Mucosal transmissibility, disease induction and coreceptor switching of R5 SHIVSF162P3Nmolecular clones in rhesus macaques
© Ren et al.; licensee BioMed Central Ltd. 2013
Received: 26 September 2012
Accepted: 25 January 2013
Published: 31 January 2013
Mucosally transmissible and pathogenic CCR5 (R5)-tropic simian-human immunodeficiency virus (SHIV) molecular clones are useful reagents to identity neutralization escape in HIV-1 vaccine experiments and to study the envelope evolutionary process and mechanistic basis for coreceptor switch during the course of natural infection.
We observed progression to AIDS in rhesus macaques infected intrarectally with molecular clones of the pathogenic R5 SHIVSF162P3N isolate. Expansion to CXCR4 usage was documented in one diseased macaque that mounted a neutralizing antibody response and in another that failed to do so, with the latter displaying a rapid progressor phenotype. V3 loop envelop glycoprotein gp120 sequence changes that are predictive of a CXCR4 (X4)-using phenotype in HIV-1 subtype B primary isolates, specifically basic amino acid substations at positions 11 (S11R), 24 (G24R) and 25 (D25K) of the loop were detected in the two infected macaques. Functional assays showed that envelopes with V3 S11R or D25K mutation were dual-tropic, infecting CD4+ target cells that expressed either the CCR5 or CXCR4 coreceptor. And, consistent with findings of coreceptor switching in macaques infected with the pathogenic isolate, CXCR4-using variant was first detected in the lymph node of the chronically infected rhesus monkey several weeks prior to its presence in peripheral blood. Moreover, X4 emergence in this macaque coincided with persistent peripheral CD4+ T cell loss and a decline in neutralizing antibody titer that are suggestive of immune deterioration, with macrophages as the major virus-producing cells at the end-stage of disease.
The data showed that molecular clones derived from the R5 SHIVSF162P3N isolate are mucosally transmissible and induced disease in a manner similar to that observed in HIV-1 infected individuals, providing a relevant and useful animal infection model for in-depth analyses of host selection pressures and the env evolutionary changes that influence disease outcome, coreceptor switching and vaccine escape.
KeywordsR5 SHIV molecular clone Coreceptor switch Antiviral antibody response Macrophage infection
The human immunodeficiency virus (HIV) enters target cells through binding of its envelope glycoprotein gp120 to the human CD4 receptor and a coreceptor, either CXCR4 (X4 HIV) or CCR5 (R5 HIV) . Over 80% of HIV-1 transmissions are initiated with R5 viruses [2–6], with X4 or dual-tropic viruses that use both CCR5 and CXCR4 (R5X4) emerging and coexisting with R5 viruses in 40-50% of non-treated subtype B and D infected individuals late in infection, but less often in subtype A and C infected patients [2, 7–11]. Emergence of CXCR4-using viruses is frequently accompanied by rapid peripheral CD4+ T cell loss and progression to end-stage disease , but the mechanism(s) underlying their expansion is not well understood. It has been suggested that X4 viruses evolve from transmitted R5 viruses to broaden target cell populations , transitioning via an intermediate stage with respect to the envelope sequence and phenotypic characteristics [14, 15]. However, early X4 presence has been documented in HIV-1 infection both phenotypically and genotypically [11, 16–23], the latest from analysis of transmission clusters , with suppression of their replication following the development of HIV-specific immunity [16–18]. The finding that recently emerged CXCR4-using variants in some HIV-1 infected individuals are more neutralization sensitive than coexisting R5 viruses further supports a role of immune-mediated selection pressure against X4 virus . Transmission and selection against CXCR4-using SIVsm in vivo , as well as emergence in the chronic phase of infection of variants that were transmitted but were not maintained at detectable levels early in infection have also been reported in SIV  and SHIV  infected monkeys. Thus, the possibility exists that R5-to-X4 conversion late in infection is the result of re-emergence of co-transmitted X4 or R5X4 variants when the immune system collapses.
Experimental infection of Asian macaques with simian (SIV) or simian-human immunodeficiency (SHIV) viruses are recognized as playing a critical role in advancing our understanding of HIV-1 transmission, pathogenesis, as well as basic vaccine, prevention and treatment concepts [29–31]. SHIVs that express the HIV-1 envelope glycoproteins (Envs) are particularly suited as challenge viruses to evaluate the role of viral tropism in AIDS pathogenesis and neutralizing antibody protection in the macaque model. The early pathogenic SHIVs primarily expressed CXCR4-using Envs and induced a disease course that differed from those observed in HIV-1 infected individuals and SIV-infected rhesus [32–35]. Subsequently, several clade B and C R5 SHIVs have been constructed that showed varying degree of mucosal transmissibility, replication competence and pathogenicity [34, 36–40], but R5 SHIV molecular clones that induce a HIV-1 like pathology including coreceptor switch coincident with peripheral CD4 decline and predominance of macrophage tropism at end-stage disease have not been described. We developed a model of experimental infection of Asian macaques with the late-stage SHIVSF162P3N isolate that exhibited many similarities to HIV-1 infection in humans including CCR5 coreceptor usage, mucosal transmissibility, acute depletion of mucosal memory CD4+ T cells, persistent infection, and progression to AIDS over a period of several months to years in a proportion of the infected animals [41, 42]. Moreover, expansion or switch to CXCR4 was observed in ~50% of R5 SHIVSF162P3N-infected AIDS macaques, with viruses that can function with both coreceptors serving as intermediates [43, 44]. Similar to HIV-1, the main determinants for coreceptor usage of SHIVSF162P3N are located in the third (V3) variable loop of Env [43, 44]. However, whereas coreceptor switch in most HIV-1 infected individuals occurred following the development of antiviral antibodies, the macaques in which coreceptor switch was observed were primarily rapid progressors (RPs) that failed to mount or sustain an antiviral antibody response. Thus, there is concern that the selective pressures for phenotypic conversion in the RP macaques might not fully reflect the human situation. Furthermore, because the populations in the SHIVSF162P3N virus stock are comprised of related but not identical variants, the presence of low-level X4 viruses in the inoculum that were initially co-transmitted and remaining cryptic until immune selective pressure is sufficiently diminished cannot be excluded. For these reasons, we generated molecular clones of R5 SHIVSF162P3N for intrarectal inoculation, with the objective of documenting disease development and a shift in coreceptor preference during the course of natural infection. Mucosally transmissible, highly replication competent and pathogenic R5 SHIV molecular clones would also be very useful as challenge viruses in vaccine efficacy and escape studies.
Envelope sequence and function of SHIVSF162P3Nmolecular clones
R5 SHIVSF162P3Nmolecular clones are infectious by the intrarectal route and induce disease
Severe lymph node CD4 T cell loss in macaques infected with R5 SHIVSF162P3Nmolecular clones that developed disease
Coreceptor switch in macaques infected with R5 SHIVSF162P3Nmolecular clones
Peripheral lymph nodes were found to be the preferred sites of X4 emergence and expansion in macaques infected with R5 SHIVs [42, 51, 52]. Because longitudinal samples (w8, 46, 54, 84) from the inguinal lymph node (Ing LN) of FF94 were available, we analyzed this tissue compartment as well as the PBMC and serum samples collected at the corresponding time points for X4 emergence in this animal. We found that the dual-tropic D25K V3 variant was detected by clonal analysis at low frequency (1 of 21 clones sequenced) in the Ing LN of FF94 sampled at 46 wpi. Representation of this variant in the Ing LN increased over time, with all 24 clones sequenced harboring this mutation at 84 wpi (Figure 4C). The D25K variant was not detectable in the blood by clonal sequence at 46 and 50 wpi, emerging in plasma and PBMCs sampled four weeks later. Representation of the V3 variant increased over time in blood cells, becoming the major Env species from 68 wpi until the time necropsy. In contrast, the D25K variant co-existed with R5 viruses in the plasma at all time points analyzed. Collectively, the results confirm and extend our findings with the isolate , demonstrating mutational pathways to CXCR4-usage that overlapped with those seen in HIV-1 infected individuals, with X4 evolution and emergence first in secondary lymph nodes.
