Simian immunodeficiency virus SIVrcm, a unique CCR2-tropic virus, selectively depletes memory CD4+ T cells in pigtailed macaques through expanded coreceptor usage in vivo

Abstract

Simian immunodeficiency virus SIVrcm, which naturally infects red-capped mangabeys (RCMs), is the only SIV that uses CCR2 as its main coreceptor due to the high frequency of a CCR5 deletion in RCMs. We investigated the dynamics of SIVrcm infection to identify specific pathogenic mechanisms associated with this major difference in SIV biology. Four pigtailed macaques (PTMs) were infected with SIVrcm, and infection was monitored for over 2 years. The dynamics of in vivo SIVrcm replication in PTMs was similar to that of other pathogenic and nonpathogenic lymphotropic SIVs. Plasma viral loads (VLs) peaked at 10(7) to 10(9) SIVrcm RNA copies/ml by day 10 postinoculation (p.i.). A viral set point was established by day 42 p.i. at 10(3) to 10(5) SIVrcm RNA copies/ml and lasted up to day 180 p.i., when plasma VLs decreased below the threshold of detection, with blips of viral replication during the follow-up. Intestinal SIVrcm replication paralleled that of plasma VLs. Up to 80% of the CD4(+) T cells were depleted by day 28 p.i. in the gut. The most significant depletion (>90%) involved memory CD4(+) T cells. Partial CD4(+) T-cell restoration was observed in the intestine at later time points. Effector memory CD4(+) T cells were the least restored. SIVrcm strains isolated from acutely infected PTMs used CCR2 coreceptor, as reported, but expansion of coreceptor usage to CCR4 was also observed. Selective depletion of effector memory CD4(+) T cells is in contrast with predicted in vitro tropism of SIVrcm for macrophages and is probably due to expansion of coreceptor usage. Taken together, these findings emphasize the importance of understanding the selective forces driving viral adaptation to a new host.

FIG. 1.

Flow-cytometric identification of CCR2 expression on blood and intestine of pigtailed macaques. CCR2 expression is restricted to CD14⁺ monocytes in blood, with the T-cell population (CD3⁺) and CD4⁺ T cells (gated on CD3⁺) in periphery (a) and intestine (b) expressing low levels of CCR2.

FIG. 2.

Dynamics of anti-SIVrcm gp41 binding antibodies by SIVrcm-specific ELISA. SIVrcm-infected PTMs seroconverted between days 21 and 42 p.i. Transmembrane ELISA results are presented as crude OD values. Cutoff was arbitrarily established at 0.15. Animal EC01, •; BU62, ▾; EC23, ▪; EC25, ♦.

FIG. 3.

Dynamics of SIVrcm replication and burden in SIVrcm-infected PTMs. Viral replication was assessed by quantifying SIVrcm RNA loads in plasma (a), PBMCs (b), intestine (c), and LNs (d). The dynamics of proviral DNA burden was quantified in PBMCs (e). Plasma viral loads are expressed as SIVrcm RNA copies/ml of plasma; detection limit of the assay is 10² copies/ml. SIVrcm RNA/proviral DNA levels in tissues are expressed per 10⁶ cells with a detection limit of 10 copies. Animal EC01, •; BU62, ▾; EC23, ▪; EC25, ♦.

FIG. 4.

Impact of SIVrcm replication on major blood populations in PTMs. No change in CCR2⁺ monocyte population in periphery during acute or chronic SIVrcm infection of PTMs (a). Significant declines in CD4⁺ T cells in periphery (b), intestine (c), and LNs (d) were observed after the peak of viremia. Partial restoration of CD4⁺ T cells occurred at later time points. CD4⁺ T cells never reached preinfection levels. Expansion of CD8⁺ T cells was observed during chronic SIVrcm infection (e). Animal EC01, •; BU62, ▾; EC23, ▪; EC25, ♦.

FIG. 5.

Immunohistochemical assessment of CD4⁺ T-cell depletion during SIVrcm infection of pigtailed macaques. Massive CD4⁺ T-cell depletion compared to baseline levels was observed after acute SIVrcm infection, followed by partial restoration during chronic infection. D, day.

FIG. 6.

Changes in CD4⁺ CCR5⁺ T cells in response to SIVrcm infection. Modest declines of CD4⁺ CCR5⁺ T cells in periphery (a) and a near total loss of CD4⁺ CCR5⁺ T cells in intestine (b) by day 28 p.i. Minimal restoration was noticed in intestine during the course of infection. Animal EC01, •; BU62, ▾; EC23, ▪; EC25, ♦.

FIG. 7.

Dynamics of CD4⁺ T-cell subsets in periphery and intestine during SIVrcm infection. Significant depletion with partial restoration of naïve CD4⁺ T cells (CD28⁺ CD95⁻) (a and b) was observed in periphery and intestine. Memory CD4⁺ T cells (CD28⁻ CD95⁺) showed massive depletion (c to f), with the least restoration being observed for the effector memory CD4⁺ T-cell population in periphery as well as intestine of SIVrcm-infected PTMs (e and f). Animal EC01, •; BU62, ▾; EC23, ▪; EC25, ♦.

FIG. 8.

Changes in the expression levels of CCR2 mRNA in PBMCs during SIVrcm infection of PTMs, as assessed by quantitative real-time PCR (a). The gene expression is shown as the absolute number of CCR2 mRNA copies per 10⁶ cells. Quantification of SIVrcm RNA levels as well as that of proviral DNA showed that SIVrcm preferentially replicates in CD4⁺ T cells and not in monocytes (b).

FIG. 9.

Coreceptor usage of SIVrcm isolated from acutely infected PTMs. Coreceptor usage of SIVrcm involves CCR2, as previously reported, but expanded to CCR4 in all four infected PTMs. SIVmac239 served as a control. Ag, antigen.

FIG. 10.

Comparison of CCR4 expression on CD14⁺ monocytes and CD4⁺ T cells in blood from an uninfected PTM. CCR4 expression was significantly lower in CD14⁺ monocytes than in CD4⁺ T cells. Naïve CD4⁺ T cells (CD28⁺ CD95⁻) showed lower CCR4 expression while memory (CD95⁺) cells expressed significantly higher levels of CCR4 on their surfaces.

FIG. 11.

Alignment of envelope protein sequences from multiple clones of SIVrcm isolated from all four infected PTMs at different time points postinfection. PCR-derived env nucleotide sequences were translated and aligned with previously reported SIVrcm (NCBI database reference sequence AF382829). V1/V5 regions showed distinct changes in clones derived from infected PTMs compared to parental SIVrcm.