Sherry M. Owen

Adaptation to promiscuous usage of CC and CXC-chemokine coreceptors in vivo correlates with HIV-1 disease progression.

Objective:To study coreceptor usage of sequential primary HIV-1 isolates in a longitudinal follow-up cohort of HIV-1-infected men to understand its contribution to pathogenesis of HIV disease. Design:Viral coreceptor usage of sequential primary isolates from HIV-1-infected individuals was examined at various timepoints and data was compared with CD4 cell counts, rates of disease progression and β-chemokine production. Methods:Fifty-eight sequential primary isolates were obtained from four rapid progressors, six late progressors, and three long-term nonprogressors (LTNP) and their coreceptor usage was examined by infection of peripheral blood mononuclear cells (PBMC) from donors with wild-type or non-functional CC-chemokine receptor (CCR)-5, and by infection of GHOST4 cells expressing CD4 and various chemokine receptors [CCR-1–CCR-5, CXC-chemokine receptor (CXCR)-4, BOB/GPR15, BONZO/STRL33]. Production of RANTES and macrophage inflammatory protein (MIP)-1β was examined using unstimulated or phytohemagglutinin (PHA)-stimulated PBMC isolated from these individuals at multiple timepoints during infection. Results:A switch from single CCR-5 coreceptor usage to multiple coreceptor usage occurred in all four rapid progressors and three out of six late progressors. In addition to the commonly used coreceptors CXCR-4, CCR-5, and CCR-3, some of the viruses isolated from patients in the terminal stage of infection also used CCR-1, CCR-2b, CCR-4, and BOB as coreceptors. The emergence of viral variants capable of utilizing multiple coreceptors generally preceded CD4 cell decline to < 200 × 106/l and correlated with the onset of AIDS. In contrast, three LTNP maintained exclusive usage of CCR-5 over a period of 7–12 years post-infection. Endogenous production of RANTES and MIP-1β by PBMC from LTNP was not significantly different from rapid and late progressors. However, PHA-driven production of both chemokines was significantly higher in LTNP, suggesting that in vivo activating stimuli might curtail HIV replication by inducing these chemokines. Conclusions:Viral variants capable of utilizing a broad range of coreceptors correlated with HIV-1 disease progression. In contrast, LTNP maintain exclusive usage of CCR-5 and produce higher levels of β-chemokines. Thus, both viral and host determinants leading to the emergence of viral variants capable of using an expanded range of coreceptors may be likely determinants of disease progression.

Simian Immunodeficiency Viruses of Diverse Origin Can Use CXCR4 as a Coreceptor for Entry into Human Cells

ABSTRACT Primary simian immunodeficiency virus (SIV) isolated from sooty mangabey (SIVsm [n = 6]), stumptail (SIVstm [n = 1]), mandrill (SIVmnd [n = 1]), and African green (SIVagm [n = 1]) primates were examined for their ability to infect human cells and for their coreceptor requirements. All isolates infected human peripheral blood mononuclear cells (PBMCs) from a CCR5+/+ donor, and seven of eight isolates tested also infected CCR5−/− PBMCs. Analysis of coreceptor utilization using GHOST and U87 cell lines revealed that all of the isolates tested used CCR5 and the orphan receptors STRL33 and GPR15. Coreceptors such as CCR2b, CCR3, CCR8, and CX3CR1 were also utilized by some primary SIV isolates. More importantly, we found that CXCR4 was used as a coreceptor by the SIVstm, the SIVagm, and four of the SIVsm isolates in GHOST and U87 cells. These data suggest that primary SIV isolates from diverse primate species can utilize CXCR4 for viral entry, similar to what has been described for human immunodeficiency viruses.