Tropism switch following the development of a neutralizing antibody response
Tissue macrophages sustain virus production in FF94
The origin of CXCR4-using variants has been investigated extensively. This is because X4 presence is known to be associated with poorer clinical prognosis, and is a major concern in the clinical use of CCR5 inhibitors [54–58]. Several lines of investigation support the hypothesis that X4 and R5X4 viruses evolved from pre-existing R5 variants which are the founder viruses in most cases of HIV-1 transmission. However, the bulk of the evidence in support of early R5 dominance in humans is made using blood from subjects who manifested clinical signs of acute infection syndrome, several weeks after the initial transmission event. Thus, the possibility that coreceptor switch during HIV-1 infection is the result of re-emergence of co-transmitted X4 or R5X4 viruses that reside in tissue sites that are not sampled or exist at levels below the threshold of detection cannot be excluded. Using a relevant nonhuman primate model of HIV-1 pathogenesis, we demonstrate in this study that R5 SHIVSF162P3N molecular clones are efficiently transmitted to macaques via intrarectal inoculation, with disease induction and switch in coreceptor tropism following the development of a neutralizing antibody response. Furthermore, we obtain evidence suggestive of a role of antibody selective pressure in counteracting X4 evolution and expansion.
Molecular clones of subtype B  and subtype C [60, 61] R5 SHIVs that are mucosally transmissible, highly replication competent and capable of inducing AIDS in rhesus macaques have been described, but expansion or conversion to CXCR4 usage has not been observed. In this regard, we show that both R5 SHIVSF162P3N molecular clones exhibited coreceptor switching that followed the 11/25 rule derived from subtype B HIV-1 . In the RP macaque EN31, serine at position 11 of the V3 loop was substituted with arginine, while in FF94, the chronic progressor that mounted an autologous neutralizing antibody response, the aspartic residue at position 25 was replaced with either lysine or arginine residues (Figure 4A). In 17/24 clones amplified from LN of FF94 at end-stage, this substitution at position 25 was accompanied by introduction of a charged residue at position 24 of the V3 loop that had been reported to improve the predictive value of X4 presence in humans . Moreover, the D25K V3 mutational event in FF94 occurred first in the lymph node where naïve T cells that express high levels of CXCR4 are enriched, in agreement with data published by us and others that peripheral LNs are the prefer sites of X4 evolution and amplification [42, 51, 52]. Notably, emergence of the dual-tropic D25K variant in the LN was in the presence of neutralizing antibody (ID50 titers of ~150 against the inoculating clone 8 virus at 46 wpi; Figure 5), with clonal analysis of the viral quasispecies showing increasing dominance of the D25K V3 variant in LN and blood cells, but not in the plasma (Figure 4C). These findings are consistent with observations in HIV-1 infected individuals of a higher prevalence of X4 viruses in PBMCs compared to serum [62–64], and support the notion that X4 viruses emerge and predominate in body compartments with lower antibody pressure than in the plasma, spreading via cell-to-cell transmission that is less susceptible to antibody neutralization. Recently emerged CXCR4-using variants in some HIV-1 infected patients have been reported to be more neutralization sensitive than coexisting R5 viruses , implying that systemic dissemination is possible only with immune system erosion that decreases the selection pressure. X4 detection in peripheral blood of FF94 at 54 wpi coincides with persistent CD4+ T cell loss to levels <200 cells/μl blood (Figure 3A) and a decline in neutralizing titers against the homologous inoculating virus that are suggestive of immune erosion (Figure 5). It will be of interest to investigate neutralization sensitivity of the emerging and late CXCR4-using variants in FF94 to contemporary sera to link a decline in autologous neutralization antibody response with X4 emergence.
The infectious molecular clones were obtained by transfection of 293T cells but cultured and propagated in activated rhesus PBMC to generate virus stocks. This expansion process in vitro necessarily introduces mutation and diversity, raising the possibility that the V3 mutations identified with tropism switch in the infected animals might have already been present in the inoculum. Deep sequencing of V3 region in the SHIVSF162P3N clone 11 virus stock indicated that this is indeed the case. Fifteen of over 21,000 sequences analyzed (0.07%) harbored the S11R mutation, raising the possibility that the R5X4 variant could have been co-transmitted and eventually outgrew in macaque EN31 which failed to mount a neutralizing antibody response. In contrast, while the S11R variant was also present in the SHIVSF162P3N clone 8 virus stock (0.11%), it was not detected by clonal sequence analysis in the conventional macaque FF94 at terminal disease stage. It is conceivable that the S11R variant was not co-transmitted in this animal. Alternatively, it was co-transmitted but remained cryptic. Further studies are needed to examine these possibilities. But importantly, deep sequencing failed to reveal the presence of D25K mutation associated with tropism switch in FF94 in the clone 8 virus inoculum, consistent with findings that multiple long-term cultures of clonal virus variants on PBMCs results in only very few mutations in the V3-V4 regions . We conclude, therefore, that the V3 mutations that confer CXCR4 usage in FF94 evolved from pre-existing R5 variants. Analysis of recombinant V3 mutant viruses showed that while the S11R mutation conferred efficient CXCR4 usage, the D25K V3 mutant entered CXCR4-expressing cells less efficiently than cells expressing CCR5, in agreement with findings in infected individuals that changes in V3 position 25 alone are not highly predictive of coreceptor switching. Besides the V3 loop, mutations in V1V2 domain of gp120 can also influence coreceptor choice [66–73], consistent with structural studies in which both V3 and the stem of the V1V2 loop were shown to participate in coreceptor binding [74–76]. Furthermore, in vitro studies suggested that the order of occurrence of mutations associated with coreceptor switching is critical for survival of the intermediates, with mutations in V1/V2 preceding those of V3 to permit virus survival . Detailed analysis of env sequence changes over time in FF94, in particular the V1/V2 domain of gp120, and the relationship of these changes to autologous neutralizing antibody response and viral fitness should provide important insights into the requirements and constraints for evolution from CCR5 to CXCR4 use in vivo.
We previously reported sustained viremia and progression to disease over a one-year infection period in ten of eleven rhesus macaques infected intrarectally with high dose of the R5 SHIVSF162P3N isolate, with a RP phenotype and coreceptor switching observed in four and five of the eleven monkeys with AIDS respectively . In this regard, although the results with the molecular clones confirm and extend findings with the isolate, showing similar evolutionary pathways, dynamics and sites of X4 emergence [42–44], infection with the molecular clones is less pathogenic. The decrease in replicative capacity and pathogenicity of R5 SHIVSF162P3N molecular clones may be related to the fact that they are less diverse than the isolate. Indeed, transmission of multiple viral variants has been suggested to influence viral persistence and rates of disease development through recombination to generate intrahost phenotypic and pathogenic diversities to escape early host selective pressures and increase fitness [79–82]. And, although the number of animals used is small, the findings suggest that SHIVSF162P3N clone 8 may be more pathogenic than clone 11. A higher proportion of the clone 8-infected macaques sustained viremia (3 of 5) and progressed to disease (2 of 5) over a two-year observation period as compared to only one of five of the clone 11-infected monkeys. The envelope glycoproteins of the two clones differed in sCD4 sensitivity and as well as infection of macrophages that are long-lived viral reservoirs . Macrophages have been reported to be the principal reservoir and sustain high viral loads in rhesus monkeys after the depletion of CD4+ T cells by highly pathogenic X4 SHIVs , and following coreceptor switch in R5 SHIVSF162P3N-infected RPs . Infected macrophages are relatively resistant to CD8+ T cell-mediated suppression [86, 87] and macrophage internal HIV-1 is protected from neutralization antibodies . Combined in situ hybridization and immunohistochemical analysis of LNs obtained at the time of necropsy indicated higher viral burden in the LNs of FF94 than EN31, with a majority (>80%) of infected cells in the former co-staining with lba-1, a macrophage marker. Thus, it is tempting to speculate that the difference in macrophage infection by the two molecular clones contributed to their differences in viral persistence. Studies in additional animals will be needed to address this. Regardless, the data support the use of both SHIVSF162P3N molecular clones to assess the efficacy of vaccines in preventing HIV-1 acquisition or in reducing peak viral load and virus-induced depletion of gut CD4+ T cells, but clone 8 may be more useful than clone 11 in judging the effects of vaccines in dampening the intensity of virus infection.
