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. 2010 Dec;84(23):12245-54.
doi: 10.1128/JVI.01603-10. Epub 2010 Sep 29.

Genetic Identity and Biological Phenotype of a Transmitted/Founder Virus Representative of Nonpathogenic Simian Immunodeficiency Virus Infection in African Green Monkeys

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Genetic Identity and Biological Phenotype of a Transmitted/Founder Virus Representative of Nonpathogenic Simian Immunodeficiency Virus Infection in African Green Monkeys

Clement Wesley Gnanadurai et al. J Virol. .
Free PMC article

Abstract

Understanding the lack of disease progression in nonpathogenic simian immunodeficiency virus (SIV) infections is essential for deciphering the immunopathogenesis of human AIDS. Yet, in vivo studies have been hampered by a paucity of infectious molecular clones (IMCs) of SIV suitable to dissect the viral and host factors responsible for the nonpathogenic phenotype. Here, we describe the identification, cloning, and biological analysis of the first transmitted/founder (T/F) virus representing a nonpathogenic SIV infection. Blood was collected at peak viremia from an acutely infected sabaeus monkey (Chlorocebus sabaeus) inoculated intravenously with an African green monkey SIV (SIVagm) strain (Sab92018) that had never been propagated in vitro. To generate IMCs, we first used conventional (bulk) PCR to amplify full-length viral genomes from peripheral blood mononuclear cell (PBMC) DNA. Although this yielded two intact SIVagmSab genomes, biological characterization revealed that both were replication defective. We then performed single-genome amplification (SGA) to generate partially overlapping 5' (n = 10) and 3' (n = 13) half genomes from plasma viral RNA. Analysis of these amplicons revealed clusters of nearly identical viral sequences representing the progeny of T/F viruses. Synthesis of the consensus sequence of one of these generated an IMC (Sab92018ivTF) that produced infectious CCR5-tropic virions and replicated to high titers in Molt-4 clone 8 cells and African green monkey PBMCs. Sab92018ivTF also initiated productive infection in sabaeus monkeys and faithfully recapitulated the replication kinetics and nonpathogenic phenotype of the parental Sab92018 strain. These results thus extend the T/F virus concept to nonpathogenic SIV infections and provide an important new tool to define viral determinants of disease nonprogression.

Figures

FIG. 1.
FIG. 1.
Molecular cloning of a SIVagmSab transmitted/founder virus. (A) Single-genome amplification was used to generate 5′ (n = 10)- and 3′ (n = 13)-half-genome sequences (drawn to scale) from the plasma of a sabaeus monkey acutely infected with a high-titer stock of isolate SIVagmSab92018. (B) Phylogenetic trees and Highlighter plots of 5′ (top row)- and 3′ (bottom row)-half-genome sequences. Tick marks indicate differences compared to the top sequence, which also represents the inferred transmitted/founder sequence (red, T; green, A; blue, C; and orange, G). Trees were inferred by the neighbor-joining method and are midpoint rooted. The scale bar represents 0.002 substitution per site. Discrete low-diversity lineages representing the progeny of a transmitted founder virus are indicated by brackets (C) The consensus sequence of low-diversity lineages was used to synthesize the Sab92018ivTF genome as proviral halves. Flanking NotI and MluI restriction sites and an internal BglI site allowed cloning into the pCR-XL TOPO vector.
FIG. 2.
FIG. 2.
Phylogenetic relationship of the SIVagmSab transmitted/founder virus to other primate lentiviruses. The tree was inferred from Env amino acid sequences. Numbers at nodes are percent bootstrap support (only values of 70% or greater are shown). The scale represents 0.2 substitution per site.
FIG. 3.
FIG. 3.
Functional characterization of the transmitted/founder Sab92018ivTF clone. (A) TZM-bl indicator cells were infected with the indicated SIVagm molecular clones. Infections were performed with virus stocks containing 10 ng of p27 antigen. Panels A and D show average values of triplicate infections ± standard deviations (SD). ivTF, SIVagmSab92018ivTF. (B) Replication of SIVagmSab92018ivTF in Molt-4 clone 8 cells. OD, optical density. (C) Western blot analysis of cellular extracts of 293T cells transfected with the indicated molecular clones of SIVagmSab (left) and virions pelleted from the culture supernatant (right). (D) TZM-bl cells were left untreated (control) or were pretreated with specific inhibitors of CCR5 (Maraviroc), CXCR4 (AMD3100), or a combination thereof prior to infection with the indicated molecular clones of HIV-1 or SIV. (E) Coreceptor usage by SIVagmSab92018ivTF and the indicated control viruses tested in GHOST cells expressing CD4 alone (parental) or together with CCR5, CXCR4, or the orphan receptors BOB/GPR15 and BONZO/STRL33. All infectivity data were confirmed in one or two independent experiments.
FIG. 4.
FIG. 4.
Replication of SIVagmSab molecular clones in AGM PBMCs. PBMCs (left) or PBMCs depleted of CD8+ T cells (right) derived from four AGMs were infected with the parental SIVagmSab92018 strain or the 92018ivTF molecular clone. Virus production was monitored by p27 antigen ELISA.
FIG. 5.
FIG. 5.
Replication of the SIVagmSab92018ivTF clone in African green monkeys. (A) Viral RNA loads in one AGM infected with SIVagmSab92018ivTF and five animals that received the uncloned parental SIVagmSab92018 strain at the TNPRC. (B and C) Levels of plasma viremia (B) and CD4+ T-cell counts (C) in three sabaeus monkeys infected with SIVagmSab92018ivTF at the German Primate Center. Individual animal numbers are indicated by five-digit numbers. The vertical line marks the time point of infection. The limit of viral RNA detection is approximately 100 copies/ml of plasma. Values were determined as described in Materials and Methods.

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