A Novel CCR5 Mutation in Sooty Mangabeys Reveals SIVsmm Infection of CCR5-null Natural Hosts: Examining the Potential Roles of Alternative Entry Pathways in HIV and SIV Infection

Natural hosts of SIV, such as sooty mangabeys (SM), maintain high levels of virus replication, but do not typically develop CD4+ T cell loss and immunodeficiency. Understanding the virus/host relationship in natural hosts will enable better understanding of pathogenic HIV infection of humans. Host cell targeting in vivo is an important determinant of pathogenesis, and is defined mainly by expression of coreceptors used by the virus for entry, in conjunction with CD4. Established dogma holds that, with rare exceptions, SIV uses CCR5 for entry. However, SM and other natural hosts express extremely low CCR5 levels on CD4+ T cells. I identified a novel SM-CCR5 mutant allele containing a two base-pair deletion (CCR5-d2) that results in a non-functional protein. Using PCR screening and direct sequence confirmation in a large captive SM colony, I found an allelic frequency of 26% for CCR5-d2, along with 3% for a previously-described CCR5-d24 null allele. Notably, SM-CCR5-d2 was also present in West African wild-living SM. Approximately 8% of captive SM were homozygous for CCR5-null alleles. Surprisingly, SIVsmm infection was not significantly less prevalent in SM lacking functional CCR5 compared to CCR5-expressing animals, and CCR5-null animals displayed high-level viremia. Primary PBMC from SM were permissive for SIVsmm replication in both the absence and presence of CCR5 in vitro. Pseudotype virions carrying Envs from both CCR5-null and wild-type infected SM used CXCR6 and GPR15 efficiently for entry in transfected cells, suggesting likely alternative entry molecules. In preliminary studies using human cells, I found that CD4+ T cell expression of CXCR6 and GPR15 was predominantly on central and effector memory subsets, although expression patterns on SM cells will require antibodies that can detect SM proteins. This finding challenges the long-standing notion that CCR5 is the exclusive coreceptor used for SIV entry in vivo, and reveals that SIVsmm can infect target cells in vivo using non-CCR5 entry pathways. Since CD4/coreceptor interactions determine viral tropism and cell targeting, these results suggest that infection in natural host SM may involve target cells defined by the distribution and use of alternative coreceptors CXCR6 and GPR15. Combined with restricted expression of CCR5 on critical target cells previously described, alternative coreceptor-dependent targeting may allow virus replication in more expendable cells without loss of immune cell homeostasis. We hypothesize that SM natural hosts may have evolved to restrict expression of CXCR6 and GPR15, in addition to CCR5, to dispensable cell subsets, thus allowing for high viral replication without causing disease. Future studies comparing CXCR6 and GPR15 expression profiles in natural and non-natural hosts will be of importance in determining the role of alternative coreceptors in natural hosts in vivo

A Novel CCR5 Mutation Common in Sooty Mangabeys Reveals SIVsmm Infection of CCR5-Null Natural Hosts and Efficient Alternative Coreceptor Use In Vivo

In contrast to HIV infection in humans and SIV in macaques, SIV infection of natural hosts including sooty mangabeys (SM) is non-pathogenic despite robust virus replication. We identified a novel SM CCR5 allele containing a two base pair deletion (Δ2) encoding a truncated molecule that is not expressed on the cell surface and does not support SIV entry in vitro. The allele was present at a 26% frequency in a large SM colony, along with 3% for a CCR5Δ24 deletion allele that also abrogates surface expression. Overall, 8% of animals were homozygous for defective CCR5 alleles and 41% were heterozygous. The mutant allele was also present in wild SM in West Africa. CD8+ and CD4+ T cells displayed a gradient of CCR5 expression across genotype groups, which was highly significant for CD8+ cells. Remarkably, the prevalence of natural SIVsmm infection was not significantly different in animals lacking functional CCR5 compared to heterozygous and homozygous wild-type animals. Furthermore, animals lacking functional CCR5 had robust plasma viral loads, which were only modestly lower than wild-type animals. SIVsmm primary isolates infected both homozygous mutant and wild-type PBMC in a CCR5-independent manner in vitro, and Envs from both CCR5-null and wild-type infected animals used CXCR6, GPR15 and GPR1 in addition to CCR5 in transfected cells. These data clearly indicate that SIVsmm relies on CCR5-independent entry pathways in SM that are homozygous for defective CCR5 alleles and, while the extent of alternative coreceptor use in SM with CCR5 wild type alleles is uncertain, strongly suggest that SIVsmm tropism and host cell targeting in vivo is defined by the distribution and use of alternative entry pathways in addition to CCR5. SIVsmm entry through alternative pathways in vivo raises the possibility of novel CCR5-negative target cells that may be more expendable than CCR5+ cells and enable the virus to replicate efficiently without causing disease in the face of extremely restricted CCR5 expression seen in SM and several other natural host species