In summary, this report documents coreceptor switch in macaques infected with R5 SHIV molecular clones, supporting R5 evolution to X4. Coreceptor switch in macaques infected with R5 SHIVSF162P3N molecular clones required genetic adaptations similar to those seen in humans, and occurred in rapid as well as conventional progressors that mounted a neutralizing antibody response. This animal model, in which the envelope sequence and functional properties of the inoculating virus are known, and where detailed samplings of blood and tissue samples are possible, provides the unique opportunity to uncover in detail the genetic requirement, obstacles and constraints for virus phenotype evolution in vivo. It can also be used to study neutralization escape during the course of infection and determine the role of humoral immunity in X4 virus emergence. Lastly, because the molecular clones express R5 HIV-1 envelope glycoproteins, main targets for neutralizing antibodies, they are better suited than the X4 SHIVs and SIVs that differ antigenically from HIV-1 as challenge viruses for antibody-based vaccine testing and development.
Materials and methods
293T cells, TZM-bl cells expressing CD4, CCR5 and CXCR4, and containing integrated reporter genes for firefly luciferase and β-galactosidase under control of the HIV-1 LTR  were propagated in DMEM supplemented with 10% fetal bovine serum (FBS), penicillin, streptomycin and L-glutamine. U87 cells stably expressing CD4 and one of the chemokine receptors  were maintained in DMEM supplemented with 10% FBS, antibiotics, 1 μg/ml puromycin (Sigma-Aldrich, St. Louis, MO) and 300 μg/ml G418 (Geneticin; Invitrogen, Carlsbad, CA). Rhesus peripheral blood mononuclear cells (PBMCs) were prepared by Ficoll gradient centrifugation, stimulated with staphylococcal enterotoxin B (SEB, 3 μg/ml ; Sigma-Aldrich), and cultured in RPMI medium containing 10% FCS, penicillin, streptomycin, L-glutamine and 20U/ml interleukin-2 (Norvatis, Emeryville, CA). Monocytes were enriched by centrifugation of human PBMCs through a 40% percoll cushion followed by plastic adherence, and cultured in RPMI 1640 medium supplemented with 10% FCS and 5% human AB serum for 5–7 days to allow for differentiation into macrophages .
Construction of R5 SHIVSF162P3Nmolecular clones
Full-length gp160 coding sequences of the R5 SHIVSF162P3N inoculum were obtained and confirmed as previously described , and subcloned into the corresponding region of the 3’SHIVSF162 genome  using the unique Kpn I and Xho I sites. Infectious molecular clones were recovered by cotransfection of 293T cells with a ligation product of the 3’ SHIVSF162 P3N gp160 and the 5’ SIV hemigenomes, followed by cocultivation with SEB-stimulated rhesus PBMCs. Stocks of SHIVSF162P3N molecular clones were propagated and tittered in rhesus PBMCs.
Animal inoculation and clinical assessments
All inoculations were carried out in adult rhesus monkeys of Indian origin (Macaca mulatta) housed at the Tulane National Primate Research Center (TNRPC) in compliance with the Guide for the Care and Use of Laboratory Animals. Animals were confirmed to be serologically and virologically negative for simian type D retrovirus, and serologically negative for SIV and simian T-cell lymphotropic virus prior to infection, and were screened for the presence of the Mamu-A*01, Mamu-B*17 and Mamu-B*08 class I alleles previously shown to be associated with control of pathogenic SIVmac239 replication using standard PCR with allele-specific primers . Macaques received a single intrarectal (ir) inoculation with 5 x 103 50% tissue culture infectious dose (TCID50) of the cell free challenge stocks of SHIVSF162P3N molecular clones. Whole blood from the inoculated animals was collected weekly for the first eight weeks, biweekly for another 16 weeks, and monthly thereafter. Surgery was performed at peak (2–3 weeks post-infection, wpi) viremia for collection of tissues from one external and one internal lymph node, and from internal organs such as the small intestine, bone marrow, thymus and spleen. Animals were euthanized at end of study period by intramuscular administration of telazol and buprenorphine followed by an overdose of sodium pentobarbital, and tissues from multiple sites were collected. Euthanasia was considered to be AIDS related if the animal exhibited peripheral blood CD4+ T-cell depletion (<200/mm3), greater than 25% loss of body weight and combinations of the following conditions: diarrhea unresponsive to treatment, opportunistic infections, peripheral lymph node atrophy, and abnormal hematology (e.g., anemia, thrombocytopenia, or leukopenia). Plasma viremia was quantified by branched DNA analysis (Siemens Medical Solutions Diagnostic Clinical Lab, Emeryville, CA) and absolute CD4+ and CD8+ cell counts were monitored by TruCount (BDBiosciences, Palo Alto, CA). The percentages of CD4+ T cells in the tissue cells were analyzed by flow cytometry (FACScalibur) using CD3-fluorescein isothiocyanate (FITC), CD4-phycoerythrin (PE) and CD8- peridinin chlorophyll protein (PerCP) antibodies. Except for CD3-FITC (BioSource, Camarillo, CA), all antibodies were obtained from BD Biosciences.
Envelope sequence analysis
For sequence analysis of V3 variants in PBMCs and tissues, proviral DNA was extracted from 3 x 106 cells with a DNA extraction kit, and the V1 to V5 region of gp120 was amplified from the vDNA using Taq DNA polymerase (Qiagen, Chatsworth, CA) with primers ED5 and ED12 or ES7 and ES8 as previously described . For sequence analysis of variants in the plasma, viral RNA was prepared from 300–500 μl plasma using a commercially available RNA extraction kit (Qiagen) followed by reverse-transcription (RT) with Superscript III RT (Invitrogen, Carlsbad, CA) and random hexamer primers (Amersham Pharmacia, Piscataway, NJ), with amplification of the V1 to V5 region of gp120 from the RT products. The PCR products were cloned with the TOPO TA cloning kit (Invitrogen) per the manufacturer’s instructions followed by direct automated sequencing (Genewiz, South Plainfield, NJ). The sequences obtained were aligned with Clustal X , edited manually using BioEdit V7.0.9 and translated to the amino acid sequence. Deep sequencing of the inoculating virus stocks was performed using the Solexa Illumina platform by the Genomic Resource Center at the Rockefeller University.
Envelope expression plasmid construction and pseudotype virus production
The generation of envelope (Env) expression plasmids, V3 mutants and luciferase-reporter viruses have been described previously . Briefly, full length gp160 coding sequence was amplified with primers SH43 (5’-AAGACAGAATTCATGAGAGTGAAGGGGATCAGGAAG-3’) and SH44 (5’-AGAGAGGGATCCTTATAGCAAAGCCCTTTCAAAGCCCT-3’), and subcloned into the pCAGGS vector. Site-directed mutagenesis was employed to introduce specific V3 mutations into the backbone of the parental Envs, and trans-complementation assay was then used to generate luciferase reporter viruses capable of only a single round of replication. The pseudovirions were quantified for p24gag content (Beckman Coulter, Fullerton, CA).
Determination of coreceptor usage
For assessment of coreceptor usage, 7 x 103 U87.CD4.CCR5 or U87.CD4.CXCR4 cells were seeded in 96-well plates 24 hours before use and infected, in triplicate, with 5 ng p24gag equivalent of the indicated pseudovirions followed by incubation for 72 h at 37°C. At the end of the incubation period, the cells were harvested, lysed and processed for activity according to the manufacturer’s instructions (Luciferase Assay System; Promega, Madison, WI). Entry, as quantified by relative light unit (RLU), was measured with an MLX microtiter plate luminometer (Dynex Technologies, Inc., Chantilly, VA). For assessment of coreceptor utilization in rhPBMCs, blocking with CCR5 (TAK779) and CXCR4 (AMD3100) inhibitors was performed. Briefly, 5 x 106 SEB-stimulated cells were infected with 200 TCID50 of the indicated SHIV in the presence or absence of 1 μM of the chemokine receptor inhibitors. After incubation for 2 hours at 37°C, cells were washed and cultured in 1.5 ml interleukin-2 and appropriate inhibitor-supplemented RPMI medium in each well of a 24-well plate. Culture supernatants were collected over time, and p27gag antigen content was quantified according to the Manufacturer’s instructions (ZeptoMetrix, Buffalo, NY). Percentage blocking at 6 days post-infection was determined by calculating the amount of p27gag antigen production in the presence relative to that in the absence of the inhibitor.
