CD4-independent use of the CCR5 receptor by sequential primary SIVsm isolates

<p>Abstract</p> <p>Background</p> <p>CD4-independence has been taken as a sign of a more open envelope structure that is more accessible to neutralizing antibodies and may confer altered cell tropism. In the present study, we analyzed SIVsm isolates for CD4-independent use of CCR5, mode of CCR5-use and macrophage tropism. The isolates have been collected sequentially from 13 experimentally infected cynomolgus macaques and have previously been shown to use CCR5 together with CD4. Furthermore, viruses obtained early after infection were neutralization sensitive, while neutralization resistance appeared already three months after infection in monkeys with progressive immunodeficiency.</p> <p>Results</p> <p>Depending whether isolated early or late in infection, two phenotypes of CD4-independent use of CCR5 could be observed. The inoculum virus (SIVsm isolate SMM-3) and reisolates obtained early in infection often showed a pronounced CD4-independence since virus production and/or syncytia induction could be detected directly in NP-2 cells expressing CCR5 but not CD4 (CD4-independent-HIGH). Conversely, late isolates were often more CD4-dependent in that productive infection in NP-2/CCR5 cells was in most cases weak and was revealed only after cocultivation of infected NP-2/CCR5 cells with peripheral blood mononuclear cells (CD4-independent-LOW). Considering neutralization sensitivity of these isolates, newly infected macaques often harbored virus populations with a CD4-independent-HIGH and neutralization sensitive phenotype that changed to a CD4-independent-LOW and neutralization resistant virus population in the course of infection. Phenotype changes occurred faster in progressor than long-term non-progressor macaques. The phenotypes were not reflected by macrophage tropism, since all isolates replicated efficiently in macrophages. Infection of cells expressing CCR5/CXCR4 chimeric receptors revealed that SIVsm used the CCR5 receptor in a different mode than HIV-1.</p> <p>Conclusion</p> <p>Our results show that SIVsm isolates use CCR5 independently of CD4. While the degree of CD4 independence and neutralization sensitivity vary over time, the ability to productively infect monocyte-derived macrophages remains at a steady high level throughout infection. The mode of CCR5 use differs between SIVsm and HIV-1, SIVsm appears to be more flexible than HIV-1 in its receptor requirement. We suggest that the mode of CCR5 coreceptor use and CD4-independence are interrelated properties.</p

Frequent CXCR4 tropism of HIV-1 subtype A and CRF02_AG during late-stage disease - indication of an evolving epidemic in West Africa

<p>Abstract</p> <p>Background</p> <p>HIV-1 is one of the fastest evolving pathogens, and is distinguished by geographic and genetic variants that have been classified into different subtypes and circulating recombinant forms (CRFs). Early in infection the primary coreceptor is CCR5, but during disease course CXCR4-using HIV-1 populations may emerge. This has been correlated with accelerated disease progression in HIV-1 subtype B. Basic knowledge of HIV-1 coreceptor tropism is important due to the recent introduction of coreceptor antagonists in antiretroviral therapy, and subtype-specific differences regarding how frequently HIV-1 CXCR4-using populations appear in late-stage disease need to be further investigated. To study how frequently CXCR4-using populations appear in late-stage disease among HIV-1 subtype A and CRF02_AG, we evaluated the accuracy of a recombinant virus phenotypic assay for these subtypes, and used it to determine the HIV-1 coreceptor tropism of plasma samples collected during late-stage disease in Guinea-Bissau. We also performed a genotypic analysis and investigated subtype-specific differences in the appearance of CXCR4 tropism late in disease.</p> <p>Results</p> <p>We found that the recombinant virus phenotypic assay accurately predicted HIV-1 coreceptor tropism of subtype A and CRF02_AG. Over the study period (1997-2007), we found an increasing and generally high frequency of CXCR4 tropism (86%) in CRF02_AG. By sequence analysis of the V3 region of our samples we developed a novel genotypic rule for predicting CXCR4 tropism in CRF02_AG, based on the combined criteria of the total number of charged amino acids and net charge. This rule had higher sensitivity than previously described genotypic rules and may be useful for development of future genotypic tools for this CRF. Finally, we conducted a literature analysis, combining data of 498 individuals in late-stage disease, and found high amounts of CXCR4 tropism for all major HIV-1 subtypes (60-77%), except for subtype C (15%).</p> <p>Conclusions</p> <p>The increase in CXCR4 tropism over time suggests an evolving epidemic of CRF02_AG. The results of the literature analysis demonstrate the need for further studies investigating subtype-specific emergence for CXCR4-tropism; this may be particularly important due to the introduction of CCR5-antagonists in HIV treatment regimens.</p

