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Defining the fitness of HIV-1 isolates with dual/mixed co-receptor usage.

Nankya IL, Tebit DM, Abraha A, Kyeyune F, Gibson R, Jegede O, Nickel G, Arts EJ - AIDS Res Ther (2015)

Bottom Line: To determine replicative fitness, these primary and chimeric dual tropic HIV-1 were then competed against HIV-1 reference isolates in U87.CD4 cells expressing CXCR4 or CCR5 or in PBMCs ± entry inhibitors.In the primary HIV-1 isolates (C19 and C27), subtype C dual tropic clones dominated over x4 clones while pure r5 clones were absent.The dual tropic C19 and C27 HIV-1 isolates outcompeted r5 primary HIV-1 isolates, B2 and C3 in PBMCs.

View Article: PubMed Central - PubMed

Affiliation: Division of Infectious Diseases, Department of Medicine, Case Western Reserve University, 10900 Euclid Ave., Cleveland, OH 44106 USA ; Virology Program, Department of Molecular Biology and Microbiology, Case Western Reserve University, Cleveland, OH USA ; Joint Clinical Research Centre, Kampala, Uganda.

ABSTRACT

Background: CCR5-using (r5) HIV-1 predominates during asymptomatic disease followed by occasional emergence of CXCR4-using (x4) or dual tropic (r5x4) virus. We examined the contribution of the x4 and r5 components to replicative fitness of HIV-1 isolates.

Methods: Dual tropic r5x4 viruses were predicted from average HIV-1 env sequences of two primary subtype C HIV-1 isolates (C19 and C27) and from two patient plasma samples (B12 and B19). Chimeric Env viruses with an NL4-3 backbone were constructed from the B12 and B19 env sequences. To determine replicative fitness, these primary and chimeric dual tropic HIV-1 were then competed against HIV-1 reference isolates in U87.CD4 cells expressing CXCR4 or CCR5 or in PBMCs ± entry inhibitors. Contribution of the x4 and r5 clones within the quasispecies of these chimeric or primary HIV-1 isolates were then compared to the frequency of x4, r5, and dual tropic clones within the quasispecies as predicted by phenotypic assays, clonal sequencing, and 454 deep sequencing.

Results: In the primary HIV-1 isolates (C19 and C27), subtype C dual tropic clones dominated over x4 clones while pure r5 clones were absent. In two subtype B chimeric viruses (B12 and B19), r5 clones were >100-fold more abundant than x4 or r5/x4 clones. The dual tropic C19 and C27 HIV-1 isolates outcompeted r5 primary HIV-1 isolates, B2 and C3 in PBMCs. When AMD3100 was added or when only U87.CD4.CCR5 cells were used, the B2 and C3 reference viruses now out-competed the r5 component of the dual tropic C19 and C27. In contrast, the same replicative fitness was observed with dualtropic B12 and B19 HIV-1 isolates relative to x4 HIV-1 A8 and E6 or the r5 B2 and C3 viruses, even when the r5 or x4 component was inhibited by maraviroc (or AMD3100) or in U87.CD4.CXCR4 (or CCR5) cells.

Conclusions: In the dual tropic HIV-1 isolates, the x4 replicative fitness is higher than r5 clones but the x4 or x4/r5 clones are typically at low frequency in the intrapatient virus population. Ex vivo HIV propagation promotes outgrowth of the x4 clones and provides an over-estimate of x4 dominance in replicative fitness within dual tropic viruses.

No MeSH data available.


Related in: MedlinePlus

Fitness of the dual tropic HIV-1 isolate when blocking the virus component using CCR5 or CXCR4. a The primary dual tropic HIV-1 isolates were competed against the x4 [50] titereds (all titered on U87.CD4.CXCR4 cells) in U87.CD4.CXCR4 cultures or in PHA-stimulated, IL-2 treated PBMC cultures with or without maraviroc. The fitness differences are plotted as described in Fig. 2 and in the “Methods”. b The level of maraviroc inhibition of the monoinfections or dual infections was measured by relative RT activity in the cell free supernatant and plotted as a percentage of the no drug control. c The primary dual tropic HIV-1 isolates and r5 reference strains were also titered on U87.CD4.CCR5 cells and then competed together in PHA-stimulated, IL-2 treated PBMC cultures with or without AMD3100. The same dual infection with the same MOI (0.004) was repeated in U87.CD4.CCR5 cultures. The fitness differences are plotted as described in Fig. 2 and “Methods”. d The level of AMD3100 inhibition of the monoinfections or dual infections was measured by relative RT activity in the cell free supernatant and plotted as a percentage of the no drug control. All experiments were performed in triplicate with the exception of c, d (performed in duplicate) due to limited supply of AMD3100.
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Fig4: Fitness of the dual tropic HIV-1 isolate when blocking the virus component using CCR5 or CXCR4. a The primary dual tropic HIV-1 isolates were competed against the x4 [50] titereds (all titered on U87.CD4.CXCR4 cells) in U87.CD4.CXCR4 cultures or in PHA-stimulated, IL-2 treated PBMC cultures with or without maraviroc. The fitness differences are plotted as described in Fig. 2 and in the “Methods”. b The level of maraviroc inhibition of the monoinfections or dual infections was measured by relative RT activity in the cell free supernatant and plotted as a percentage of the no drug control. c The primary dual tropic HIV-1 isolates and r5 reference strains were also titered on U87.CD4.CCR5 cells and then competed together in PHA-stimulated, IL-2 treated PBMC cultures with or without AMD3100. The same dual infection with the same MOI (0.004) was repeated in U87.CD4.CCR5 cultures. The fitness differences are plotted as described in Fig. 2 and “Methods”. d The level of AMD3100 inhibition of the monoinfections or dual infections was measured by relative RT activity in the cell free supernatant and plotted as a percentage of the no drug control. All experiments were performed in triplicate with the exception of c, d (performed in duplicate) due to limited supply of AMD3100.