Detection of antiviral humoral response
The titers of SHIV-specific antibodies were measured by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s instructions (GS HIV-1/HIV-2 PLUS O EIA; Bio-Rad, Redmond, WA). Endpoint titers were determined as the reciprocal of the highest serum dilution that resulted in an optical density reading greater than the average values obtained with negative human sera plus three standard deviations. Autologous virus neutralization was assessed using TZM-bl cells in 96-well plates. Briefly, equal volumes (50 μl) of the inoculating virus were incubated with 4-fold serial dilutions of heat-inactivated sera from infected macaques for 1 hour at 37°C and then added to cells, in duplicate wells, for an additional 2 hours at 37°C. 100 μl of medium was then added to each well and the virus-antibody (Ab) cultures maintained for 72 hours. Control cultures received virus in the absence of SHIV sera. At the end of the culture period, the cells were lysed and processed for β-galactosidase activity. A neutralization curve was generated by plotting the percentage of neutralization vs serum dilution, and 50% inhibitory dilution (ID50) titer was determined using the Prism 4 software (GraphPad, San Diego, CA). Neutralization titers are expressed as the reciprocal of the plasma dilution that inhibited virus infection by 50% (ID50). The lowest serum dilution used in the assay was 1:20.
Immunophenotyping of SHIV-infected cells
Identification of SHIV-infected cells was accomplished with double-label immunohistochemistry performed as previously described with modifications [95, 96]. Briefly, lymph node sections were deparaffinized in xylene and rehydrated through graded ethanol to tris-buffered saline (TBS) plus tween 20. Endogenous peroxidase activity was blocked by incubation in 3% H2O2 in PBS. Antigen retrieval was accomplished by microwave heating sections at 95°C for 20 minutes in citrate buffer (Vector Laboratories, Burlingame, CA), followed by 20 minute cooling, and Dako protein block (Carpinteria, CA) for 10 minutes. The blocked sections were incubated with SIVnef antibody (clone KK75, IgG1; 1:200) overnight at 4°C then reacted with biotinylated secondary antibody (HAM-b, Dako, 1:200) for 30 minutes. Sections were detected using standard avidin-biotin peroxidase complex technique (ABC Elite, Vector Laboratories) and DAB chromagen (Dako). Sections were blocked again for 10 minutes with protein block (Dako) and incubated with Iba-1 antibody (Wako Chemicals, Richmond, VA, rabbit polyclonal, 019–19741, 1:1000) for macrophages for 30 minutes at room temperature followed by biotinylated secondary antibody (GAR-b, Dako, 1:200) for 30 minutes. Sections were detected using standard avidin-biotin alkaline phosphatase complex technique (Vectastain ABC-AP, Vector Laboratories) and Permanent Red (Dako). Slides were counterstained with Mayer’s hematoxylin, rinsed in tap water, coated with Clear Mount (Electron Microscopy Science, Hatfield, PA), air-dried overnight, then coverslipped.
The TZM-bl (catalog no. 8129 from Dr. John C. Kappes, Xiaoyun Wu and Tranzyme, Inc.), U87.CD4 indicator cell lines (catalog no. 4035 and 4036 from HongKui Deng and Dan Littman), AMD100 (catalog no. 8128) and TAK779 (catalog no. 4983) were obtained through the NIH AIDS Research and Reagent Program, Division of AIDS, NIAID, NIH. We thank Dr. Connie Zhao and Scott Dewell at the RU Genomic Resource Center for help with Illumina deep-sequencing and analysis. This work was supported by National Institutes of Health grant R01AI46980. Additional support was provided by the Tulane National Primate Research Center Base grant RR00164.
- Berger EA, Murphy PM, Farber JM: Chemokine receptors as HIV-1 coreceptors: roles in viral entry, tropism, and disease. Annu Rev Immunol. 1999, 17: 657-700. 10.1146/annurev.immunol.17.1.657.View ArticlePubMedGoogle Scholar
- Schuitemaker H, Koot M, Kootstra NA, Dercksen MW, de Goede RE, van Steenwijk RP, Lange JM, Schattenkerk JK, Miedema F, Tersmette M: 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. J Virol. 1992, 66: 1354-1360.PubMed CentralPubMedGoogle Scholar
- van't Wout AB, Kootstra NA, Mulder-Kampinga GA, Albrecht-van Lent N, Scherpbier HJ, Veenstra J, Boer K, Coutinho RA, Miedema F, Schuitemaker H: Macrophage-tropic variants initiate human immunodeficiency virus type 1 infection after sexual, parenteral, and vertical transmission. J Clin Invest. 1994, 94: 2060-2067. 10.1172/JCI117560.PubMed CentralView ArticlePubMedGoogle Scholar
- Zhu T, Mo H, Wang N, Nam DS, Cao Y, Koup RA, Ho DD: Genotypic and phenotypic characterization of HIV-1 patients with primary infection. Science. 1993, 261: 1179-1181. 10.1126/science.8356453.View ArticlePubMedGoogle Scholar
- Long EM, Rainwater SM, Lavreys L, Mandaliya K, Overbaugh J: HIV type 1 variants transmitted to women in Kenya require the CCR5 coreceptor for entry, regardless of the genetic complexity of the infecting virus. AIDS Res Hum Retroviruses. 2002, 18: 567-576. 10.1089/088922202753747914.View ArticlePubMedGoogle Scholar
- Keele BF, Giorgi EE, Salazar-Gonzalez JF, Decker JM, Pham KT, Salazar MG, Sun C, Grayson T, Wang S, Li H, et al: Identification and characterization of transmitted and early founder virus envelopes in primary HIV-1 infection. Proc Natl Acad Sci USA. 2008, 105: 7552-7557. 10.1073/pnas.0802203105.PubMed CentralView ArticlePubMedGoogle Scholar
- Scarlatti G, Tresoldi E, Bjorndal A, Fredriksson R, Colognesi C, Deng HK, Malnati MS, Plebani A, Siccardi AG, Littman DR, et al: In vivo evolution of HIV-1 co-receptor usage and sensitivity to chemokine-mediated suppression. Nat Med. 1997, 3: 1259-1265. 10.1038/nm1197-1259.View ArticlePubMedGoogle Scholar
- Connor RI, Sheridan KE, Ceradini D, Choe S, Landau NR: Change in coreceptor use coreceptor use correlates with disease progression in HIV-1–infected individuals. J Exp Med. 1997, 185: 621-628. 10.1084/jem.185.4.621.PubMed CentralView ArticlePubMedGoogle Scholar
- Esbjornsson J, Mansson F, Martinez-Arias W, Vincic E, Biague AJ, da Silva ZJ, Fenyo EM, Norrgren H, Medstrand P: Frequent CXCR4 tropism of HIV-1 subtype A and CRF02_AG during late-stage disease–indication of an evolving epidemic in West Africa. Retrovirology. 2010, 7: 23-10.1186/1742-4690-7-23.PubMed CentralView ArticlePubMedGoogle Scholar
