Tracking the Emergence of Host-Specific Simian Immunodeficiency Virus env and nef Populations Reveals nef Early Adaptation and Convergent Evolution in Brain of Naturally Progressing Rhesus Macaques

Abstract

While a clear understanding of the events leading to successful establishment of host-specific viral populations and productive infection in the central nervous system (CNS) has not yet been reached, the simian immunodeficiency virus (SIV)-infected rhesus macaque provides a powerful model for the study of human immunodeficiency virus (HIV) intrahost evolution and neuropathogenesis. The evolution of the gp120 and nef genes, which encode two key proteins required for the establishment and maintenance of infection, was assessed in macaques that were intravenously inoculated with the same viral swarm and allowed to naturally progress to simian AIDS and potential SIV-associated encephalitis (SIVE). Longitudinal plasma samples and immune markers were monitored until terminal illness. Single-genome sequencing was employed to amplify full-length env through nef transcripts from plasma over time and from brain tissues at necropsy. nef sequences diverged from the founder virus faster than gp120 diverged. Host-specific sequence populations were detected in nef (ϳ92 days) before they were detected in gp120 (ϳ182 days). At necropsy, similar brain nef sequences were found in different macaques, indicating convergent evolution, while gp120 brain sequences remained largely host specific. Molecular clock and selection analyses showed weaker clock-like behavior and stronger selection pressure in nef than in gp120, with the strongest nef selection in the macaque with SIVE. Rapid nef diversification, occurring prior to gp120 diversification, indicates that early adaptation of nef in the new host is essential for successful infection. Moreover, the convergent evolution of nef sequences in the CNS suggests a significant role for nef in establishing neurotropic strains. IMPORTANCEThe SIV-infected rhesus macaque model closely resembles HIV-1 immunopathogenesis, neuropathogenesis, and disease progression in humans. Macaques were intravenously infected with identical viral swarms to investigate evolutionary patterns in the gp120 and nef genes leading to the emergence of host-specific viral populations and potentially linked to disease progression. Although each macaque exhibited unique immune profiles, macaque-specific nef sequences evolving under selection were consistently detected in plasma samples at 3 months postinfection, significantly earlier than in gp120 macaque-specific sequences. On the other hand, nef sequences in brain tissues, collected at necropsy of two animals with detectable infection in the central nervous system (CNS), revealed convergent evolution. The results not only indicate that early adaptation of nef in the new host may be essential for successful infection, but also suggest that specific nef variants may be required for SIV to efficiently invade CNS macrophages and/or enhance macrophage migration, resulting in HIV neuropathology. Aclear understanding of the events leading to the establishment of human immunodeficiency virus type 1 (HIV-1) infection in a new host, including the central nervous system (C...

Evolution of Neuroadaptation in the Periphery and Purifying Selection in the Brain Contribute to Compartmentalization of Simian Immunodeficiency Virus (SIV) in the Brains of Rhesus Macaques with SIV-Associated Encephalitis

“…Procedures on macaques with an intact immune system (Mac251-NP), which were allowed to progress naturally to simian AIDS (SAIDS), were conducted according to the standards of the American Association for Accreditation of Laboratory Animal Care and IACUC protocol 04802, and treatment of these animals was in accordance with the Guide for the Care and Use of Laboratory Animals (25). Further detailed information on the handling and supervisional guidelines for these animals can be found in Lamers et al (26). Plasma viral loads were monitored as previously described by quantitative PCR (qPCR) methods targeting a conserved sequence in gag (27,28).…”

“…Sequence alignment was performed as previously described (26). Briefly, following sequence assembly in Geneious R7 (available from BioMatters, Auckland, New Zealand), sequences (SIVmac239 coordinates 6706 to 8049) were aligned using the Clustal X algorithm (37) implemented in BioEdit (38; available from http://www.mbio .ncsu.edu/bioedit/bioedit.html); the alignment was further modified by a manual optimization protocol taking into account conserved glycosylation motifs (39).…”

“…Viral RNA isolated from both the inoculating viral swarm and plasma samples were reverse transcribed using the provided oligo(dT) 20 primer. Total RNA from peripheral CD3 ϩ and CD14 ϩ cells, BAL fluid, and bone marrow aspirate samples was reverse transcribed using the KOUTR primer (5=-TGTAATAAATCCCTTC CAGTCCCCCC-3=) (9479 to 9454 bp of SIVmac239) (33), whereas total RNA from brain tissue and meninges was reverse transcribed using the cULTR primer (5=-ATGGCAGCTTTATTGAAGAGG-3=) (10125 to 10145 bp of SIVmac239) (26), a primer that binds in the SIV 3= long terminal repeat (3= LTR) and 5= LTR regions. A modified cDNA synthesis protocol was followed for the inoculating viral swarm, plasma, and brain tissue RNA to maximize the length of the cDNA (26).…”

“…Total RNA from peripheral CD3 ϩ and CD14 ϩ cells, BAL fluid, and bone marrow aspirate samples was reverse transcribed using the KOUTR primer (5=-TGTAATAAATCCCTTC CAGTCCCCCC-3=) (9479 to 9454 bp of SIVmac239) (33), whereas total RNA from brain tissue and meninges was reverse transcribed using the cULTR primer (5=-ATGGCAGCTTTATTGAAGAGG-3=) (10125 to 10145 bp of SIVmac239) (26), a primer that binds in the SIV 3= long terminal repeat (3= LTR) and 5= LTR regions. A modified cDNA synthesis protocol was followed for the inoculating viral swarm, plasma, and brain tissue RNA to maximize the length of the cDNA (26). Total RNA from the remaining tissues/cell populations was reverse transcribed according to the manufacturer's recommendations as follows: RNA was incubated at 65°C for 5 min with deoxynucleoside triphosphates (dNTPs) (0.5 mM [each]) and 5 M KOUTR and then cooled quickly to 4°C.…”

Insights into the Impact of CD8+Immune Modulation on Human Immunodeficiency Virus Evolutionary Dynamics in Distinct Anatomical Compartments by Using Simian Immunodeficiency Virus-Infected Macaque Models of AIDS Progression

“…Procedures involving the Mac251-NP animals, which were housed at the New England Primate Research Center, were conducted according to the standards of the American Association for Accreditation of Laboratory Animal Care and IACUC protocol 04802, and treatment of all animals was in accordance with the Guide for the Care and Use of Laboratory Animals (60). Further detailed information on the handling and supervisory guidelines for the Mac251-NP cohort was published previously (61). All possible measures were taken to minimize discomfort of the animals, and the guidelines for humane euthanasia of rhesus macaques were followed.…”