A SIV molecular clone that targets the CNS and induces neuroAIDS in rhesus macaques.

Despite effective control of plasma viremia with the use of combination antiretroviral therapies (cART), minor cognitive and motor disorders (MCMD) persist as a significant clinical problem in HIV-infected patients. Non-human primate models are therefore required to study mechanisms of disease progression in the central nervous system (CNS). We isolated a strain of simian immunodeficiency virus (SIV), SIVsm804E, which induces neuroAIDS in a high proportion of rhesus macaques and identified enhanced antagonism of the host innate factor BST-2 as an important factor in the macrophage tropism and initial neuro-invasion of this isolate. In the present study, we further developed this model by deriving a molecular clone SIVsm804E-CL757 (CL757). This clone induced neurological disorders in high frequencies but without rapid disease progression and thus is more reflective of the tempo of neuroAIDS in HIV-infection. NeuroAIDS was also induced in macaques co-inoculated with CL757 and the parental AIDS-inducing, but non-neurovirulent SIVsmE543-3 (E543-3). Molecular analysis of macaques infected with CL757 revealed compartmentalization of virus populations between the CNS and the periphery. CL757 exclusively targeted the CNS whereas E543-3 was restricted to the periphery consistent with a role for viral determinants in the mechanisms of neuroinvasion. CL757 would be a useful model to investigate disease progression in the CNS and as a model to study virus reservoirs in the CNS

Clinical and pathologic endpoints in macaques inoculated with CL757.

<p>(A.) The cumulative survival of macaques infected with CL757 (Red) and E543-3 (Black) are shown as Kaplan Meier curves and compared by Log-Rank test (P = 0.0032). Asterisks below the graph indicate the death of animals with SIVE. B. SIV specific <i>in situ</i> hybridization of the brain parenchyma of a representative animal with SIVE (H880) showing characteristic glial nodules containing multinucleated giant cells (indicated by arrows). Dark blue pigment (NBT) indicate the presence of viral RNA associated with lesions.</p

Phylogenetic analysis of envelope sequences amplified from the brain, CSF, plasma and axillary lymph node of animals infected with CL757 with sequences from all three animals combined in a radial tree demonstrates that viruses in the CSF/Brain from individual macaques were more closely related to one another than to the autologous plasma/lymph node sequences.

<p>Phylogenetic analysis of envelope sequences amplified from the brain, CSF, plasma and axillary lymph node of animals infected with CL757 with sequences from all three animals combined in a radial tree demonstrates that viruses in the CSF/Brain from individual macaques were more closely related to one another than to the autologous plasma/lymph node sequences.</p

Replication of CL757 in rhesus macaques <i>in vivo</i>.

<p>(A) Plasma and (B) CSF viral RNA loads, and circulating (C) CD4+ T cells and (D) memory CD4+ T cells, of animals infected with CL757. Red symbols indicate animals that developed SIVE, and black symbols indicate animals that died with AIDS but without SIVE.</p

Clinical, virologic and neurological outcome in macaques inoculated with CL757.

<p>Clinical, virologic and neurological outcome in macaques inoculated with CL757.</p

Compartmentalization of viruses in the CNS of animals inoculated with CL757 that developed SIVE.

<p>Phylogenetic analysis of envelope sequences amplified from the brain, CSF, plasma and axillary lymph node of animals (A) H880, (B) H882, (C) H886.</p

<i>In vivo</i> replication in animals co-inoculated with SIVsm804E-CL757 and SIVsmE543-3.

<p>(A) Plasma and (B) CSF viral RNA loads, and circulating (C) CD4+ T cells and (D) memory CD4+ T cells, of animals infected with SIVsm804E-CL757.</p