties of env genes from long-term survivors of human immunodeficiency virus type 1 infection.

A small number of persons infected with human immunodeficiency virus type 1 (HIV-1) remain clinically and immunologically healthy for more than a decade after infection. Recent reports suggest that these individuals may be infected with an attenuated strain of HIV-1; however, a common genetic basis for viral attenuation has not been found in all cases. In the present study, we examined the functional properties of the HIV-1 env genes from six long-term survivors. env clones were generated by PCR amplification of proviral env sequences, followed by cloning of the amplified regions into expression vectors. Eight to ten clones from each subject were screened by transient transfection for expression of the envelope precursor glycoprotein, gp160. Those clones expressing gp160 were then cotransfected with an HIV-1 luciferase reporter vector, pNL4-3Env(-)LUC(+) and evaluated for their ability to mediate infection of phytohemagglutinin-activated peripheral blood mononuclear cells in single-cycle infectivity assays. Clones expressing gp160 were identified for all six long-term survivors, indicating the presence of proviral env genes with intact open reading frames. For two subjects, D and DH, the encoded envelope glycoproteins yielded high levels of luciferase activity when pseudotyped onto HIV-1 virions and tested in single-cycle infectivity assays. In contrast, envelope glycoproteins cloned from four other long-term survivors were poorly processed and failed to mediate infection. Sequencing of the gp120/41 cleavage site and conserved gp41 cysteine residues of these clones did not reveal any obvious mutations to explain the functional defects. The functional activity of env clones from long-term survivors D and DH was comparable to that seen with several primary HIV-1 env genes cloned from individuals with disease progression and AIDS. These results suggest that the long-term survival of subjects D and DH is not associated with overt functional defects in env; however, functional abnormalities in env may contribute to maintaining a long-term asymptomatic state in the other four cases we studied.     

Pathogenesis of primary R5 human immunodeficiency virus type 1 clones in SCID-hu mice

We studied the replication and cytopathicity in SCID-hu mice of R5 human immunodeficiency virus type 1 (HIV-1) biological clones from early and late stages of infection of three patients who never developed MT-2 cell syncytium-inducing (SI; R5X4 or X4) viruses. Several of the late-stage non-MT-2 cell syncytium-inducing (NSI; R5) viruses from these patients depleted human CD4(+) thymocytes from SCID-hu mice. Earlier clones from the same patients did not deplete CD4(+) thymocytes from SCID-hu mice as well as later clones. We studied three R5 HIV-1 clones from patient ACH142 in greater detail. Two of these clones were obtained prior to the onset of AIDS; the third was obtained following the AIDS diagnosis. In GHOST cell infection assays, all three ACH142 R5 HIV-1 clones could infect GHOST cells expressing CCR5 but not GHOST cells expressing any of nine other HIV coreceptors tested. Furthermore, these patient clones efficiently infected stimulated peripheral blood mononuclear cells from a normal donor but not those from a homozygous CCR5Delta32 individual. Statistical analyses of data obtained from infection of SCID-hu mice with patient ACH142 R5 clones revealed that only the AIDS-associated clone significantly depleted CD4(+) thymocytes from SCID-hu mice. This clone also replicated to higher levels in SCID-hu mice than the two earlier clones, and a significant correlation between viral replication and CD4(+) thymocyte depletion was observed. Our results indicate that an intrinsic property of AIDS-associated R5 patient clones causes their increased replication and cytopathic effects in SCID-hu mice and likely contributes to the development of AIDS in patients who harbor only R5 quasispecies of HIV-1.     

Gp120 V3-dependent impairment of R5 HIV-1 infectivity due to virion-incorporated CCR5

Entry of R5 human immunodeficiency virus type 1 (HIV-1) into target cells requires sequential interactions of the envelope glycoprotein gp120 with the receptor CD4 and the coreceptor CCR5. We investigated replication of 45 R5 viral clones derived from the HIV-1JR-FLan library carrying 0-10 random amino acid substitutions in the gp120 V3 loop. It was found that 6.7% (3/45) of the viruses revealed >or=10-fold replication suppression in PM1/CCR5 cells expressing high levels of CCR5 compared with PM1 cells expressing low levels of CCR5. In HIV-1V3L#08, suppression of replication was not associated with entry events and viral production but with a marked decrease in infectivity of nascent progeny virus. HIV-1V3L#08, generated from infected PM1/CCR5 cells, was 98% immunoprecipitated by anti-CCR5 monoclonal antibody T21/8, whereas the other infectious viruses were only partially precipitated, suggesting that incorporation of larger amounts of CCR5 into the virions caused impairment of viral infectivity in HIV-1V3L#08. The results demonstrate the implications of an alternative influence of CCR5 on HIV-1 replication.     

