A1 Vertaisarvioitu alkuperäisartikkeli tieteellisessä lehdessä
Functional characterization of the human immunodeficiency virus type 1 genome by genetic footprinting
Tekijät: Laurent LC, Olsen MN, Crowley RA, Savilahti H, Brown PO
Kustantaja: AMER SOC MICROBIOLOGY
Julkaisuvuosi: 2000
Lehti:: Journal of Virology
Tietokannassa oleva lehden nimi: JOURNAL OF VIROLOGY
Lehden akronyymi: J VIROL
Vuosikerta: 74
Numero: 6
Aloitussivu: 2760
Lopetussivu: 2769
Sivujen määrä: 10
ISSN: 0022-538X
DOI: https://doi.org/10.1128/JVI.74.6.2760-2769.2000
Tiivistelmä
We present a detailed and quantitative analysis of the functional characteristics of the 1,000-nucleotide segment at the 5' end of the human immunodeficiency virus type 1 (HIV-1) RNA genome. This segment of the viral genome contains several important cis-acting sequences, including the TAR, polyadenylation, viral att site, minus-strand primer-binding site, and 5' splice donor sequences, as well as coding sequences for the matrix protein and the N-terminal half of the capsid protein. The genetic footprinting technique was used to determine quantitatively the abilities of 134 independent insertion mutations to (i) make stable viral RNA, (ii) assemble and release viral RNA-containing viral particles, and (iii) enter host cells, complete reverse transcription, enter the nuclei of host cells, and generate proviruses in the host genome by integration. All of the mutants were constructed and analyzed en masse, greatly decreasing the labor typically involved in mutagenesis studies, The results confirmed the presence of several previously known functional features in this region of the HIV genome and provided evidence for several novel features, including nea ly identified cis-acting sequences that appeared to contribute to (i) the formation of stable viral transcripts, (ii) viral RNA packaging, and (iii) an early step in viral replication, The results also pointed to an unanticipated trans-acting role for the N-terminal portion of matrix in the formation of stable viral RNA transcripts. Finally, in contrast to previous reports, the results of this study suggested that detrimental mutations in the matrix and capsid proteins principally interfered with viral assembly.
We present a detailed and quantitative analysis of the functional characteristics of the 1,000-nucleotide segment at the 5' end of the human immunodeficiency virus type 1 (HIV-1) RNA genome. This segment of the viral genome contains several important cis-acting sequences, including the TAR, polyadenylation, viral att site, minus-strand primer-binding site, and 5' splice donor sequences, as well as coding sequences for the matrix protein and the N-terminal half of the capsid protein. The genetic footprinting technique was used to determine quantitatively the abilities of 134 independent insertion mutations to (i) make stable viral RNA, (ii) assemble and release viral RNA-containing viral particles, and (iii) enter host cells, complete reverse transcription, enter the nuclei of host cells, and generate proviruses in the host genome by integration. All of the mutants were constructed and analyzed en masse, greatly decreasing the labor typically involved in mutagenesis studies, The results confirmed the presence of several previously known functional features in this region of the HIV genome and provided evidence for several novel features, including nea ly identified cis-acting sequences that appeared to contribute to (i) the formation of stable viral transcripts, (ii) viral RNA packaging, and (iii) an early step in viral replication, The results also pointed to an unanticipated trans-acting role for the N-terminal portion of matrix in the formation of stable viral RNA transcripts. Finally, in contrast to previous reports, the results of this study suggested that detrimental mutations in the matrix and capsid proteins principally interfered with viral assembly.
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