Mentions: In the competitions performed in PBMC cultures with PBMC-derived titers, C19 and C27 could replicate and compete with either the r5 (B2 and C3) or the x4 (A8 and E6) viruses (Fig. 3). In Fig. 4, we performed the same competitions as in Fig. 3 but prevented replication of either the “CXCR4”-using component (panel A) or the “CCR5”-using component (panel C) of the dual tropic C19 and C27 viruses. First, the C19+A8, C19+E6, C27+A8, and C27+E6 dual infections were added in equal “x4” titers (determined on U87.CD4.CXCR4 cells) to PBMC cultures in the presence of high maraviroc (MVC) concentrations. As described in Fig. 4b, the IC99 concentration of MVC, a CCR5 antagonist [50, 51], resulted in complete inhibition of the r5 B2 and C3 viruses but no apparent inhibition of C19 and C27 in PBMCs. Lack of C19 and C27 inhibition by MVC suggests that the majority of the HIV-1 clones in these quasispecies may be dual tropic (r5/x4) and fully capable of infecting CXCR4+ cells in the PBMC cultures such that MVC does not inhibit any virus replication. The composition of the quaispecies in terms of co-receptor usage is described below (Fig. 1). Interestingly, there was no significant difference in the replicative fitness of C19 or C27 (versus control x4 A8 or E6 HIV-1) when comparing results of competitions performed in PBMCs, PBMCs + MVC, or in U87.CD4.CXCR4 cells (Fig. 4a). The latter condition would only support replication of the control x4 viruses and the “CXCR4”-using component of the dual tropic viruses. The reference viruses A8 and E6 were significantly more fit than the “CXCR4”-using component of the dual tropic C19 and C27 viruses regardless of the above conditions (Fig. 3a).Fig. 4


Defining the fitness of HIV-1 isolates with dual/mixed co-receptor usage.

Nankya IL, Tebit DM, Abraha A, Kyeyune F, Gibson R, Jegede O, Nickel G, Arts EJ - AIDS Res Ther (2015)

Fitness of the dual tropic HIV-1 isolate when blocking the virus component using CCR5 or CXCR4. a The primary dual tropic HIV-1 isolates were competed against the x4 [50] titereds (all titered on U87.CD4.CXCR4 cells) in U87.CD4.CXCR4 cultures or in PHA-stimulated, IL-2 treated PBMC cultures with or without maraviroc. The fitness differences are plotted as described in Fig. 2 and in the “Methods”. b The level of maraviroc inhibition of the monoinfections or dual infections was measured by relative RT activity in the cell free supernatant and plotted as a percentage of the no drug control. c The primary dual tropic HIV-1 isolates and r5 reference strains were also titered on U87.CD4.CCR5 cells and then competed together in PHA-stimulated, IL-2 treated PBMC cultures with or without AMD3100. The same dual infection with the same MOI (0.004) was repeated in U87.CD4.CCR5 cultures. The fitness differences are plotted as described in Fig. 2 and “Methods”. d The level of AMD3100 inhibition of the monoinfections or dual infections was measured by relative RT activity in the cell free supernatant and plotted as a percentage of the no drug control. All experiments were performed in triplicate with the exception of c, d (performed in duplicate) due to limited supply of AMD3100.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4592561&req=5