- Connell BJ, Michler K, Capovilla A, Venter WD, Stevens WS, Papathanasopoulos MA: Emergence of X4 usage among HIV-1 subtype C: evidence for an evolving epidemic in South Africa. AIDS. 2008, 22: 896-899. 10.1097/QAD.0b013e3282f57f7a.View ArticlePubMedGoogle Scholar
- Koot M, Keet IP, Vos AH, de Goede RE, Roos MT, Coutinho RA, Miedema F, Schellekens PT, Tersmette M: Prognostic value of HIV-1 syncytium-inducing phenotype for rate of CD4+ cell depletion and progression to AIDS. Ann Intern Med. 1993, 118: 681-688.View ArticlePubMedGoogle Scholar
- Richman DD, Bozzette SA: The impact of the syncytium-inducing phenotype of human immunodeficiency virus on disease progression. J Infect Dis. 1994, 169: 968-974. 10.1093/infdis/169.5.968.View ArticlePubMedGoogle Scholar
- Regoes RR, Bonhoeffer S: The HIV coreceptor switch: a population dynamical perspective. Trends Microbiol. 2005, 13: 269-277. 10.1016/j.tim.2005.04.005.View ArticlePubMedGoogle Scholar
- Pastore C, Ramos A, Mosier DE: Intrinsic obstacles to human immunodeficiency virus type 1 coreceptor switching. J Virol. 2004, 78: 7565-7574. 10.1128/JVI.78.14.7565-7574.2004.PubMed CentralView ArticlePubMedGoogle Scholar
- Simmons G, Wilkinson D, Reeves JD, Dittmar MT, Beddows S, Weber J, Carnegie G, Desselberger U, Gray PW, Weiss RA, Clapham PR: Primary, syncytium-inducing human immunodeficiency virus type 1 isolates are dual-tropic and most can use either Lestr or CCR5 as coreceptors for virus entry. J Virol. 1996, 70: 8355-8360.PubMed CentralPubMedGoogle Scholar
- Cornelissen M, Mulder-Kampinga G, Veenstra J, Zorgdrager F, Kuiken C, Hartman S, Dekker J, van der Hoek L, Sol C, Coutinho R, et al: Syncytium-inducing (SI) phenotype suppression at seroconversion after intramuscular inoculation of a non-syncytium-inducing/SI phenotypically mixed human immunodeficiency virus population. J Virol. 1995, 69: 1810-1818.PubMed CentralPubMedGoogle Scholar
- Lathey JL, Pratt RD, Spector SA: Appearance of autologous neutralizing antibody correlates with reduction in virus load and phenotype switch during primary infection with human immunodeficiency virus type 1. J Infect Dis. 1997, 175: 231-232. 10.1093/infdis/175.1.231.View ArticlePubMedGoogle Scholar
- Tersmette M, Miedema F: Interactions between HIV and the host immune system in the pathogenesis of AIDS. AIDS. 1990, 4 (Suppl 1): S57-66.PubMedGoogle Scholar
- Pratt RD, Shapiro JF, McKinney N, Kwok S, Spector SA: Virologic characterization of primary human immunodeficiency virus type 1 infection in a health care worker following needlestick injury. J Infect Dis. 1995, 172: 851-854. 10.1093/infdis/172.3.851.View ArticlePubMedGoogle Scholar
- Abbate I, Vlassi C, Rozera G, Bruselles A, Bartolini B, Giombini E, Corpolongo A, D'Offizi G, Narciso P, Desideri A, et al: Detection of quasispecies variants predicted to use CXCR4 by ultra-deep pyrosequencing during early HIV infection. AIDS. 2011, 25: 611-617. 10.1097/QAD.0b013e328343489e.View ArticlePubMedGoogle Scholar
- Nielsen C, Pedersen C, Lundgren JD, Gerstoft J: Biological properties of HIV isolates in primary HIV infection: consequences for the subsequent course of infection. AIDS. 1993, 7: 1035-1040. 10.1097/00002030-199308000-00002.View ArticlePubMedGoogle Scholar
- Fiore JR, Bjorndal A, Peipke KA, Di Stefano M, Angarano G, Pastore G, Gaines H, Fenyo EM, Albert J: The biological phenotype of HIV-1 is usually retained during and after sexual transmission. Virology. 1994, 204: 297-303. 10.1006/viro.1994.1534.View ArticlePubMedGoogle Scholar
- Karlsson A, Parsmyr K, Sandstrom E, Fenyo EM, Albert J: MT-2 cell tropism as prognostic marker for disease progression in human immunodeficiency virus type 1 infection. J Clin Microbiol. 1994, 32: 364-370.PubMed CentralPubMedGoogle Scholar
- Chalmet K, Dauwe K, Foquet L, Baatz F, Seguin-Devaux C, Van Der Gucht B, Vogelaers D, Vandekerckhove L, Plum J, Verhofstede C: Presence of CXCR4-using HIV-1 in patients with recently diagnosed infection: correlates and evidence for transmission. J Infect Dis. 2012, 205: 174-184. 10.1093/infdis/jir714.View ArticlePubMedGoogle Scholar
- Bunnik EM, Quakkelaar ED, van Nuenen AC, Boeser-Nunnink B, Schuitemaker H: Increased neutralization sensitivity of recently emerged CXCR4-using human immunodeficiency virus type 1 strains compared to coexisting CCR5-using variants from the same patient. J Virol. 2007, 81: 525-531. 10.1128/JVI.01983-06.PubMed CentralView ArticlePubMedGoogle Scholar
- Vodros D, Thorstensson R, Biberfeld G, Schols D, De Clercq E, Fenyo EM: Coreceptor usage of sequential isolates from cynomolgus monkeys experimentally Infected with simian immunodeficiency virus (SIVsm). Virology. 2001, 291: 12-21. 10.1006/viro.2001.1164.View ArticlePubMedGoogle Scholar
- Felber BK, Valentin A, von Gegerfelt A, Jalah R, Patel V, Kulkarni V, Alicea C, Rosati M, Khan A, Draghia-Akli R, Pavlakis GN: P18-09. Persistent virological benefit in SIV-infected macaques upon therapeutic vaccination upon vaccination with DNA vectors. Retrovirology. 2009, 6 (Suppl 3):Google Scholar
- Varela M, Landskron L, Lai RP, McKinley TJ, Bogers WM, Verschoor EJ, Dubbes R, Barnett SW, Frost SD, Heeney JL: Molecular evolution analysis of the human immunodeficiency virus type 1 envelope in simian/human immunodeficiency virus-infected macaques: implications for challenge dose selection. J Virol. 85: 10332-10345.Google Scholar
- Morgan C, Marthas M, Miller C, Duerr A, Cheng-Mayer C, Desrosiers R, Flores J, Haigwood N, Hu SL, Johnson RP, et al: The use of nonhuman primate models in HIV vaccine development. PLoS Med. 2008, 5: e173-10.1371/journal.pmed.0050173.PubMed CentralView ArticlePubMedGoogle Scholar
- Veazey RS, Shattock RJ, Klasse PJ, Moore JP: Animal models for microbicide studies. Curr HIV Res. 2012, 10: 79-87. 10.2174/157016212799304715.PubMed CentralView ArticlePubMedGoogle Scholar
- Van Rompay KK: The use of nonhuman primate models of HIV infection for the evaluation of antiviral strategies. AIDS Res Hum Retroviruses. 2012, 28: 16-35. 10.1089/aid.2011.0234.View ArticlePubMedGoogle Scholar
- Joag SV, Li Z, Foresman L, Stephens EB, Zhao LJ, Adany I, Pinson DM, McClure HM, Narayan O: Chimeric simian/human immunodeficiency virus that causes progressive loss of CD4+ T cells and AIDS in pig-tailed macaques. J Virol. 1996, 70: 3189-3197.PubMed CentralPubMedGoogle Scholar
- Reimann KA, Li JT, Veazey R, Halloran M, Park IW, Karlsson GB, Sodroski J, Letvin NL: A chimeric simian/human immunodeficiency virus expressing a primary patient human immunodeficiency virus type 1 isolate env causes an AIDS-like disease after in vivo passage in rhesus monkeys. J Virol. 1996, 70: 6922-6928.PubMed CentralPubMedGoogle Scholar