CD4-independent use of the CCR5 receptor by sequential primary SIVsm isolates-0

<p><b>Copyright information:</b></p><p>Taken from "CD4-independent use of the CCR5 receptor by sequential primary SIVsm isolates"</p><p>http://www.retrovirology.com/content/4/1/50</p><p>Retrovirology 2007;4():50-50.</p><p>Published online 23 Jul 2007</p><p>PMCID:PMC1950888.</p><p></p>RT/well. The day after infection cultures were washed extensively and fresh medium was added. Infected NP-2/CCR5 cells were followed for syncytia induction up to seven days after infection. RT was analyzed in supernatants from NP-2 cells at day 1 after wash and before start of cocultivation. Cocultivation of NP-2/CCR5 cells with hPBMC was started seven days after infection and virus production was measured after additional 6 days. CD4-independent-HIGH, virus production and/or syncytia induction could be detected directly in NP-2/CCR5 cells (dark grey). CD4-independent-LOW, productive infection in NP-2/CCR5 cells revealed only after cocultivation of infected NP-2/CCR5 cells with hPBMC (light grey). RT was analyzed with undiluted supernatants and therefore values above 1000 pg RT/ml cannot be separated. Detection limit for RT was 50 pg/ml. Values are means of duplicate infections

CD4-independent use of the CCR5 receptor by sequential primary SIVsm isolates-3

<p><b>Copyright information:</b></p><p>Taken from "CD4-independent use of the CCR5 receptor by sequential primary SIVsm isolates"</p><p>http://www.retrovirology.com/content/4/1/50</p><p>Retrovirology 2007;4():50-50.</p><p>Published online 23 Jul 2007</p><p>PMCID:PMC1950888.</p><p></p>ell (in 88% of the cultures) and RT production was measured in MDM 15 days after infection. Values are means from at least two experiments with MDM from different donors

CD4-independent use of the CCR5 receptor by sequential primary SIVsm isolates-4

<p><b>Copyright information:</b></p><p>Taken from "CD4-independent use of the CCR5 receptor by sequential primary SIVsm isolates"</p><p>http://www.retrovirology.com/content/4/1/50</p><p>Retrovirology 2007;4():50-50.</p><p>Published online 23 Jul 2007</p><p>PMCID:PMC1950888.</p><p></p>time in 13 macaques. Neutralization sensitivity of three isolates (A, 2-week isolates, B, 3 or 4-month isolates and C, late isolates) from each macaque was tested with 1:20 dilution of serum [30]. Neutralization was also performed with autologous serum which gave similar results (data not shown). Neutralization sensitivity was measured using the GHOST(3) cell plaque reduction assay which has a cut-off for neutralization at 30% (marked with a line), that is results below 30% are negative [67]. The majority of newly infected macaques harbored virus populations with a CD4-independent-HIGH (dark grey bars) and neutralization sensitive phenotype. This phenotype gradually changed to become a CD4-independent-LOW (light grey bars) and neutralization resistant (below 30%) virus population. CD4-dependent isolates (white bars) were seen in both neutralization sensitive and neutralization resistant populations. Values are mean neutralization (+/- SD) of two independent assays performed in triplicates

