Adoption of an “Open” Envelope Conformation Facilitating CD4 Binding and Structural Remodeling Precedes Coreceptor Switch in R5 SHIV-Infected Macaques

A change in coreceptor preference from CCR5 to CXCR4 towards the end stage disease in some HIV-1 infected individuals has been well documented, but the reasons and mechanisms for this tropism switch remain elusive. It has been suggested that envelope structural constraints in accommodating amino acid changes required for CXCR4 usage is an obstacle to tropism switch, limiting the rate and pathways available for HIV-1 coreceptor switching. The present study was initiated in two R5 SHIVSF162P3N-infected rapid progressor macaques with coreceptor switch to test the hypothesis that an early step in the evolution of tropism switch is the adoption of a less constrained and more “open” envelope conformation for better CD4 usage, allowing greater structural flexibility to accommodate further mutational changes that confer CXCR4 utilization. We show that, prior to the time of coreceptor switch, R5 viruses in both macaques evolved to become increasingly sCD4-sensitive, suggestive of enhanced exposure of the CD4 binding site and an “open” envelope conformation, and this correlated with better gp120 binding to CD4 and with more efficient infection of CD4low cells such as primary macrophages. Moreover, significant changes in neutralization sensitivity to agents and antibodies directed against functional domains of gp120 and gp41 were seen for R5 viruses close to the time of X4 emergence, consistent with global changes in envelope configuration and structural plasticity. These observations in a simian model of R5-to-X4 evolution provide a mechanistic basis for the HIV-1 coreceptor switch

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▿

Fitness disadvantage of the transitional intermediates compared to the initial R5 viruses has been suggested to constitute one of the blockades to coreceptor switching, explaining the late appearance of X4 viruses. Using a simian model for human immunodeficiency virus type 1 (HIV-1) coreceptor switching, we demonstrate in this study that similar molecular evolutionary pathways to coreceptor switch occur in more than one R5 simian/human immunodeficiency virus (SHIV)SF162P3N-infected macaque. In infected animals where multiple pathways for expansion or switch to CXCR4 coexist, fitness of the transitional intermediates in coreceptor usage efficiency influences their outgrowth and representation in the infecting virus population. Dualtropic and X4 viruses appear at different disease stages, but they have lower entry efficiency than the coexisting R5 strains, which may explain why they do not outcompete the R5 viruses. Similar observations were made in two infected macaques with coreceptor switch, providing in vivo evidence that fitness disadvantage is an obstacle to X4 emergence and expansion

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

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

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