Temporal relationship between V1V2 variation, macrophage replication, and coreceptor adaptation during HIV-1 disease progression

Background: Specific mutations in VPR and V2 potentially restrict HIV-1 replication in macrophages. Such restriction could potentially limit HIV replication in long-term non-progressors (LTNP), thus accounting for low viral load and delayed progression to AIDS. Objective: To examine whether a specific VPR phenotype (truncated versus non-truncated) correlates with disease progression and whether elongated V2 restricts viral replication in macrophages or alters viral tropism. Methods: Sequence analysis was carried for VPR and V1-V3 env from four rapid progressors (RPs), six late progressors (LPs), and three LTNPs in cohort of HIV-1-infected homosexual men. The replication kinetics of sequential isolates was examined in primary CD4 cells and macrophages and coreceptor usage was determined by GHOST infection assays. Results: No differences were found in the VPR protein from RP and LTNP isolates. Analysis of the V2 region revealed that all RPs maintained similar V2 lengths (40 aa), whereas LPs and LTNPs acquired additional amino acids (2-13 aa) in the V2 region. Coreceptor specificity revealed that RP switch from CCR5 to multiple coreceptor usage, whereas LTNPs maintained R5 viruses. Sequential isolates from each group revealed comparable replication efficiencies in both T-cells and macrophages, regardless of the V2 length or coreceptor utilization. In addition, cross-section analysis of six LTNPs from Australia revealed extended V2 with consistent usage of CCR5 coreceptor. Conclusion: The present results suggest that acquisition of a V2 extension over time in HIV-1-infected LPs/LTNPs appears to correlate with maintenance of CCR5 usage among LTNPs. These findings may be important for a better understanding of the host interactions and disease progression.

Resistance mutation in HIV entry inhibitors

BackgroundTwo of the fusion inhibitors T-20 and 5-helix polypeptide have been shown to be potent inhibitors of cell-to-cell fusion and are currently under investigation as therapy for HIV-1. ObjectivesTo examine variability of HIV-1 gp41 heptads repeat regions (HR1 and HR2), with special emphasis on the presence of T-20 resistance mutations and 5-helix variability at critical epitopes, in treatment-naive patients infected with diverse HIV-1 subtypes from different geographic regions. MethodsA total of 150 specimens representing HIV-1 group M subtypes (A–G) from persons naive to HIV-1 viral entry inhibitor therapy were used to amplify and sequence a 506 bp segment of transmembrane protein. ResultsIn general, both HR1 (a.a. 540–593) and HR2 (a.a. 628–673) domains were highly conserved. Sequence analysis of the T-20 resistant domain (a.a. 547–549, GIV) revealed that 99% of the specimens (149 of 150) carried a T-20 sensitive genotype. The critical epitopes involved in the 5-helix interaction include residues at positions 628W, 631W, 635I, 638Y, 642I, 645L, 649S, 652Q, 656N, and 659E. Analysis of the 150 specimens revealed that all had identical residues at six of these positions, whereas two positions had minor variations (635 and 649) and two (645 and 659) appeared to have subtype-specific substitutions. ConclusionsThis data indicates that there is limited resistance to T-20 in these worldwide populations and that the critical epitopes for effective 5-helix binding are highly conserved across all subtypes. Taken together, these data suggest that T-20 and 5-helix should provide useful additives to current antiretroviral therapy for clinical management of HIV disease.BACKGROUND Two of the fusion inhibitors T-20 and 5-helix polypeptide have been shown to be potent inhibitors of cell-to-cell fusion and are currently under investigation as therapy for HIV-1. OBJECTIVES To examine variability of HIV-1 gp41 heptads repeat regions (HR1 and HR2), with special emphasis on the presence of T-20 resistance mutations and 5-helix variability at critical epitopes, in treatment-naive patients infected with diverse HIV-1 subtypes from different geographic regions. METHODS A total of 150 specimens representing HIV-1 group M subtypes (A-G) from persons naive to HIV-1 viral entry inhibitor therapy were used to amplify and sequence a 506 bp segment of transmembrane protein. RESULTS In general, both HR1 (a.a. 540-593) and HR2 (a.a. 628-673) domains were highly conserved. Sequence analysis of the T-20 resistant domain (a.a. 547-549, GIV) revealed that 99% of the specimens (149 of 150) carried a T-20 sensitive genotype. The critical epitopes involved in the 5-helix interaction include residues at positions 628W, 631W, 635I, 638Y, 642I, 645L, 649S, 652Q, 656N, and 659E. Analysis of the 150 specimens revealed that all had identical residues at six of these positions, whereas two positions had minor variations (635 and 649) and two (645 and 659) appeared to have subtype-specific substitutions. CONCLUSIONS This data indicates that there is limited resistance to T-20 in these worldwide populations and that the critical epitopes for effective 5-helix binding are highly conserved across all subtypes. Taken together, these data suggest that T-20 and 5-helix should provide useful additives to current antiretroviral therapy for clinical management of HIV disease.

