Mutational analysis of the 5' noncoding region of human immunodeficiency virus type 1 genome.
Retrovirus particles are released by budding from the membranes of infected cells. In the course of virus production, particularly during the late stage, viral genomic RNA is incorporated specifically into virion particles. This specific incorporation of the genomic RNA requires a packaging signal sequence. A region that functions as the packaging signal was mapped to a location upstream of the gag open reading frame on the HIV-1 viral genome. In addition of this packaging signal, other cis-acting elements that are scattered throughout the genome are also required for efficient packaging. The region upstream of the splice donor site is probably important for dimer formation. Therefore, we focused on one region located between the 3' end of the primer binding site and the 5' splice donor site of HIV-1. Experiments were conducted to investigate how deletions or point mutations in this region affect both dimerization in vitro and the production of infectious virus particles. A series of RNAs of varying lengths containing the 5' noncoding region were generated, and genomic dimerization of the altered viral RNA was analyzed in vitro. One RNA construct which consisted of 112 nucleotides (nt) from nt 639 to nt 750 formed a heterodimeric complex with the RNA which consisted of 200 nucleotides from nt 551 to nt 750. We then constructed proviruses with mutations in the 639 to 750 nt region and assayed for virus production. Several mutants that lacked the complementarity necessary to form a possible stem-loop structure in this region showed decreased production of infectious virus particles. Moreover, both deletion of this region and randomization of its nucleotide sequence completely impaired infectious virus production. Thus, the way that this region affects infectious virus production may be through its RNA secondary structure.
Hirota
M
M
Virology Division, National Cancer Center Research Institute, Tokyo.
Koyanagi
Y
Y
An
D S
DS
Iwanaga
Y
Y
Yamamoto
N
N
Shimotohno
K
K
D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D004252:DNA Mutational Analysis / Q000379:methods; D004279:DNA, Viral / Q000378:metabolism; D019281:Dimerization; D015750:Genes, gag; D016679:Genome, Viral; D015497:HIV-1 / Q000235:genetics* / Q000472:pathogenicity / Q000502:physiology; D006801:Humans; D008156:Luciferases / Q000096:biosynthesis; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D011533:Proviruses; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012045:Regulatory Sequences, Nucleic Acid; D014162:Transfection; D014779:Virus Replication
D004279:DNA, Viral; D012367:RNA, Viral; D011993:Recombinant Fusion Proteins; D008156:Luciferases