Fig4: Fitness of the dual tropic HIV-1 isolate when blocking the virus component using CCR5 or CXCR4. a The primary dual tropic HIV-1 isolates were competed against the x4 [50] titereds (all titered on U87.CD4.CXCR4 cells) in U87.CD4.CXCR4 cultures or in PHA-stimulated, IL-2 treated PBMC cultures with or without maraviroc. The fitness differences are plotted as described in Fig. 2 and in the “Methods”. b The level of maraviroc inhibition of the monoinfections or dual infections was measured by relative RT activity in the cell free supernatant and plotted as a percentage of the no drug control. c The primary dual tropic HIV-1 isolates and r5 reference strains were also titered on U87.CD4.CCR5 cells and then competed together in PHA-stimulated, IL-2 treated PBMC cultures with or without AMD3100. The same dual infection with the same MOI (0.004) was repeated in U87.CD4.CCR5 cultures. The fitness differences are plotted as described in Fig. 2 and “Methods”. d The level of AMD3100 inhibition of the monoinfections or dual infections was measured by relative RT activity in the cell free supernatant and plotted as a percentage of the no drug control. All experiments were performed in triplicate with the exception of c, d (performed in duplicate) due to limited supply of AMD3100.
Mentions: In the competitions performed in PBMC cultures with PBMC-derived titers, C19 and C27 could replicate and compete with either the r5 (B2 and C3) or the x4 (A8 and E6) viruses (Fig. 3). In Fig. 4, we performed the same competitions as in Fig. 3 but prevented replication of either the “CXCR4”-using component (panel A) or the “CCR5”-using component (panel C) of the dual tropic C19 and C27 viruses. First, the C19+A8, C19+E6, C27+A8, and C27+E6 dual infections were added in equal “x4” titers (determined on U87.CD4.CXCR4 cells) to PBMC cultures in the presence of high maraviroc (MVC) concentrations. As described in Fig. 4b, the IC99 concentration of MVC, a CCR5 antagonist [50, 51], resulted in complete inhibition of the r5 B2 and C3 viruses but no apparent inhibition of C19 and C27 in PBMCs. Lack of C19 and C27 inhibition by MVC suggests that the majority of the HIV-1 clones in these quasispecies may be dual tropic (r5/x4) and fully capable of infecting CXCR4+ cells in the PBMC cultures such that MVC does not inhibit any virus replication. The composition of the quaispecies in terms of co-receptor usage is described below (Fig. 1). Interestingly, there was no significant difference in the replicative fitness of C19 or C27 (versus control x4 A8 or E6 HIV-1) when comparing results of competitions performed in PBMCs, PBMCs + MVC, or in U87.CD4.CXCR4 cells (Fig. 4a). The latter condition would only support replication of the control x4 viruses and the “CXCR4”-using component of the dual tropic viruses. The reference viruses A8 and E6 were significantly more fit than the “CXCR4”-using component of the dual tropic C19 and C27 viruses regardless of the above conditions (Fig. 3a).Fig. 4

Bottom Line: To determine replicative fitness, these primary and chimeric dual tropic HIV-1 were then competed against HIV-1 reference isolates in U87.CD4 cells expressing CXCR4 or CCR5 or in PBMCs ± entry inhibitors.In the primary HIV-1 isolates (C19 and C27), subtype C dual tropic clones dominated over x4 clones while pure r5 clones were absent.The dual tropic C19 and C27 HIV-1 isolates outcompeted r5 primary HIV-1 isolates, B2 and C3 in PBMCs.

View Article: PubMed Central - PubMed

Affiliation: Division of Infectious Diseases, Department of Medicine, Case Western Reserve University, 10900 Euclid Ave., Cleveland, OH 44106 USA ; Virology Program, Department of Molecular Biology and Microbiology, Case Western Reserve University, Cleveland, OH USA ; Joint Clinical Research Centre, Kampala, Uganda.

ABSTRACT

Background: CCR5-using (r5) HIV-1 predominates during asymptomatic disease followed by occasional emergence of CXCR4-using (x4) or dual tropic (r5x4) virus. We examined the contribution of the x4 and r5 components to replicative fitness of HIV-1 isolates.

Methods: Dual tropic r5x4 viruses were predicted from average HIV-1 env sequences of two primary subtype C HIV-1 isolates (C19 and C27) and from two patient plasma samples (B12 and B19). Chimeric Env viruses with an NL4-3 backbone were constructed from the B12 and B19 env sequences. To determine replicative fitness, these primary and chimeric dual tropic HIV-1 were then competed against HIV-1 reference isolates in U87.CD4 cells expressing CXCR4 or CCR5 or in PBMCs ± entry inhibitors. Contribution of the x4 and r5 clones within the quasispecies of these chimeric or primary HIV-1 isolates were then compared to the frequency of x4, r5, and dual tropic clones within the quasispecies as predicted by phenotypic assays, clonal sequencing, and 454 deep sequencing.

Results: In the primary HIV-1 isolates (C19 and C27), subtype C dual tropic clones dominated over x4 clones while pure r5 clones were absent. In two subtype B chimeric viruses (B12 and B19), r5 clones were >100-fold more abundant than x4 or r5/x4 clones. The dual tropic C19 and C27 HIV-1 isolates outcompeted r5 primary HIV-1 isolates, B2 and C3 in PBMCs. When AMD3100 was added or when only U87.CD4.CCR5 cells were used, the B2 and C3 reference viruses now out-competed the r5 component of the dual tropic C19 and C27. In contrast, the same replicative fitness was observed with dualtropic B12 and B19 HIV-1 isolates relative to x4 HIV-1 A8 and E6 or the r5 B2 and C3 viruses, even when the r5 or x4 component was inhibited by maraviroc (or AMD3100) or in U87.CD4.CXCR4 (or CCR5) cells.

Conclusions: In the dual tropic HIV-1 isolates, the x4 replicative fitness is higher than r5 clones but the x4 or x4/r5 clones are typically at low frequency in the intrapatient virus population. Ex vivo HIV propagation promotes outgrowth of the x4 clones and provides an over-estimate of x4 dominance in replicative fitness within dual tropic viruses.

No MeSH data available.


Related in: MedlinePlus