Human immunodeficiency virus type 1 clones chimeric for the envelope V3 domain differ in syncytium formation and replication capacity.

Chimeric human immunodeficiency virus type 1 (HIV-1) molecular clones differing only in the envelope V3 region were constructed. The V3 regions were derived from two HIV-1 isolates with a non-syncytium-inducing, non-T-cell-tropic phenotype and from four HIV-1 isolates with a syncytium-inducing, T-cell-tropic phenotype. When assayed in SupT1 cells, the two chimeric viruses with a V3 region derived from the non-syncytium-inducing isolates did not induce syncytia and showed a low level of replication. The four chimeric viruses with a V3 region derived from the syncytium-inducing isolates did induce syncytia and replicated efficiently in SupT1 cells. In A3.01 cells, which do not support syncytium formation, the V3 loop affected replication similarly. Upon prolonged culture in SupT1 cells, the phenotype of a non-syncytium-inducing, low-replicating chimeric HIV-1 converted into a syncytium-inducing, high-replicating phenotype. Mutations within the usually conserved GPGR tip of the loop, which were shown to be responsible for the conversion into the syncytium-inducing, high-replicating phenotype, had occurred. In vitro mutagenesis showed that coupled changes of amino acids at both sides of the tip of the V3 loop were able to convert the viral phenotype from non-syncytium-inducing, low replicating into syncytium inducing, high replicating. Our data show that the V3 loop is involved in both syncytium forming and replicative capacity of HIV-1.     

Association of human immunodeficiency virus type 1 envelope glycoprotein with particles depends on interactions between the third variable and conserved regions of gp120.

Many regions within the envelope of human immunodeficiency virus type 1 (HIV-1) that affect its structure and function have been identified. We have previously reported that the interaction of the second conserved (C2) and third variable (V3) regions of gp120 influences the ability of HIV-1 to establish a productive infection in susceptible cells. To better understand the basis for this interaction, we have conducted structure-function analyses of envelope expressed from molecular proviral clones of HIV-1 containing defined mutations in C2 and V3 that individually and in combination differentially affect envelope function. The substitution of a glutamine for an asparagine residue (Q-267) at a potential asparagine-linked glycosylation site in C2, which severely impairs virus infectivity, reduces intracellular processing of gp160 into gp120, the association of gp120 with virions, and the ability of gp120 to bind to the HIV-1 cell surface receptor protein, CD4. The change of an arginine to an isoleucine codon in V3 (I-308), in the presence of the Q-267 mutation, restores virus infectivity to near wild-type levels by increasing the amount of gp120 associated with virions as compared with the Q-267 mutant but does not compensate for the Q-267-induced processing defect. The I-308 change in the context of the wild-type HIV-1 has no affect on processing, association, or CD4 binding. These results indicate that the impaired infectivity of the Q-267 mutant virus is due to a marked reduction in the amount of virion gp120 and suggest that the interaction of C2 and V3 stabilizes the association of gp120 with gp41.     

Autologous antibody response against the principal neutralizing domain of human immunodeficiency virus type 1 isolated from infected humans.

High titers of neutralizing antibodies in human immunodeficiency virus type 1 (HIV-1) infection are directed primarily against the third hypervariable domain (V3) of the virion envelope glycoprotein gp120. This region has been designated the principal neutralizing domain of HIV-1. Because the frequency and significance of autologous V3 antibodies in natural infection are not fully clarified, we have cloned, sequenced, and expressed the V3 domain from virus of HIV-1-infected patients to test the autologous and heterologous V3 antibody response. The resulting recombinant Escherichia coli V3 fusion proteins reacted strongly with both autologous and heterologous patient antibodies in Western blots. Thirty-one different V3 fragments were cloned from 24 hemophiliac patients with different immunological and clinical statuses. Antibody reactivity against the autologous V3 fusion proteins was detected in all serum samples except one; moreover, all serum samples contained antibody reactivity against a vast majority of heterologous fusion proteins despite significant amino acid variability in V3. The results suggest that V3 antibodies are highly prevalent; further, we find no association between the stage of the HIV-1 infection and the presence of V3 antibodies.     

