Takehisa Jun, Kraus Matthias H, Decker Julie M, Li Yingying, Keele Brandon F, Bibollet-Ruche Fréderic, Zammit Kenneth P, Weng Zhiping, Santiago Mario L, Kamenya Shadrack, Wilson Michael L, Pusey Anne E, Bailes Elizabeth, Sharp Paul M, Shaw George M, Hahn Beatrice H
Department of Medicine, University of Alabama at Birmingham, 720 20th Street South, Kaul 816, Birmingham, AL 35294, USA.
J Virol. 2007 Jul;81(14):7463-75. doi: 10.1128/JVI.00551-07. Epub 2007 May 9.
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.
对濒危灵长类宿主中的猿猴免疫缺陷病毒（SIV）进行研究具有明显的医学和公共卫生重要性，但在技术上具有挑战性。尽管在灵长类粪便样本中已检测到SIV特异性抗体和核酸，但尚未从此类样本中成功分离出具有复制能力的病毒。在此，我们报告了从粪便病毒共有序列构建SIVcpz感染性分子克隆的过程。从三只野生黑猩猩的粪便RNA中扩增出包含完整前病毒的亚基因组片段，并直接进行测序。使用重叠延伸PCR将一组扩增子连接起来。所得克隆（TAN1.24）包含完整的基因和调控区域，但复制存在缺陷，并与粪便共有序列相差76个核苷酸。逐步消除所有错义突变产生了几个具有恢复复制潜力的构建体。产生最具感染性病毒的克隆（TAN1.910）在蛋白质和长末端重复序列上与共有序列相同。通过直接合成粪便共有序列构建了另外两个SIVcpz克隆。其中一个（TAN3.1）产生了完全具有感染性的病毒，而另一个（TAN2.69）需要在病毒pol基因的一个模糊位点进行修饰才能具有生物学活性。所有三个重建的前病毒都产生了可在人和黑猩猩CD4(+) T细胞中复制的感染性病毒粒子，具有CCR5嗜性，并且在中和表型上类似于原发性人类免疫缺陷病毒1型分离株。这些结果提供了首个直接证据，表明自然存在的SIVcpz毒株已经具备了持续感染人类所需的许多生物学特性，包括对CD4和CCR5的依赖性以及中和抗性。此外，它们概述了一种获取迄今为止尚未得到研究但具有医学重要性的“SIV分离株”的新策略。需要此类分离株来鉴定有助于跨物种传播和宿主适应的病毒决定因素。
