title: Generation of infectious molecular clones of simian immunodeficiency virus from fecal consensus sequences of wild chimpanzees.
abstract: Studies of simian immunodeficiency viruses (SIVs) in their endangered primate hosts are of obvious medical and public health importance, but technically challenging. Although SIV-specific antibodies and nucleic acids have been detected in primate fecal samples, recovery of replication-competent virus from such samples has not been achieved. Here, we report the construction of infectious molecular clones of SIVcpz from fecal viral consensus sequences. Subgenomic fragments comprising a complete provirus were amplified from fecal RNA of three wild-living chimpanzees and sequenced directly. One set of amplicons was concatenated using overlap extension PCR. The resulting clone (TAN1.24) contained intact genes and regulatory regions but was replication defective. It also differed from the fecal consensus sequence by 76 nucleotides. Stepwise elimination of all missense mutations generated several constructs with restored replication potential. The clone that yielded the most infectious virus (TAN1.910) was identical to the consensus sequence in both protein and long terminal repeat sequences. Two additional SIVcpz clones were constructed by direct synthesis of fecal consensus sequences. One of these (TAN3.1) yielded fully infectious virus, while the second one (TAN2.69) required modification at one ambiguous site in the viral pol gene for biological activity. All three reconstructed proviruses produced infectious virions that replicated in human and chimpanzee CD4(+) T cells, were CCR5 tropic, and resembled primary human immunodeficiency virus type 1 isolates in their neutralization phenotype. These results provide the first direct evidence that naturally occurring SIVcpz strains already have many of the biological properties required for persistent infection of humans, including CD4 and CCR5 dependence and neutralization resistance. Moreover, they outline a new strategy for obtaining medically important "SIV isolates" that have thus far eluded investigation. Such isolates are needed to identify viral determinants that contribute to cross-species transmission and host adaptation.
authors.affiliation: Department of Medicine, University of Alabama at Birmingham, 720 20th Street South, Kaul 816, Birmingham, AL 35294, USA.
authors.forename: Jun
authors.initials: J
authors.lastname: Takehisa
authors.forename: Matthias H
authors.initials: MH
authors.lastname: Kraus
authors.forename: Julie M
authors.initials: JM
authors.lastname: Decker
authors.forename: Yingying
authors.initials: Y
authors.lastname: Li
authors.forename: Brandon F
authors.initials: BF
authors.lastname: Keele
authors.forename: Fréderic
authors.initials: F
authors.lastname: Bibollet-Ruche
authors.forename: Kenneth P
authors.initials: KP
authors.lastname: Zammit
authors.forename: Zhiping
authors.initials: Z
authors.lastname: Weng
authors.forename: Mario L
authors.initials: ML
authors.lastname: Santiago
authors.forename: Shadrack
authors.initials: S
authors.lastname: Kamenya
authors.forename: Michael L
authors.initials: ML
authors.lastname: Wilson
authors.forename: Anne E
authors.initials: AE
authors.lastname: Pusey
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Bailes
authors.forename: Paul M
authors.initials: PM
authors.lastname: Sharp
authors.forename: George M
authors.initials: GM
authors.lastname: Shaw
authors.forename: Beatrice H
authors.initials: BH
authors.lastname: Hahn
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D016384:Consensus Sequence; D017931:DNA Primers; D005243:Feces / Q000821:virology*; D005260:Female; D015684:Gene Products, pol / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D009500:Neutralization Tests; D002679:Pan troglodytes; D010802:Phylogeny; D017386:Sequence Homology, Amino Acid; D015302:Simian Immunodeficiency Virus / Q000235:genetics / Q000472:pathogenicity / Q000502:physiology*; D014779:Virus Replication / Q000235:genetics
chemical_list: D017931:DNA Primers; D015684:Gene Products, pol