Human immunodeficiency virus env expression becomes Rev-independent if the env region is not defined as an intron

137

Replication of human immunodeficiency virus type 1 requires expression of the viral trans activator Rev. Rev binds to a highly structured RNA, the Rev response element, which is present in singly spliced and unspliced genomic viral RNAs. Although Rev helps to transport these transcripts from the nucleus to the cytoplasm, the mechanism(s) involved is not fully understood. Using the yeast two-hybrid system, we isolated a murine protein (YL2) that interacts with the basic domain of Rev, which is essential for the function of Rev in vivo and for the inhibitory splicing activity of Rev in vitro. YL2 has 92% identity to a human 32-kDa protein (p32), which copurifies with alternative splicing factor SF2/ASF. Furthermore, we found that whereas expression of YL2 greatly potentiated the activity of Rev, antisense YL2 transcripts blocked the effects of Rev in mammalian cells. YL2 also increased the activities of Rex on the Rex response element and of hybrid Rev proteins fused to Tat and the coat protein of bacteriophage MS2 on their respective RNAs. Thus, YL2 or p32 is a cellular protein that modulates the function of human immunodeficiency virus type 1 Rev.Rev is one of the earliest proteins expressed by human immunodeficiency virus type 1 (HIV-1) and is involved in the posttranscriptional regulation of viral RNAs (6,11,27,28,46). It is a 19-kDa nuclear protein that contains four functional domains (38). Of these, the basic domain binds to the Rev response element (RRE) (22,30,31,39,49,52), which forms highly structured RNA stem-loops (41). Since this basic domain also inhibits RNA splicing in vitro (32, 33) and a basic sequence is required for the optimal function of Rev even when it is tethered to RNA via a heterologous RNA-binding protein such as the coat protein of bacteriophage MS2 (45, 50), the basic region of Rev performs functions in addition to RNA binding. Furthermore, the two regions flanking this basic domain are required for multimerization of Rev on the RRE (38,42). Finally, five leucines near the C terminus form the activation domain of this protein (38). Splicing and transport of primary HIV-1 transcripts are complex. In the absence of Rev, only multiply spliced viral transcripts that direct the synthesis of Tat, Rev, and Nef are exported from the nucleus (29,48). These proteins perform important regulatory functions but are not themselves packaged into virions. In the presence of Rev, singly spliced and unspliced genomic viral transcripts also appear in the cytoplasm (7,10,19,29). Whereas singly spliced RNA codes for envelope glycoproteins gp120 and gp41, the unspliced RNA codes for Gag proteins, integrase, reverse transcriptase, and protease, which together with this RNA are packaged into the budding virion. Thus, Rev plays an essential role in the life cycle of HIV-1 (7).However, the mechanism of trans activation by Rev is not well understood. Since singly spliced and unspliced viral transcripts are detected in the nucleus but not in the cytoplasm in the absence of Rev, Rev might itself transport viral...

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 97%

Cellular protein modulates effects of human immunodeficiency virus type 1 Rev

Luo

Peterlin

1994

137

“…2). The constructs described here are intronless, and this feature may alleviate the Rev requirement for efficient expression (21). The pLdk3lacZ-RREpA plasmid contains unique HindIII and BamHI sites between the HIV-1 LTR promoter and the lacZ gene.…”

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confidence: 99%

Subcellular localization of avian sarcoma virus and human immunodeficiency virus type 1 integrases

Kukolj

Jones

Skalka

1997

The composition and subcellular trafficking of subviral preintegration complexes are reported to vary among the different retroviruses. The process by which the avian sarcoma virus (ASV) preintegration complex gains access to target chromatin remains unknown. Here we report that ASV integrase (IN) expressed as a fusion to ␤-galactosidase accumulates in the nuclei of transfected COS-1 cells. In contrast, human immunodeficiency type 1 (HIV-1) IN-␤-galactosidase fusions expressed similarly are predominantly cytoplasmic. To identify the region of ASV IN that specifies nuclear localization, various subdomains of the protein were expressed as ␤-galactosidase fusions and their subcellular locations were assessed cytochemically and by indirect immunofluorescence. These analyses showed that the ASV IN protein possesses a functional nuclear localization signal that spans amino acids 206 to 235 and displays limited homology with known nuclear transport signals.

“…It has been shown that inhibition is caused by the presence on the mRNAs of inhibitory RNA sequences located in the coding sequences of Gag, Pol, and Env (5,8,41,45,48,52). Unutilized 5Ј and 3Ј splice sites have also been implicated in posttranscriptional regulation of HIV-1 expression (6,28,40). When functional levels of Rev accumulate in the infected cell, production of the structural proteins Gag, Pol, and Env is induced while production of the early regulatory proteins Tat and Rev is repressed through a feedback mechanism (17).…”

mentioning

confidence: 99%

The Rev protein of human immunodeficiency virus type 1 counteracts the effect of an AU-rich negative element in the human papillomavirus type 1 late 3' untranslated region

Tan

Schwartz

1995

We have identified a sequence in the late 3 untranslated region of human papillomavirus type 1 mRNAs that acts posttranscriptionally to repress gene expression. Deletion analysis localized the inhibitory element to an AU-rich sequence between nucleotides 6958 and 6984 on the human papillomavirus type 1 genome. This sequence inhibits gene expression in an orientation-dependent manner. Upon transfection of eucaryotic cells with plasmids containing this sequence, approximately 4-fold-lower cytoplasmic mRNA levels and 64-to 128-fold-lower protein levels were produced compared with those produced by plasmids lacking the inhibitory sequence. Interestingly, providing the constitutive transport element of simian retrovirus type 1 in sense orientation counteracted inhibition exerted by the human papillomavirus type 1 sequence. Inhibition could also be overcome by the presence of human immunodeficiency virus type 1 Rev protein in trans and its target sequence, the Rev-responsive element, in cis. Rev is a nuclear protein and acts by promoting nuclear export of human immunodeficiency virus type 1 mRNAs encoding structural proteins. Our results are consistent with a model for human papillomavirus type 1 late-gene expression in which mRNAs containing human papillomavirus type 1 inhibitory sequences enter a nonproductive route in the nucleus, resulting in inefficient mRNA utilization. Rev directs mRNA containing inhibitory sequences to a productive route by interacting with the Rev-responsive element.