Effect of polypurine tract (PPT) mutations on human immunodeficiency virus type 1 replication: a virus with a completely randomized PPT retains low infectivity.
We introduced polypurine tract (PPT) mutations, which we had previously tested in an in vitro assay, into the viral clone NL4-3KFSdelta 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.
Miles
Lesa R
LR
Department of Microbiology, Biochemistry and Immunology, Morehouse School of Medicine, 720 Westview Dr. S. W., Atlanta, GA 30310, USA.
Agresta
Beth E
BE
Khan
Mahfuz B
MB
Tang
Shixing
S
Levin
Judith G
JG
Powell
Michael D
MD
D001483:Base Sequence; D002460:Cell Line; D004270:DNA, Circular / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D016325:HIV Long Terminal Repeat; D015497:HIV-1 / Q000235:genetics* / Q000472:pathogenicity / Q000502:physiology*; D006801:Humans; D009154:Mutation; D014774:Virulence / Q000235:genetics; D014779:Virus Replication / Q000235:genetics*
D004270:DNA, Circular; D004279:DNA, Viral