- Luciw PA, Pratt-Lowe E, Shaw KE, Levy JA, Cheng-Mayer C: Persistent infection of rhesus macaques with T-cell-line-tropic and macrophage-tropic clones of simian/human immunodeficiency viruses (SHIV). Proc Natl Acad Sci USA. 1995, 92: 7490-7494. 10.1073/pnas.92.16.7490.PubMed CentralView ArticlePubMedGoogle Scholar
- Igarashi T, Endo Y, Englund G, Sadjadpour R, Matano T, Buckler C, Buckler-White A, Plishka R, Theodore T, Shibata R, Martin M: Emergence of a highly pathogenic simian/human immunodeficiency virus in a rhesus macaque treated with anti-CD8 mAb during a primary infection with a nonpathogenic virus. Proc Natl Acad Sci USA. 1999, 96: 14049-14054. 10.1073/pnas.96.24.14049.PubMed CentralView ArticlePubMedGoogle Scholar
- Chen Z, Zhao X, Huang Y, Gettie A, Ba L, Blanchard J, Ho DD: CD4+ lymphocytopenia in acute infection of Asian macaques by a vaginally transmissible subtype-C, CCR5-tropic Simian/Human Immunodeficiency Virus (SHIV). J Acquir Immune Defic Syndr. 2002, 30: 133-145.View ArticlePubMedGoogle Scholar
- Harouse JM, Gettie A, Tan RC, Blanchard J, Cheng-Mayer C: Distinct pathogenic sequela in rhesus macaques infected with CCR5 or CXCR4 utilizing SHIVs. Science. 1999, 284: 816-819. 10.1126/science.284.5415.816.View ArticlePubMedGoogle Scholar
- Humbert M, Rasmussen RA, Song R, Ong H, Sharma P, Chenine AL, Kramer VG, Siddappa NB, Xu W, Else JG, et al: SHIV-1157i and passaged progeny viruses encoding R5 HIV-1 clade C env cause AIDS in rhesus monkeys. Retrovirology. 2008, 5: 94-10.1186/1742-4690-5-94.PubMed CentralView ArticlePubMedGoogle Scholar
- Pal R, Taylor B, Foulke JS, Woodward R, Merges M, Praschunus R, Gibson A, Reitz M: Characterization of a simian human immunodeficiency virus encoding the envelope gene from the CCR5-tropic HIV-1 Ba-L. J Acquir Immune Defic Syndr. 2003, 33: 300-307. 10.1097/00126334-200307010-00003.View ArticlePubMedGoogle Scholar
- Gautam R, Nishimura Y, Lee WR, Donau O, Buckler-White A, Shingai M, Sadjadpour R, Schmidt SD, LaBranche CC, Keele BF, et al: Pathogenicity and mucosal transmissibility of the R5-tropic simian/human immunodeficiency virus SHIV(AD8) in rhesus macaques: implications for use in vaccine studies. J Virol. 2012, 86: 8516-8526. 10.1128/JVI.00644-12.PubMed CentralView ArticlePubMedGoogle Scholar
- Ho SH, Tasca S, Shek L, Li A, Gettie A, Blanchard J, Boden D, Cheng-Mayer C: Coreceptor switch in R5-tropic simian/human immunodeficiency virus-infected macaques. J Virol. 2007, 81: 8621-8633. 10.1128/JVI.00759-07.PubMed CentralView ArticlePubMedGoogle Scholar
- Ren W, Tasca S, Zhuang K, Gettie A, Blanchard J, Cheng-Mayer C: Different tempo and anatomic location of dual-tropic and X4 virus emergence in a model of R5 simian-human immunodeficiency virus infection. J Virol. 84: 340-351.Google Scholar
- Tasca S, Ho SH, Cheng-Mayer C: R5X4 viruses are evolutionary, functional, and antigenic intermediates in the pathway of a simian-human immunodeficiency virus coreceptor switch. J Virol. 2008, 82: 7089-7099. 10.1128/JVI.00570-08.PubMed CentralView ArticlePubMedGoogle Scholar
- Shakirzyanova M, Ren W, Zhuang K, Tasca S, Cheng-Mayer C: Fitness disadvantage of transitional intermediates contributes to dynamic change in the infecting-virus population during coreceptor switch in R5 simian/human immunodeficiency virus-infected macaques. J Virol. 2010, 84: 12862-12871. 10.1128/JVI.01478-10.PubMed CentralView ArticlePubMedGoogle Scholar
- Hartley O, Klasse PJ, Sattentau QJ, Moore JP: V3: HIV's switch-hitter. AIDS Res Hum Retroviruses. 2005, 21: 171-189. 10.1089/aid.2005.21.171.View ArticlePubMedGoogle Scholar
- De Jong JJ, De Ronde A, Keulen W, Tersmette M, Goudsmit J: Minimal requirements for the human immunodeficiency virus type 1 V3 domain to support the syncytium-inducing phenotype: analysis by single amino acid substitution. J Virol. 1992, 66: 6777-6780.PubMed CentralPubMedGoogle Scholar
- Fouchier RA, Groenink M, Kootstra NA, Tersmette M, Huisman HG, Miedema F, Schuitemaker H: Phenotype-associated sequence variation in the third variable domain of the human immunodeficiency virus type 1 gp120 molecule. J Virol. 1992, 66: 3183-3187.PubMed CentralPubMedGoogle Scholar
- Jensen MA, Li FS, van 't Wout AB, Nickle DC, Shriner D, He HX, McLaughlin S, Shankarappa R, Margolick JB, Mullins JI: Improved coreceptor usage prediction and genotypic monitoring of R5-to-X4 transition by motif analysis of human immunodeficiency virus type 1 env V3 loop sequences. J Virol. 2003, 77: 13376-13388. 10.1128/JVI.77.24.13376-13388.2003.PubMed CentralView ArticlePubMedGoogle Scholar
- De Wolf F, Hogervorst E, Goudsmit J, Fenyo EM, Rubsamen-Waigmann H, Holmes H, Galvao-Castro B, Karita E, Wasi C, Sempala SD, et al: Syncytium-inducing and non-syncytium-inducing capacity of human immunodeficiency virus type 1 subtypes other than B: phenotypic and genotypic characteristics. WHO Network for HIV Isolation and Characterization. AIDS Res Hum Retroviruses. 1994, 10: 1387-1400. 10.1089/aid.1994.10.1387.View ArticlePubMedGoogle Scholar
- Cardozo T, Kimura T, Philpott S, Weiser B, Burger H, Zolla-Pazner S: Structural basis for coreceptor selectivity by the HIV type 1 V3 loop. AIDS Res Hum Retroviruses. 2007, 23: 415-426. 10.1089/aid.2006.0130.View ArticlePubMedGoogle Scholar
- Nishimura Y, Shingai M, Willey R, Sadjadpour R, Lee WR, Brown CR, Brenchley JM, Buckler-White A, Petros R, Eckhaus M, et al: Generation of the pathogenic R5-tropic simian/human immunodeficiency virus SHIVAD8 by serial passaging in rhesus macaques. J Virol. 2010, 84: 4769-4781. 10.1128/JVI.02279-09.PubMed CentralView ArticlePubMedGoogle Scholar
- Ince WL, Zhang L, Jiang Q, Arrildt K, Su L, Swanstrom R: Evolution of the HIV-1 env gene in the Rag2−/− gammaC−/− humanized mouse model. J Virol. 2010, 84: 2740-2752. 10.1128/JVI.02180-09.PubMed CentralView ArticlePubMedGoogle Scholar
- Richman DD, Wrin T, Little SJ, Petropoulos CJ: Rapid evolution of the neutralizing antibody response to HIV type 1 infection. Proc Natl Acad Sci USA. 2003, 100: 4144-4149. 10.1073/pnas.0630530100.PubMed CentralView ArticlePubMedGoogle Scholar
- Gulick RM, Su Z, Flexner C, Hughes MD, Skolnik PR, Wilkin TJ, Gross R, Krambrink A, Coakley E, Greaves WL, et al: Phase 2 study of the safety and efficacy of vicriviroc, a CCR5 inhibitor, in HIV-1-Infected, treatment-experienced patients: AIDS clinical trials group 5211. J Infect Dis. 2007, 196: 304-312. 10.1086/518797.View ArticlePubMedGoogle Scholar
