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. 2012 Dec 13:9:106.
doi: 10.1186/1742-4690-9-106.

Identification of interdependent variables that influence coreceptor switch in R5 SHIV(SF162P3N)-infected macaques

Ke Zhuang  1 , Andres Finzi, Jonathan Toma, Arne Frantzell, Wei Huang, Joseph Sodroski, Cecilia Cheng-Mayer
Affiliations

Identification of interdependent variables that influence coreceptor switch in R5 SHIV(SF162P3N)-infected macaques

Ke Zhuang et al. Retrovirology. .

Abstract

Background: We previously reported that adoption of an "open" envelope glycoprotein (Env) to expose the CD4 binding site for efficient receptor binding and infection of cell targets such as macrophages that express low levels of the receptor represents an early event in the process of coreceptor switch in two rapidly progressing (RP) R5 SHIV(SF162P3N)-infected rhesus macaques, releasing or reducing Env structural constraints that have been suggested to limit the pathways available for a change in coreceptor preference. Here we extended these studies to two additional RP monkeys with coreceptor switch and three without to confirm and identify additional factors that facilitated the process of phenotypic conversion.

Results: We found that regardless of coreceptor switching, R5 viruses in SHIV(SF162P3N)-infected RP macaques evolved over time to infect macrophages more efficiently; this was accompanied by increased sCD4 sensitivity, with structural changes in the CD4 binding site, the V3 loop and/or the fusion domain of their Envs that are suggestive of better CD4 contact, CCR5 usage and/or virus fusion. However, sCD4-sensitive variants with improved CD4 binding were observed only in RPs with coreceptor switch. Furthermore, cumulative viral load was higher in RPs with than in those without phenotypic switch, with the latter maintaining a longer period of seroconversion.

Conclusions: Our data suggest that the increased virus replication in the RPs with R5-to-X4 conversion increased the rate of virus evolution and reduction in the availability of target cells with optimal CD4 expression heightened the competition for binding to the receptor. In the absence of immunological restrictions, variants that adopt an "open" Env to expose the CD4 binding site for better CD4 use are selected, allowing structural changes that confer CXCR4-use to be manifested. Viral load, change in target cell population during the course of infection and host immune response therefore are interdependent variables that influence R5 virus evolution and coreceptor switch in SHIV(SF162P3N)-infected rhesus macaques. Because an "open" Env conformation also renders the virus more susceptible to antibody neutralization, our findings help to explain the infrequent and late appearance of X4 virus in HIV-1 infection when the immune system deteriorates.

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Figures

Figure 1
Figure 1
Viral load and CD4+ T cell count in SHIVSF162P3N–infected macaques with (A) and without (B) coreceptor switch. (A) Macaque DE86 was infected by the intravenous route and DG08 was inoculated intrarectally. Dashed line designates the time of dual-tropic and X4 virus emergence in these animals. (B) Macaques BT78, CC39 were infected intravenously while DN57 was challenged intrarectally. Time to euthanasia for the five RPs is: w12 (DE86), w20 (DG08), w13 (BT78), w12 (CC39) and w30 (DN57).
Figure 2
Figure 2
Entry efficiency, PSC-RANTES and sCD4 sensitivity of R5 viruses evolving over time in DE86 and DG08. Entry of luciferase reporter viruses expressing CCR5-using envelopes into TZM-bl cells expressed as relative light unit (RLU) (A), and susceptibility of the reporter viruses to neutralization with PSC-RANTES (B) and sCD4 (C) were determined. The dashed vertical line indicates time of tropism switch in DE86 (12 wpi), and DG08 (13 wpi). The numbers in the brackets indicate the number of clones analyzed at each time point. Absolute CD4+ T cell count in the animal over the course of infection is shown in (C) for reference and values above the bars indicate fold increase in sCD4 sensitivity relative to that of the w2 viruses. * P<0.05 (Mann-Whitney U test). Data are representative of 2-3 independent experiments (error bars, s.d.).
Figure 3
Figure 3
(A) Relationship between sCD4 sensitivity, CD4-Ig binding and infection of primary macrophages (mΦ) of DE86 and DG08 viruses. Values above the bars indicate fold increase in sCD4 sensitivity of evolving viruses compared to early viruses, and the vertical dashed line indicates the time of coreceptor switching. sgp120 binding to CD4-Ig was normalized to that of sgp120 binding to polyclonal serum from HIV-1 infected individuals. Infectivity in mΦ that express low levels of CD4 was expressed as a ratio of infectivity in autologous PBMCs that express high levels of CD4 and CCR5. The shaded area highlights the time prior and during coreceptor switch. For sgp120 CD4-Ig binding, data are the means and standard deviations from at least two independent experiments. For infection of macrophages, data are representative of at least 3 independent experiments (error bars, s.d.). * indicates statistically significant differences between the early and the evolving R5 viruses. (B) Changes in neutralization sensitivity of R5 viruses evolving over time in macaques DE86 and DG08. Susceptibility of R5 pseudoviruses to neutralization with IgG1b12, 447-52D and T20 was determined. The vertical dashed line indicates the time of coreceptor switching, and the shaded area designates the period of marked envelope conformational changes. Data are representative of at least two independent experiments (error bars, s.d.). * above the bars indicate IC50 values that are statistically different between the acute and the evolving R5 viruses, P<0.05 (Mann-Whitney U test).
Figure 4
Figure 4
Entry efficiency, PSC-RANTES and sCD4 sensitivity of R5 viruses evolving over time in BT78, CC39 and DN57. Entry of luciferase reporter viruses expressing CCR5-using Envs into TZM-bl cells expressed as relative light unit (RLU) (A), and susceptibility of the reporter viruses to neutralization with PSC-RANTES (B) and sCD4 (C) were determined. The numbers in the brackets indicate the number of clones analyzed at each time point and values above the bars indicate fold increase in sCD4 sensitivity relative to that of the w2 viruses. * P<0.05 (Mann-Whitney U test). Data are representative of 2-3 independent experiments (error bars, s.d.).
Figure 5
Figure 5
Enhanced macrophage infection accompanied by Env structural changes in R5 viruses evolving over time in macaques BT78, CC39 and DN57. The relationship between sCD4 sensitivity, binding of sgp120 to CD4-Ig, infectivity of primary macrophages (mΦ) (A), and neutralization susceptibility (B) of pseudoviruses bearing CCR5-using Envs amplified over time from BT78, CC39 and DN57 is shown. Data are representative of at least two independent experiments (error bars, s.d.). * above bars indicate differences in sCD4 sensitivity, CD4-Ig binding and susceptibility to agents and antibodies between the acute (w2) and the evolving R5 viruses that are statistically significant, * P<0.05 (Mann-Whitney U test).
Figure 6
Figure 6
Comparison of cumulative viral load in R5 SHIVSF162P3N-infected RPs with (n=4) and without (n=3) coreceptor switch. Cumulative log10 RNA copies/ml of plasma viremia up to the time of euthanasia were compared using unpaired t tests. Time to euthanasia for the four RPs with coreceptor switch is: w24 (BR24), w15 (CA28), w12 (DE86), w20 (DG08), and for the three RPs without coreceptor switch is: w13 (BT78), w12 (CC39) and w30 (DN57). P value of <0.05 was considered significant.
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