An infectious chimeric human immunodeficiency virus type 2 (HIV-2) expressing the HIV-1 principal neutralizing determinant.

The human immunodeficiency virus type 1 strain MN (HIV-1MN) principal neutralizing determinant (PND, V3 loop) was introduced into infectious molecular clones HIV-2KR and simian immunodeficiency virus mm239 (SIVmm239) by hybridization PCR, replacing the corresponding HIV-2 or SIV envelope cysteine loops with the HIV-1 coding sequence. The HIV-2 chimera (HIV-2KR-MNV3) was found to be capable of infecting a number of T-cell lymphoblastic cell lines as well as primary peripheral blood mononuclear cells. In contrast, the SIV chimera (SIV239MNV3) was not replication competent. Envelope produced by HIV-2KR-MNV3 but not the parental HIV-2KR was recognized by V3-specific and HIV-1-specific polyclonal antisera in radioimmunoprecipitation assays. HIV-2-specific antisera recognized both the chimeric and parental virus but not HIV-1MN. The chimeric HIV-2KR-MNV3 virus proved to be exquisitely susceptible to neutralization by HIV-1-specific and V3-specific antisera, suggesting the potential for use in animal models designed to test HIV-1 vaccine candidates which target the PND.     

Characterization of primary isolate-like variants of simian-human immunodeficiency virus

Several different strains of simian-human immunodeficiency virus (SHIV) that contain the envelope glycoproteins of either T-cell-line-adapted (TCLA) strains or primary isolates of human immunodeficiency virus type 1 (HIV-1) are now available. One of the advantages of these chimeric viruses is their application to studies of HIV-1-specific neutralizing antibodies in preclinical AIDS vaccine studies in nonhuman primates. In this regard, an important consideration is the spectrum of antigenic properties exhibited by the different envelope glycoproteins used for SHIV construction. The antigenic properties of six SHIV variants were characterized here in neutralization assays with recombinant soluble CD4 (rsCD4), monoclonal antibodies, and serum samples from SHIV-infected macaques and HIV-1-infected individuals. Neutralization of SHIV variants HXBc2, KU2, 89.6, and 89.6P by autologous and heterologous sera from SHIV-infected macaques was restricted to an extent that these viruses may be considered heterologous to one another in their major neutralization determinants. Little or no variation was seen in the neutralization determinants on SHIV variants 89.6P, 89.6PD, and SHIV-KB9. Neutralization of SHIV HXBc2 by sera from HXBc2-infected macaques could be blocked with autologous V3-loop peptide; this was less true in the case of SHIV 89.6 and sera from SHIV 89.6-infected macaques. The poorly immunogenic but highly conserved epitope for monoclonal antibody IgG1b12 was a target for neutralization on SHIV variants HXBc2, KU2, and 89.6 but not on 89.6P and KB9. The 2G12 epitope was a target for neutralization on all five SHIV variants. SHIV variants KU2, 89.6, 89.6P, 89.6PD, and KB9 exhibited antigenic properties characteristic of primary isolates by being relatively insensitive to neutralization in peripheral blood mononuclear cells with serum samples from HIV-1-infected individuals and 12-fold to 38-fold less sensitive to inhibition with recombinant soluble CD4 than TCLA strains of HIV-1. The utility of nonhuman primate models in AIDS vaccine development is strengthened by the availability of SHIV variants that are heterologous in their neutralization determinants and exhibit antigenic properties shared with primary isolates.     

