Effect of Polypurine Tract (PPT) Mutations on Human Immunodeficiency Virus Type 1 Replication: a Virus with a Completely Randomized PPT Retains Low Infectivity

传染性 生物 病毒学 突变体 病毒 病毒复制 突变 克隆(Java方法) 分子生物学 病毒定量 基因 遗传学
作者
Lesa Miles,Beth E. Agresta,Mahfuz Khan,Shixing Tang,Judith G. Levin,Michael D. Powell
出处
期刊:Journal of Virology [American Society for Microbiology]
卷期号:79 (11): 6859-6867 被引量:28
标识
DOI:10.1128/jvi.79.11.6859-6867.2005
摘要

ABSTRACT We introduced polypurine tract (PPT) mutations, which we had previously tested in an in vitro assay, into the viral clone NL4-3KFSΔ nef . Each mutant was tested for single-round infectivity and virion production. All of the PPT mutations had an effect on replication; however, mutation of the 5′ end appeared to have less of an effect on infectivity than mutation of the 3′ end of the PPT sequence. Curiously, a mutation in which the entire PPT sequence was randomized (PPTSUB) retained 12% of the infectivity of the wild type (WT) in a multinuclear activation of galactosidase indicator assay. Supernatants from these infections contained viral particles, as evidenced by the presence of p24 antigen. Two-long terminal repeat (2-LTR) circle junction analysis following PPTSUB infection revealed that the mutant could form a high percentage of normal junctions. Quantification of the 2-LTR circles using real-time PCR revealed that number of 2-LTR circles from cells infected with the PPTSUB mutant was 3.5 logs greater than 2-LTR circles from cells infected with WT virus. To determine whether the progeny virions from a PPTSUB infection could undergo further rounds of replication, we introduced the PPTSUB mutation into a replication-competent virus. Our results show that the mutant virus is able to replicate and that the infectivity of the progeny virions increases with each passage, quickly reverting to a WT PPT sequence. Together, these experiments confirm that the 3′ end of the PPT is important for plus-strand priming and that a virus that completely lacks a PPT can replicate at a low level.
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