Exon 1 leader sequences downstream of U5 are important for efficient human immunodeficiency virus type 1 gene expression

Abstract

In previous studies, little attention has been paid to maintaining the native HIV-1 leader sequence in reporter constructs analyzing the human immunodeficiency virus type 1 (HIV-1) promoter activity. To investigate a possible influence of the leader sequence on HIV-1-driven gene expression in the presence as well as in the absence of Tat, an expression vector was designed for transcripts consisting of the native HIV-1 tat 1.4 mRNA leader followed by the open reading frame for the bacterial chloramphenicol acetyltransferase (CAT). Deletion mutants with mutations within the leader sequence downstream of U5 (lsdU5) were constructed, as well as a mutant containing a mutation with a reverse orientation of this region. Quantification of CAT protein in HeLa-T4+ cells transiently transfected with wild-type and mutant leader constructs showed that the exon 1-derived lsdU5 region has an influence on basal as well as Tat-induced protein expression. The dramatic decrease in the level of CAT protein upon deletion of lsdU5 was paralleled by a drop in the steady-state level of CAT mRNA. Deletion of the exon 1-derived lsdU5 region also decreased the expression of mRNAs containing authentic HIV-1 sequences instead of CAT. The effect observed with the reporter constructs was not due to the loss of binding sites for nuclear factors, as could be shown with DBF1 and Sp1 mutant constructs. Nuclear run-on transcription assays showed that the presence or absence of lsdU5 did not influence the rate of transcription. This indicates that the exon 1 lsdU5 element functions at the posttranscriptional level in the processing, nucleocytoplasmic export, or stabilization of HIV-1 transcripts.

Compensatory Point Mutations in the Human Immunodeficiency Virus Type 1 Gag Region That Are Distal from Deletion Mutations in the Dimerization Initiation Site Can Restore Viral Replication

“…In addition to the foregoing, we have also shown that the MP2 mutation leads to increased protein production in mutated LD3-MP2 virus. This result is consistent with recent reports that noncoding sequences in the leader region between the primer binding site and the major splice donor are required for efficient viral gene expression (26,29). Although the SL1 region may play a posttranscriptional role in the expression of unspliced viral RNA, it is also possible that the LD3 deletion reduced overall HIV gene expression and, hence, resulted in diminished levels of Gag proteins, as shown in previous studies (29).…”

Mutations within Four Distinct Gag Proteins Are Required To Restore Replication of Human Immunodeficiency Virus Type 1 after Deletion Mutagenesis within the Dimerization Initiation Site

“…The primer binding site is an 18-nucleotide (18-nt) segment that binds to tRNA 3 Lys , which serves as the cognate primer of reverse transcription. The exon 1 leader sequences downstream of U5 include the splice donor site and are also involved in HIV-1 gene transcription as well as the dimerization and selective encapsidation of full-length viral genomic RNA (3,6,23,(27)(28)(29)31).…”