Dynamic antibody specificities and virion concentrations in circulating immune complexes in acute to chronic HIV-1 infection

Understanding the interactions between human immunodeficiency virus type 1 (HIV-1) virions and antibodies (Ab) produced during acute HIV-1 infection (AHI) is critical for defining antibody antiviral capabilities. Antibodies that bind virions may prevent transmission by neutralization of virus or mechanically prevent HIV-1 migration through mucosal layers. In this study, we quantified circulating HIV-1 virion-immune complexes (ICs), present in approximately 90% of AHI subjects, and compared the levels and antibody specificity to those in chronic infection. Circulating HIV-1 virions coated with IgG (immune complexes) were in significantly lower levels relative to the viral load in acute infection than in chronic HIV-1 infection. The specificities of the antibodies in the immune complexes differed between acute and chronic infection (anti-gp41 Ab in acute infection and anti-gp120 in chronic infection), potentially suggesting different roles in immunopathogenesis for complexes arising at different stages of infection. We also determined the ability of circulating IgG from AHI to bind infectious versus noninfectious virions. Similar to a nonneutralizing anti-gp41 monoclonal antibody (MAb), purified plasma IgG from acute HIV-1 subjects bound both infectious and noninfectious virions. This was in contrast to the neutralizing antibody 2G12 MAb that bound predominantly infectious virions. Moreover, the initial antibody response captured acute HIV-1 virions without selection for different HIV-1 envelope sequences. In total, this study demonstrates that the composition of immune complexes are dynamic over the course of HIV-1 infection and are comprised initially of antibodies that nonselectively opsonize both infectious and noninfectious virions, likely contributing to the lack of efficacy of the antibody response during acute infection.     

Phenotype-associated sequence variation in the third variable domain of the human immunodeficiency virus type 1 gp120 molecule.

The third variable (V3) domain has been implicated in determining the human immunodeficiency virus (HIV) phenotype, including fusion capacity and monocytotropism. In a large set of primary HIV type 1 (HIV-1) isolates, V3 sequence analysis revealed that fast-replicating, syncytium-inducing isolates contained V3 sequences with a significantly higher positive charge than those of slow-replicating, non-syncytium-inducing monocytotropic isolates. It appeared that these differences in charge could be attributed to highly variable amino acid residues located on either side of the V3 loop, midway between the cysteine residues and the central GPG motif. In non-syncytium-inducing monocytotropic isolates, these residues were negatively charged or uncharged, whereas in syncytium-inducing nonmonocytotropic isolates, either one or both were positively charged. The substitutions at these positions result in changes in the predicted secondary structure of the V3 loop. Our data suggest that two amino acid residues in the highly variable V3 domain are responsible for phenotype differences and point to conformational differences in V3 loops from phenotypically distinct HIV-1 isolates.     

Infectious Virion Capture by HIV-1 gp120-Specific IgG from RV144 Vaccinees

The detailed examination of the antibody repertoire from RV144 provides a unique template for understanding potentially protective antibody functions. Some potential immune correlates of protection were untested in the correlates analyses due to inherent assay limitations, as well as the need to keep the correlates analysis focused on a limited number of endpoints to achieve statistical power. In an RV144 pilot study, we determined that RV144 vaccination elicited antibodies that could bind infectious virions (including the vaccine strains HIV-1 CM244 and HIV-1 MN and an HIV-1 strain expressing transmitted/founder Env, B.WITO.c). Among vaccinees with the highest IgG binding antibody profile, the majority (78%) captured the infectious vaccine strain virus (CM244), while a smaller proportion of vaccinees (26%) captured HIV-1 transmitted/founder Env virus. We demonstrated that vaccine-elicited HIV-1 gp120 antibodies of multiple specificities (V3, V2, conformational C1, and gp120 conformational) mediated capture of infectious virions. Although capture of infectious HIV-1 correlated with other humoral immune responses, the extent of variation between these humoral responses and virion capture indicates that virion capture antibodies occupy unique immunological space.     

Pseudotyping with human T-cell leukemia virus type I broadens the human immunodeficiency virus host range.

