Changes in neutralization sensitivity of R5 viruses evolving over time in macaque BR24.

<p>Susceptibility of BR24 R5 pseudoviruses to neutralization with b12, 447-52D and T20 was determined, with sensitivity of variants from the inoculating virus SHIV_SF162P3N (P3N) shown for reference. The vertical dashed line indicates the time of coreceptor switching, and the dotted 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 (w2) and the evolving R5 viruses.</p

Relationship between sCD4 sensitivity, CD4-Ig binding, infection of CD4^low cells and sCD4-induced gp120 release of BR24 viruses.

<p>(A) The relationship between sgp120 binding to CD4-Ig, sCD4 sensitivity, infection of RC49 cells and primary macrophages (mΦ) of BR24 dervied viruses is illustrated. Values above the bars indicate fold increase in sCD4 sensitivity of BR24 viruses compared to viruses in the SHIV_SF162P3N inoculum (P3N). (B) Extent of sCD4-induced gp120 from surface of 293T cells transiently expressing BR24-derived envelope glycoproteins. Percentage difference in gp120 release in the presence of sCD4 relative to that in the absence of sCD4 is shown. The data are the means and standard deviations of two independent experiments. The vertical dashed line in (A) and (B) indicates the time of coreceptor switching, and the dotted area highlights the time when the relationship between sCD4 sensitivity, sgp120 binding to CD4-Ig and infection of CD4^low cells dissipates.</p

Entry efficiency, PSC-RANTES and sCD4 sensitivity of R5 viruses evolving over time in CA28.

<p>Entry of luciferase reporter viruses expressing CCR5-using envelopes into TZM-bl cells (A), and susceptibility of the reporter viruses to neutralization with PSC-RANTES (B) and sCD4 (C) were determined. The solid and dashed vertical lines indicate the two switch events in CA28 leading to the emergence of distinct dual-tropic and X4 viruses, respectively. The numbers in the brackets denote the number of envelope clones analyzed at each time point. Absolute CD4+ T-cell count in the animal over the course of infection is shown in (C), and values above the bars indicate fold increase in sCD4 sensitivity of CA28 viruses compared to viruses in the SHIV_SF162P3N inoculum (P3N). *<i>P</i><0.05 (Mann-Whitney <i>U</i> test). Data are representative of at least three independent experiments (error bars, s.d.).</p

Entry efficiency, PSC-RANTES and sCD4 sensitivity of R5 viruses evolving over time in BR24.

<p>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 BR24 (20 wpi), and the numbers in the brackets indicate the number of clones analyzed at each time point. Envelope clones from the SHIV_SF162P3N inoculum (P3N) were also included in the characterization for comparison. 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 <i>U</i> test). Data are representative of 2–3 independent experiments (error bars, s.d.).</p

SHIV-infected macrophages identified with double-label SIVnef and Iba-1 immunohistochemistry.

<p>Tissue macrophages are the primary SHIV infected cells at end stage disease in BR24 (A) and CA28 (B). Double-labeled immunohistochemical staining for SIVnef (brown) and the macrophage marker lba-1 (red) was performed. Arrows mark representative double-positive cells.</p

sgp120 CD4-Ig binding and infection of CD4^low cells with BR24 viruses.

<p>The binding of sgp120 to CD4-Ig together with the fold-increase in sCD4 sensitivity (A), infectivity of HeLa RC49 cells (B) and primary macrophages (mΦ; C) that express low levels of CD4 with pseudotyped viruses bearing CCR5-using Envs amplified over time from BR24 were determined. Properties of four envelope clones in the SHIV_SF162P3N inoculum (P3N) were also determined and shown for reference. sgp120 binding to CD4-Ig (A) was normalized to that of sgp120 binding to polyclonal serum from HIV-1 infected individuals. Infectivity in RC49 cells (B) and macrophages (C) that express low levels of CD4 was expressed as a ratio of infectivity in JC53 cells and autologous PBMCs that express high levels of CD4 and CCR5, respectively. The dashed vertical line indicates time of tropism switch. For sgp120 CD4-Ig binding, data are the means and standard deviations from at least two independent experiments. For infection of CD4^low cells, data are representative of at least 3 independent experiments (error bars, s.d.). * above the bars indicates normalized CD4-Ig binding and CD4^low cell infectivity ratios that are statistically different between the acute (w2) and the evolving R5 viruses.</p