- Lalezari J, Thompson M, Kumar P, Piliero P, Davey R, Patterson K, Shachoy-Clark A, Adkison K, Demarest J, Lou Y, et al: Antiviral activity and safety of 873140, a novel CCR5 antagonist, during short-term monotherapy in HIV-infected adults. AIDS. 2005, 19: 1443-1448. 10.1097/01.aids.0000183633.06580.8a.View ArticlePubMedGoogle Scholar
- Westby M, Lewis M, Whitcomb J, Youle M, Pozniak AL, James IT, Jenkins TM, Perros M, van der Ryst E: Emergence of CXCR4-using human immunodeficiency virus type 1 (HIV-1) variants in a minority of HIV-1-infected patients following treatment with the CCR5 antagonist maraviroc is from a pretreatment CXCR4-using virus reservoir. J Virol. 2006, 80: 4909-4920. 10.1128/JVI.80.10.4909-4920.2006.PubMed CentralView ArticlePubMedGoogle Scholar
- Saag M, Goodrich J, Fatkenheuer G, Clotet B, Clumeck N, Sullivan J, Westby M, van der Ryst E, Mayer H: A double-blind, placebo-controlled trial of maraviroc in treatment-experienced patients infected with non-R5 HIV-1. J Infect Dis. 2009, 199: 1638-1647. 10.1086/598965.View ArticlePubMedGoogle Scholar
- Wilkin TJ, Su Z, Krambrink A, Long J, Greaves W, Gross R, Hughes MD, Flexner C, Skolnik PR, Coakley E, et al: Three-year safety and efficacy of vicriviroc, a CCR5 antagonist, in HIV-1-infected treatment-experienced patients. J Acquir Immune Defic Syndr. 2010, 54: 470-476. 10.1097/QAI.0b013e3181e2cba0.PubMed CentralView ArticlePubMedGoogle Scholar
- Hsu M, Ho SH, Balfe P, Gettie A, Harouse J, Blanchard J, Cheng-Mayer C: A CCR5-tropic simian-HIV molecular clone capable of inducing AIDS in rhesus macaques. J Acquir Immune Defic Syndr. 2005, 40: 383-387. 10.1097/01.qai.0000184857.39318.4f.View ArticlePubMedGoogle Scholar
- Song RJ, Chenine AL, Rasmussen RA, Ruprecht CR, Mirshahidi S, Grisson RD, Xu W, Whitney JB, Goins LM, Ong H, et al: Molecularly cloned SHIV-1157ipd3N4: a highly replication- competent, mucosally transmissible R5 simian-human immunodeficiency virus encoding HIV clade C Env. J Virol. 2006, 80: 8729-8738. 10.1128/JVI.00558-06.PubMed CentralView ArticlePubMedGoogle Scholar
- Ho O, Larsen K, Polacino P, Li Y, Anderson D, Song R, Ruprecht RM, Hu SL: Pathogenic infection of Macaca nemestrina with a CCR5-tropic subtype-C simian-human immunodeficiency virus. Retrovirology. 2009, 6: 65-10.1186/1742-4690-6-65.PubMed CentralView ArticlePubMedGoogle Scholar
- Verhofstede C, Vandekerckhove L, Eygen VV, Demecheleer E, Vandenbroucke I, Winters B, Plum J, Vogelaers D, Stuyver L: CXCR4-using HIV type 1 variants are more commonly found in peripheral blood mononuclear cell DNA than in plasma RNA. J Acquir Immune Defic Syndr. 2009, 50: 126-136. 10.1097/QAI.0b013e31819118fa.View ArticlePubMedGoogle Scholar
- Edo-Matas D, van Gils MJ, Bowles EJ, Navis M, Rachinger A, Boeser-Nunnink B, Stewart-Jones GB, Kootstra NA, van 't Wout AB, Schuitemaker H: Genetic composition of replication competent clonal HIV-1 variants isolated from peripheral blood mononuclear cells (PBMC), HIV-1 proviral DNA from PBMC and HIV-1 RNA in serum in the course of HIV-1 infection. Virology. 2010, 405: 492-504. 10.1016/j.virol.2010.06.029.View ArticlePubMedGoogle Scholar
- Bunnik EM, Swenson LC, Edo-Matas D, Huang W, Dong W, Frantzell A, Petropoulos CJ, Coakley E, Schuitemaker H, Harrigan PR, van 't Wout AB: Detection of inferred CCR5- and CXCR4-using HIV-1 variants and evolutionary intermediates using ultra-deep pyrosequencing. PLoS Pathog. 2011, 7: e1002106-10.1371/journal.ppat.1002106.PubMed CentralView ArticlePubMedGoogle Scholar
- Navis M, Matas DE, Rachinger A, Koning FA, van Swieten P, Kootstra NA, Schuitemaker H: Molecular evolution of human immunodeficiency virus type 1 upon transmission between human leukocyte antigen disparate donor-recipient pairs. PLoS One. 2008, 3: e2422-10.1371/journal.pone.0002422.PubMed CentralView ArticlePubMedGoogle Scholar
- Groenink M, Fouchier RA, Broersen S, Baker CH, Koot M, van't Wout AB, Huisman HG, Miedema F, Tersmette M, Schuitemaker H: Relation of phenotype evolution of HIV-1 to envelope V2 configuration. Science. 1993, 260: 1513-1516. 10.1126/science.8502996.View ArticlePubMedGoogle Scholar
- Koito A, Harrowe G, Levy JA, Cheng-Mayer C: Functional role of the V1/V2 region of human immunodeficiency virus type 1 envelope glycoprotein gp120 in infection of primary macrophages and soluble CD4 neutralization. J Virol. 1994, 68: 2253-2259.PubMed CentralPubMedGoogle Scholar
- Koito A, Stamatatos L, Cheng-Mayer C: Small amino acid sequence changes within the V2 domain can affect the function of a T-cell line-tropic human immunodeficiency virus type 1 envelope gp120. Virology. 1995, 206: 878-884. 10.1006/viro.1995.1010.View ArticlePubMedGoogle Scholar
- Carrillo A, Ratner L: Cooperative effects of the human immunodeficiency virus type 1 envelope variable loops V1 and V3 in mediating infectivity for T cells. J Virol. 1996, 70: 1310-1316.PubMed CentralPubMedGoogle Scholar
- Hoffman TL, Stephens EB, Narayan O, Doms RW: HIV type I envelope determinants for use of the CCR2b, CCR3, STRL33, and APJ coreceptors. Proc Natl Acad Sci USA. 1998, 95: 11360-11365. 10.1073/pnas.95.19.11360.PubMed CentralView ArticlePubMedGoogle Scholar
- Ogert RA, Lee MK, Ross W, Buckler-White A, Martin MA, Cho MW: N-linked glycosylation sites adjacent to and within the V1/V2 and the V3 loops of dualtropic human immunodeficiency virus type 1 isolate DH12 gp120 affect coreceptor usage and cellular tropism. J Virol. 2001, 75: 5998-6006. 10.1128/JVI.75.13.5998-6006.2001.PubMed CentralView ArticlePubMedGoogle Scholar
- Sullivan N, Thali M, Furman C, Ho DD, Sodroski J: Effect of amino acid changes in the V1/V2 region of the human immunodeficiency virus type 1 gp120 glycoprotein on subunit association, syncytium formation, and recognition by a neutralizing antibody. J Virol. 1993, 67: 3674-3679.PubMed CentralPubMedGoogle Scholar
- Wyatt R, Moore J, Accola M, Desjardin E, Robinson J, Sodroski J: Involvement of the V1/V2 variable loop structure in the exposure of human immunodeficiency virus type 1 gp120 epitopes induced by receptor binding. J Virol. 1995, 69: 5723-5733.PubMed CentralPubMedGoogle Scholar
- Kwong PD, Wyatt R, Robinson J, Sweet RW, Sodroski J, Hendrickson WA: Structure of an HIV gp120 envelope glycoprotein in complex with the CD4 receptor and a neutralizing human antibody. Nature. 1998, 393: 648-659. 10.1038/31405.View ArticlePubMedGoogle Scholar