Several epidemiologic and clinical studies suggest that patients coinfected with human immunodeficiency virus (HIV), the primary etiologic agent in AIDS, and other viruses, such as cytomegalovirus or human T-cell leukemia virus (HTLV), have a more severe clinical course than those infected with HIV alone. Cells infected with two viruses can, in some cases, give rise to phenotypically mixed virions with altered or broadened cell tropism and could therefore account for some of these findings. Such pseudotypes could alter the course of disease by infecting more tissues than are normally infected by HIV. We show here that HIV type 1 (HIV-1) efficiently incorporates the HTLV type I (HTLV-I) envelope glycoprotein and that both HIV-1 and HTLV-II accept other widely divergent envelope glycoproteins to form infectious pseudotype viruses whose cellular tropisms and relative abilities to be transmitted by cell-free virions or by cell contact are determined by the heterologous envelope. We also show that the mechanism by which virions incorporate heterologous envelope glycoproteins is independent of the presence of the homologous glycoprotein or heterologous gag proteins. These results may have important implications for the mechanism of HIV pathogenesis.     

Coreceptor tropism in human immunodeficiency virus type 1 subtype D: high prevalence of CXCR4 tropism and heterogeneous composition of viral populations

In human immunodeficiency virus type 1 (HIV-1) subtype B, CXCR4 coreceptor use ranges from approximately 20% in early infection to approximately 50% in advanced disease. Coreceptor use by non-subtype B HIV is less well characterized. We studied coreceptor tropism of subtype A and D HIV-1 collected from 68 pregnant, antiretroviral drug-naive Ugandan women (HIVNET 012 trial). None of 33 subtype A or 10 A/D-recombinant viruses used the CXCR4 coreceptor. In contrast, nine (36%) of 25 subtype D viruses used both CXCR4 and CCR5 coreceptors. Clonal analyses of the nine subtype D samples with dual or mixed tropism revealed heterogeneous viral populations comprised of X4-, R5-, and dual-tropic HIV-1 variants. In five of the six samples with dual-tropic strains, V3 loop sequences of dual-tropic clones were identical to those of cocirculating R5-tropic clones, indicating the presence of CXCR4 tropism determinants outside of the V3 loop. These dual-tropic variants with R5-tropic-like V3 loops, which we designated "dual-R," use CCR5 much more efficiently than CXCR4, in contrast to dual-tropic clones with X4-tropic-like V3 loops ("dual-X"). These observations have implications for pathogenesis and treatment of subtype D-infected individuals, for the association between V3 sequence and coreceptor tropism phenotype, and for understanding potential mechanisms of evolution from exclusive CCR5 use to efficient CXCR4 use by subtype D HIV-1.     

Genetically Related Human Immunodeficiency Virus Type 1 in Three Adults of a Family with No Identified Risk Factor for Intrafamilial Transmission

A small number of cases of human immunodeficiency virus (HIV) infection have been reported in individuals with no identified risk factors for transmission. We report on the seroconversion of the 61-year-old mother and the subsequent finding of HIV seropositivity in the 66-year-old father of a 31-year-old AIDS patient. Extensive investigation failed to identify any risk factor for intrafamilial transmission. We conducted a genetic analysis and determined the amino acid signature patterns of the V3, V4, and V5 hypervariable domains and flanking regions in the HIV-1 gp120 env gene of 26 clones derived from proviral DNA in peripheral blood mononuclear cells of the members of the family. env sequences of the viruses isolated from the patients were compared with sequences of HIV-1 subtype B viruses from Europe and local field isolates. Phylogenetic analysis revealed that the sequences of the viruses isolated from the patients were genetically related and formed an intrafamilial cluster of HIV-1 distinct from other subtype B viruses. Interindividual nucleotide variability in the C2-V3 and V4-C4-V5 domains ranged between 1.2 and 5.0% and between 2.2 and 7.5%, respectively, whereas divergence between HIV strains from the patients and control viral strains ranged from 6.6 to 29.3%. The amino acid signature patterns of viral clones from the three patients were closely related. In the C2-V3 region, two minor clones derived from the son’s virus showed less nucleotide divergence (mean, 3.5 and 3.9%) than did the clones derived from the viruses of both parents or the seven other predominant clones derived from the virus from the son (mean, 5.4%). The top of the V3 loop of the last two clones and of all viral clones from the parents exhibited an unusual GPGG sequence. This is the first report of genotypic relatedness of HIV-1 in three adults of the same family in the absence of identified risk factor for transmission between the members of the family. Our findings suggest that atypical transmission of HIV may occur.