CD4-independent use of the CCR5 receptor by sequential primary SIVsm isolates-2

<p><b>Copyright information:</b></p><p>Taken from "CD4-independent use of the CCR5 receptor by sequential primary SIVsm isolates"</p><p>http://www.retrovirology.com/content/4/1/50</p><p>Retrovirology 2007;4():50-50.</p><p>Published online 23 Jul 2007</p><p>PMCID:PMC1950888.</p><p></p>he CCR5 part is represented as grey and the CXCR4 part as black. B. Mode of CCR5-use was analyzed by infection of U87.CD4 cells expressing CCR5 or chimeric receptors. Length of bars indicates degree of infection and syncytia induction as observed in a light microscope 5 and 7 days after infection. Degree of infection follows a scale from 0 to 5 where 0 is no syncytia and RT negative; 1 was 90% of the wells. RT production was positive in grades ranging from 2 to 5 and in concordance with syncytia induction

CD4-independent use of the CCR5 receptor by sequential primary SIVsm isolates-1

<p><b>Copyright information:</b></p><p>Taken from "CD4-independent use of the CCR5 receptor by sequential primary SIVsm isolates"</p><p>http://www.retrovirology.com/content/4/1/50</p><p>Retrovirology 2007;4():50-50.</p><p>Published online 23 Jul 2007</p><p>PMCID:PMC1950888.</p><p></p>ysed by 0.001% Triton X-100 followed by one cycle of freeze-thawing step. Lysates were titrated at five-fold dilution steps on hPBMC. Supernatant culture fluids from hPBMC infections were collected at day 7 and production of RT was analyzed with undiluted supernatants. The RT cut-off detection level was 50 pg/ml and values above 1000 pg/ml could not be separated. Dark grey bars represent mean virus production in NP-2/CD4/CCR5 cells and. light grey bars represent virus production in NP-2/CCR5 cells. White bars represent virus production measured by RT in PBMC infected with cell lysates diluted 1:5 from infected NP-2/CCR5 cells. Positive syncytia induction (SI) are indicated with +. Means of RT production in duplicates of infection are indicated

Evolution of human immunodeficiency virus type 2 coreceptor usage, autologous neutralization, envelope sequence and glycosylation

To investigate why human immunodeficiency virus type 2 (HIV-2) is less virulent than HIV-1, the evolution of coreceptor usage, autologous neutralization, envelope sequence and glycosylation was studied in sequentially obtained virus isolates and sera from four HIV-2-infected individuals. Neutralization of primary HIV-2 isolates was tested by a cell line-based assay and IgG purified from patients' sera. Significant autologous neutralization was observed for the majority (39 of 54) of the HIV-2 serum-virus combinations tested, indicating that neutralization escape is rare in HIV-2 infection. Furthermore, sera from 18 HIV-2 patients displayed extensive heterologous cross-neutralization when tested against a panel of six primary HIV-2 isolates. This indicates that HIV-2 is intrinsically more sensitive to antibody neutralization than HIV-1. In line with earlier reports, HIV-2 isolates could use several alternative receptors in addition to the major coreceptors CCR5 and CXCR4. Intrapatient evolution from CCR5 use to CXCR4 use was documented for the first time. Furthermore, CXCR4 use was linked to the immunological status of the patients. Thus, all CXCR4-using isolates, except one, were obtained from patients with CD4 counts below 200 cells mu l(-1). Sequence analysis revealed an association between coreceptor usage and charge of the V3 loop of the HIV-2 envelope, as well as an association between the rate of disease progression and the glycosylation pattern of the envelope protein. Furthermore, HIV-2 isolates had fewer glycosylation sites in the V3 domain than HIV-1 (two to three versus four to five). It is proposed here that HIV-2 has a more open and accessible V3 domain than HIV-1, due to differences in glycan packing, and that this may explain its broader coreceptor usage and greater sensitivity to neutralizing antibodies