- Rizzuto CD, Wyatt R, Hernandez-Ramos N, Sun Y, Kwong PD, Hendrickson WA, Sodroski J: A conserved HIV gp120 glycoprotein structure involved in chemokine receptor binding. Science. 1998, 280: 1949-1953. 10.1126/science.280.5371.1949.View ArticlePubMedGoogle Scholar
- Kwong PD, Wyatt R, Sattentau QJ, Sodroski J, Hendrickson WA: Oligomeric modeling and electrostatic analysis of the gp120 envelope glycoprotein of human immunodeficiency virus. J Virol. 2000, 74: 1961-1972. 10.1128/JVI.74.4.1961-1972.2000.PubMed CentralView ArticlePubMedGoogle Scholar
- Pastore C, Nedellec R, Ramos A, Pontow S, Ratner L, Mosier DE: Human immunodeficiency virus type 1 coreceptor switching: V1/V2 gain-of-fitness mutations compensate for V3 loss-of-fitness mutations. J Virol. 2006, 80: 750-758. 10.1128/JVI.80.2.750-758.2006.PubMed CentralView ArticlePubMedGoogle Scholar
- Shakirzyanova M, Tsai L, Ren W, Gettie A, Blanchard J, Cheng-Mayer C: Pathogenic consequences of vaginal infection with CCR5-tropic simian-human immunodeficiency virus SHIVSF162P3N. J Virol. 2012, 86: 9432-9442. 10.1128/JVI.00852-12.PubMed CentralView ArticlePubMedGoogle Scholar
- Gottlieb GS, Nickle DC, Jensen MA, Wong KG, Grobler J, Li F, Liu SL, Rademeyer C, Learn GH, Karim SS, et al: Dual HIV-1 infection associated with rapid disease progression. Lancet. 2004, 363: 619-622. 10.1016/S0140-6736(04)15596-7.View ArticlePubMedGoogle Scholar
- Sagar M, Lavreys L, Baeten JM, Richardson BA, Mandaliya K, Chohan BH, Kreiss JK, Overbaugh J: Infection with multiple human immunodeficiency virus type 1 variants is associated with faster disease progression. J Virol. 2003, 77: 12921-12926. 10.1128/JVI.77.23.12921-12926.2003.PubMed CentralView ArticlePubMedGoogle Scholar
- Abrahams MR, Anderson JA, Giorgi EE, Seoighe C, Mlisana K, Ping LH, Athreya GS, Treurnicht FK, Keele BF, Wood N, et al: Quantitating the multiplicity of infection with human immunodeficiency virus type 1 subtype C reveals a non-poisson distribution of transmitted variants. J Virol. 2009, 83: 3556-3567. 10.1128/JVI.02132-08.PubMed CentralView ArticlePubMedGoogle Scholar
- Malim MH, Emerman M: HIV-1 sequence variation: drift, shift, and attenuation. Cell. 2001, 104: 469-472. 10.1016/S0092-8674(01)00234-3.View ArticlePubMedGoogle Scholar
- Montaner LJ, Crowe SM, Aquaro S, Perno CF, Stevenson M, Collman RG: Advances in macrophage and dendritic cell biology in HIV-1 infection stress key understudied areas in infection, pathogenesis, and analysis of viral reservoirs. J Leukoc Biol. 2006, 80: 961-964. 10.1189/jlb.0806488.View ArticlePubMedGoogle Scholar
- Igarashi T, Brown CR, Endo Y, Buckler-White A, Plishka R, Bischofberger N, Hirsch V, Martin MA: Macrophage are the principal reservoir and sustain high virus loads in rhesus macaques after the depletion of CD4+ T cells by a highly pathogenic simian immunodeficiency virus/HIV type 1 chimera (SHIV): Implications for HIV-1 infections of humans. Proc Natl Acad Sci USA. 2001, 98: 658-663. 10.1073/pnas.98.2.658.PubMed CentralView ArticlePubMedGoogle Scholar
- Zhuang K, Finzi A, Tasca S, Shakirzyanova M, Knight H, Westmoreland S, Sodroski J, Cheng-Mayer C: Adoption of an "open" envelope conformation facilitating CD4 binding and structural remodeling precedes coreceptor switch in R5 SHIV-infected macaques. PLoS One. 2011, 6: e21350-10.1371/journal.pone.0021350.PubMed CentralView ArticlePubMedGoogle Scholar
- Schutten M, van Baalen CA, Guillon C, Huisman RC, Boers PH, Sintnicolaas K, Gruters RA, Osterhaus AD: Macrophage tropism of human immunodeficiency virus type 1 facilitates in vivo escape from cytotoxic T-lymphocyte pressure. J Virol. 2001, 75: 2706-2709. 10.1128/JVI.75.6.2706-2709.2001.PubMed CentralView ArticlePubMedGoogle Scholar
- Vojnov L, Martins MA, Bean AT, Veloso de Santana MG, Sacha JB, Wilson NA, Bonaldo MC, Galler R, Stevenson M, Watkins DI: The majority of freshly sorted simian immunodeficiency virus (SIV)-specific CD8(+) T cells cannot suppress viral replication in SIV-infected macrophages. J Virol. 2012, 86: 4682-4687. 10.1128/JVI.06324-11.PubMed CentralView ArticlePubMedGoogle Scholar
- Koppensteiner H, Banning C, Schneider C, Hohenberg H, Schindler M: Macrophage internal HIV-1 is protected from neutralizing antibodies. J Virol. 2012, 86: 2826-2836. 10.1128/JVI.05915-11.PubMed CentralView ArticlePubMedGoogle Scholar
- Wei X, Decker JM, Wang S, Hui H, Kappes JC, Wu X, Salazar-Gonzalez JF, Salazar MG, Kilby JM, Saag MS, et al: Antibody neutralization and escape by HIV-1. Nature. 2003, 422: 307-312. 10.1038/nature01470.View ArticlePubMedGoogle Scholar
- Deng H, Liu R, Ellmeier W, Choe S, Unutmaz D, Burkhart M, Di Marzio P, Marmon S, Sutton RE, Hill CM, et al: Identification of a major co-receptor for primary isolates of HIV-1. Nature. 1996, 381: 661-666. 10.1038/381661a0.View ArticlePubMedGoogle Scholar
- Ho SH, Martin F, Higginbottom A, Partridge LJ, Parthasarathy V, Moseley GW, Lopez P, Cheng-Mayer C, Monk PN: Recombinant extracellular domains of tetraspanin proteins are potent inhibitors of the infection of macrophages by human immunodeficiency virus type 1. J Virol. 2006, 80: 6487-6496. 10.1128/JVI.02539-05.PubMed CentralView ArticlePubMedGoogle Scholar
- Goulder PJ, Watkins DI: Impact of MHC class I diversity on immune control of immunodeficiency virus replication. Nat Rev Immunol. 2008, 8: 619-630. 10.1038/nri2357.PubMed CentralView ArticlePubMedGoogle Scholar
- Delwart EL, Gordon CJ: Tracking changes in HIV-1 envelope quasispecies using DNA heteroduplex analysis. Methods. 1997, 12: 348-354. 10.1006/meth.1997.0489.View ArticlePubMedGoogle Scholar
- Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, et al: Clustal W and Clustal X version 2.0. Bioinformatics. 2007, 23: 2947-2948. 10.1093/bioinformatics/btm404.View ArticlePubMedGoogle Scholar
- Schwartz AJ, Alvarez X, Lackner AA: Distribution and immunophenotype of DC-SIGN-expressing cells in SIV-infected and uninfected macaques. AIDS Res Hum Retroviruses. 2002, 18: 1021-1029. 10.1089/08892220260235380.View ArticlePubMedGoogle Scholar
- Yearley JH, Kanagy S, Anderson DC, Dalecki K, Pauley DR, Suwyn C, Donahoe RM, McClure HM, O'Neil SP: Tissue-specific reduction in DC-SIGN expression correlates with progression of pathogenic simian immunodeficiency virus infection. Dev Comp Immunol. 2008, 32: 1510-1521. 10.1016/j.dci.2008.06.006.View ArticlePubMedGoogle Scholar
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