HIV-1 strains from a cohort of American subjects reveal the presence of a V2 region extension unique to slow progressors and non-progressors.

ObjectivesTo determine the molecular nature of HIV-1 quasispecies and their evolution, in vivo over time, in an American cohort of 22 homosexual men [four rapid progressors (RP), 15 slow progressors (SP) and three long-term non-progressors (LTNP)], infected with HIV-1 between 1982 and 1983, and to assess the possible role of the HIV-1 V2 region extension in HIV disease progression. DesignGenetic and phylogenetic analyses of the V3 region and the nef gene clones over time from uncultured peripheral blood mononuclear cells (PBMC) of American patients with varying HIV disease progression rates. MethodsProviral DNA from longitudinally collected uncultured PBMC were subjected to PCR amplification in the nef gene and env V2 and V3 regions, followed by cloning, sequencing and phylogenetic analysis to establish evolutionary relationships between HIV-1 strains over time. ResultsAnalysis of multiple viral clones showed nef gene deletions/insertions in 10 out of 15 SP, along with the coexistence of intact and defective nef gene lineages in the same individual over time, whereas these nef gene abnormalities were absent from HIV-1 strains from LTNP. Increasing quasispecies diversity in HIV-1 strains, over time, abrogation of a V3 region N-linked glycosylation site in > 60% of the clones, and, importantly, an extended V2 region were unique features of HIV-1 strains from SP and LTNP. ConclusionsThe V2 region extension was unique to only SP and LTNP, and so may have a role in slow progression or non-progression of HIV disease. Increasing genetic diversity in HIV-1 strains in SP and LTNP correlated with the immunocompetent status of the host.

Mother-to-Child Transmission of Human T-Lymphotropic Virus Type II (HTLV-II)

