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The strength of the HIV-1 3' splice sites affects Rev function.
Retrovirology. 2006 Dec 04; 3:89.R

Abstract

BACKGROUND

The HIV-1 Rev protein is a key component in the early to late switch in HIV-1 splicing from early intronless (e.g. tat, rev) to late intron-containing Rev-dependent (e.g. gag, vif, env) transcripts. Previous results suggested that cis-acting sequences and inefficient 5' and 3' splice sites are a prerequisite for Rev function. However, we and other groups have shown that two of the HIV-1 5' splice sites, D1 and D4, are efficiently used in vitro and in vivo. Here, we focus on the efficiency of the HIV-1 3' splice sites taking into consideration to what extent their intrinsic efficiencies are modulated by their downstream cis-acting exonic sequences. Furthermore, we delineate their role in RNA stabilization and Rev function.

RESULTS

In the presence of an efficient upstream 5' splice site the integrity of the 3' splice site is not essential for Rev function whereas an efficient 3' splice site impairs Rev function. The detrimental effect of a strong 3' splice site on the amount of Rev-dependent intron-containing HIV-1 glycoprotein coding (env) mRNA is not compensatable by weakening the strength of the upstream 5' splice site. Swapping the HIV-1 3' splice sites in an RRE-containing minigene, we found a 3' splice site usage which was variably dependent on the presence of the usual downstream exonic sequence. The most evident activation of 3' splice site usage by its usual downstream exonic sequence was observed for 3' splice site A1 which was turned from an intrinsic very weak 3' splice site into the most active 3' splice site, even abolishing Rev activity. Performing pull-down experiments with nuclear extracts of HeLa cells we identified a novel ASF/SF2-dependent exonic splicing enhancer (ESE) within HIV-1 exon 2 consisting of a heptameric sequence motif occurring twice (M1 and M2) within this short non-coding leader exon. Single point mutation of M1 within an infectious molecular clone is detrimental for HIV-1 exon 2 recognition without affecting Rev-dependent vif expression.

CONCLUSION

Under the conditions of our assay, the rate limiting step of retroviral splicing, competing with Rev function, seems to be exclusively determined by the functional strength of the 3' splice site. The bipartite ASF/SF2-dependent ESE within HIV-1 exon 2 supports cross-talk between splice site pairs across exon 2 (exon definition) which is incompatible with processing of the intron-containing vif mRNA. We propose that Rev mediates a switch from exon to intron definition necessary for the expression of all intron-containing mRNAs.

Authors+Show Affiliations

Institut für Virologie, Heinrich-Heine-Universität Düsseldorf, Universitätsstr, 1, Geb, 22,21, D-40225 Düsseldorf, Germany. suk@mb.au.dkNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

17144911

Citation

Kammler, Susanne, et al. "The Strength of the HIV-1 3' Splice Sites Affects Rev Function." Retrovirology, vol. 3, 2006, p. 89.
Kammler S, Otte M, Hauber I, et al. The strength of the HIV-1 3' splice sites affects Rev function. Retrovirology. 2006;3:89.
Kammler, S., Otte, M., Hauber, I., Kjems, J., Hauber, J., & Schaal, H. (2006). The strength of the HIV-1 3' splice sites affects Rev function. Retrovirology, 3, 89.
Kammler S, et al. The Strength of the HIV-1 3' Splice Sites Affects Rev Function. Retrovirology. 2006 Dec 4;3:89. PubMed PMID: 17144911.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - The strength of the HIV-1 3' splice sites affects Rev function. AU - Kammler,Susanne, AU - Otte,Marianne, AU - Hauber,Ilona, AU - Kjems,Jørgen, AU - Hauber,Joachim, AU - Schaal,Heiner, Y1 - 2006/12/04/ PY - 2006/09/18/received PY - 2006/12/04/accepted PY - 2006/12/6/pubmed PY - 2007/1/20/medline PY - 2006/12/6/entrez SP - 89 EP - 89 JF - Retrovirology JO - Retrovirology VL - 3 N2 - BACKGROUND: The HIV-1 Rev protein is a key component in the early to late switch in HIV-1 splicing from early intronless (e.g. tat, rev) to late intron-containing Rev-dependent (e.g. gag, vif, env) transcripts. Previous results suggested that cis-acting sequences and inefficient 5' and 3' splice sites are a prerequisite for Rev function. However, we and other groups have shown that two of the HIV-1 5' splice sites, D1 and D4, are efficiently used in vitro and in vivo. Here, we focus on the efficiency of the HIV-1 3' splice sites taking into consideration to what extent their intrinsic efficiencies are modulated by their downstream cis-acting exonic sequences. Furthermore, we delineate their role in RNA stabilization and Rev function. RESULTS: In the presence of an efficient upstream 5' splice site the integrity of the 3' splice site is not essential for Rev function whereas an efficient 3' splice site impairs Rev function. The detrimental effect of a strong 3' splice site on the amount of Rev-dependent intron-containing HIV-1 glycoprotein coding (env) mRNA is not compensatable by weakening the strength of the upstream 5' splice site. Swapping the HIV-1 3' splice sites in an RRE-containing minigene, we found a 3' splice site usage which was variably dependent on the presence of the usual downstream exonic sequence. The most evident activation of 3' splice site usage by its usual downstream exonic sequence was observed for 3' splice site A1 which was turned from an intrinsic very weak 3' splice site into the most active 3' splice site, even abolishing Rev activity. Performing pull-down experiments with nuclear extracts of HeLa cells we identified a novel ASF/SF2-dependent exonic splicing enhancer (ESE) within HIV-1 exon 2 consisting of a heptameric sequence motif occurring twice (M1 and M2) within this short non-coding leader exon. Single point mutation of M1 within an infectious molecular clone is detrimental for HIV-1 exon 2 recognition without affecting Rev-dependent vif expression. CONCLUSION: Under the conditions of our assay, the rate limiting step of retroviral splicing, competing with Rev function, seems to be exclusively determined by the functional strength of the 3' splice site. The bipartite ASF/SF2-dependent ESE within HIV-1 exon 2 supports cross-talk between splice site pairs across exon 2 (exon definition) which is incompatible with processing of the intron-containing vif mRNA. We propose that Rev mediates a switch from exon to intron definition necessary for the expression of all intron-containing mRNAs. SN - 1742-4690 UR - https://www.unboundmedicine.com/medline/citation/17144911/The_strength_of_the_HIV_1_3'_splice_sites_affects_Rev_function_ L2 - https://retrovirology.biomedcentral.com/articles/10.1186/1742-4690-3-89 DB - PRIME DP - Unbound Medicine ER -