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Journal of Virology, July 2007, p. 7463-7475, Vol. 81, No. 14
0022-538X/07/$08.00+0     doi:10.1128/JVI.00551-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Generation of Infectious Molecular Clones of Simian Immunodeficiency Virus from Fecal Consensus Sequences of Wild Chimpanzees{triangledown}

Jun Takehisa,1 Matthias H. Kraus,1 Julie M. Decker,1 Yingying Li,1 Brandon F. Keele,1 Fréderic Bibollet-Ruche,1 Kenneth P. Zammit,1 Zhiping Weng,1 Mario L. Santiago,2 Shadrack Kamenya,3 Michael L. Wilson,3 Anne E. Pusey,4 Elizabeth Bailes,5 Paul M. Sharp,5 George M. Shaw,1,6 and Beatrice H. Hahn1,6*

Departments of Medicine,1 Microbiology, University of Alabama at Birmingham, Birmingham, Alabama 35294,6 Gladstone Institute of Virology and Immunology, San Francisco, California 94158,2 Gombe Stream Research Centre, The Jane Goodall Institute, Kigoma, Tanzania,3 Department of Ecology, Evolution and Behavior, University of Minnesota, St. Paul, Minnesota 55108,4 Institute of Genetics, University of Nottingham, Queens Medical Centre, Nottingham, United Kingdom5

Received 15 March 2007/ Accepted 30 April 2007

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.

* Corresponding author. Mailing address: Department of Medicine, University of Alabama at Birmingham, 720 20th Street South, Kaul 816, Birmingham, AL 35294. Phone: (205) 934-0412. Fax: (205) 934-1580. E-mail: bhahn{at}uab.edu

{triangledown} Published ahead of print on 9 May 2007.

Journal of Virology, July 2007, p. 7463-7475, Vol. 81, No. 14
0022-538X/07/$08.00+0     doi:10.1128/JVI.00551-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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