Human T-lymphotropic virus type II (HTLV-II) is transmitted primarily by sharing contaminated needles and by sexual contact [1, 2]. Unlike for type I, vertical transmission of HTLV-II has not been documented, although breast-feeding has been suggested as a possible risk factor for HTLV-II transmission [3, 4]. Recent serologic analyses of prostitutes and Mayan Indians in Mexico identified women with HTLV-II infection [5]. We studied family members of four of these women who were positive for HTLV-II to identify the mode of transmission of HTLV-II to close family contacts. Although we could not document sexual transmission in the sexual partners of these women who were positive for HTLV-II, we report the first case of mother-to-child transmission of HTLV-II, which occurred in an 8-year-old child who was breast-fed from birth to 4 years. Methods Blood specimens were obtained from women who were positive for HTLV-II (three prostitutes [donors Y06, Y01, Y03] and one woman with a history of cervical cancer [Y08]) and their family members from Yucatan, Mexico, who consented to free testing. Information regarding sexual behavior, drug use, and breast-feeding history was obtained for each participant. The serum samples were tested for antibodies to HTLV by a modified Western blot assay, incorporating purified recombinant transmembrane protein and HTLV type-specific external glycoproteins specific for HTLV-I (rgp46I) or HTLV-II (rgp46II) protein with a whole virus lysate [6]. The amplification and detection of HTLV sequences by the nested polymerase chain reaction were performed on DNA specimens from selected persons who had Western blot profiles suggestive of HTLV infection. Nested amplification was performed in three gene regions, pol, env, and tax. Results Western blot analyses of the serum samples from family members (Table 1) showed isolated r21e reactivity, followed by some rgp46II reactivity. Three of the four sexual partners had r21e reactivity. However, the 8-year-old son (donor Y17) of a prostitute (donor Y06) had antibodies to both gag (p24) and env (rgp46II, r21e) gene products, indicating HTLV-II positivity. Human T-lymphotropic virus type II-specific genomic sequences were also detected from peripheral blood lymphocytes of donor Y17, further confirming HTLV-II infection in this 8-year-old boy (Table 1). Table 1. Demographics and Polymerase Chain Reaction Analysis of the Study Participants* Evaluation of the various risk factors for HTLV-II infection in the 8-year-old child (donor Y17) showed no history of intravenous drug abuse, blood transfusion, or use of nondisposable needles for childhood vaccinations. He is not homeless and attends an elementary school. Repeated questioning related to sexual behavior ruled out the possibility of sexual abuse. This childs only recognizable risk factor for acquisition of HTLV-II infection appears to be breast-feeding during the first 4 years of his life. In contrast, his 3-year-old brother (donor Y20), who was seronegative, was breast-fed for only 2 months. The breast-feeding of this child for up to 4 years suggests rather unusual bonding between mother and child, thus raising the possibility of incestuous behavior. Discussion A recent study showed that the HTLV-II genome can be found in the breast milk of women infected with HTLV-II [4], thus suggesting the possible transmission of this virus through breast-feeding. That the breast-feeding might be the major route of mother-to-child transmission of HTLV-II is further supported by a recent study in which no nonbreast-fed babies born to women infected with HTLV-II had the HTLV-II genome [3]. Although the exact mechanism of transmission is not known, some of the virus-infected cells could have penetrated the mucosal barriers during their passage from the oral cavity to the gastrointestinal tract. Furthermore, oral administration of HTLV-I-infected human breast milk lymphocytes to marmoset monkeys showed transmission of HTLV-I in this animal model [7]. Nested polymerase chain reaction analyses were performed using primers in the pol, env, and tax gene regions to identify the HTLV genome in those persons who had any band on Western blot analyses. Although all of the specimens with antibodies to both gag (p24) and env (rgp46 and r21e) contained the HTLV-II genome (donors Y08, Y06, Y01, and Y03), all of the specimens with isolated r21e reactivity (Y26, Y29, Y34, Y18, Y20, and Y36) and with rgp46II and r21e reactivity (Y31) were negative by nested polymerase chain reaction analysis, suggesting that isolated gag and env reactivities do not represent true HTLV infection. Serologic analyses of the spouse or partners (or both) of these women infected with HTLV-II revealed antibodies to r21e in three of the four partners; however, no HTLV-specific genomic sequences could be amplified, even by nested polymerase chain reaction analysis. Although presence of the r21e band previously represented an early marker of seroconversion [8], lack of the HTLV-I and HTLV-II genomes in all of these specimens supports the absence of true HTLV infection in the sexual partners of women infected with HTLV-II. Antibodies to r21e have also been documented in persons with no evidence of HTLV infection and presumably reflect antigenic mimicry to a closely related antigen [9]. Previous studies analyzing sexual transmission showed that HTLV-I transmission between spouses occurred more frequently from husband to wife and rarely from wife to husband [10]. A similar study of HTLV-II infection among spouses also showed predominantly male-to-female transmission [2]. However, large cohort studies are needed to confirm these findings.

Detection of Simian Immunodeficiency Virus in Diverse Species and of Human Immunodeficiency Virus Type 2 by Using Consensus Primers within the pol Region

ABSTRACT Human immunodeficiency virus type 2 (HIV-2) is the result of cross-species transmission of simian immunodeficiency virus (SIV) from sooty mangabey monkeys to humans. Primer pairs (intHIV-2/SIV) based on a region of integrase that has considerable homology across HIV-2 and SIV lineages were designed to develop a broadly cross-reactive molecular assay to detect lentivirus infection in primates. The intHIV-2/SIV primers detect HIV-2 and simian viruses SIVcpz, SIVsmm, SIVsyk, SIVagm, and SIVmnd. The primers are also capable of amplifying some HIV-1 strains. Additionally, sequences from the integrase amplicons were of sufficient genetic diversity to permit not only phylogenetic clustering of all simian viruses to their respective lineages but also HIV type and group classification. Thus, the primers described here provide a method to detect primate lentiviruses from diverse species of nonhuman primates, as well as from persons infected with HIV-1 and HIV-2.