Activation of a cryptic splice donor in human immunodeficiency virus type-1.

The human immunodeficiency virus type-1 regulatory protein Rev is absolutely required for the production of viral structural proteins. Splice sites have been seen to function as cis-acting repressor sequendes (CRS) and inhibit expression of the Rev-dependent RNAs. In order to analyze the role of a splice donor in Rev dependence, the wild-type 5' splice donor of HIV-1 was mutated in the context of other gag sequences. Following transient transfection, RNA expression by RT-PCR was analyzed. The unspliced RNA produced by the mutant construct still required Rev for the cytoplasmic accumulation of the RNA. Despite deletion of the wild-type 5' splice donor and the tat splice acceptor was used. A cryptic splice donor was identified by PCR and subsequent cloning of the spliced RNA. The cryptic site is 5/9 to the consensus sequence and located immediately downstream of the initiation codon (ATG) for Gag. Analysis of the RNA product containing the cryptic splice donor revealed that the Rev was required for the cytoplasmic accumulation of unspliced RNA, while spliced RNA was Rev independent. Transfection of a wild-type construct also demonstrated usage of the cryptic splice donor. These results indicate that a cryptic splice donor can be activated when the wild-type splice donor is inactivated and that the cryptic splice donor may retain Rev regulation. The findings also suggest the potential for cryptic splice sites to serve as CRS in the determining the Rev dependence of viral RNAs.

Effect of Rev on the intranuclear localization of HIV-1 unspliced RNA.

Human immunodeficiency virus type 1 (HIV-1) Rev is a 19-kDa regulatory protein which binds to unspliced and partially spliced HIV-1 RNAs. Export, splicing, stability, and translation of HIV-1 RNAs are influenced by Rev. To further understand the effect of Rev on HIV-1 RNA splicing, the intranuclear localization of unspliced HIV-1 RNA and a cellular splicing factor was examined in the presence and absence of Rev. Splicing component-35 (SC-35) is an essential SR protein splicing factor which localizes into 20-40 nuclear granules (Fu, X. D., and Maniatis, T. Nature 343 (6257), 437-441, 1990). Laser scanning confocal microscopy was utilized to examine the colocalization of unspliced HIV-1 RNA and SC-35-containing granules. In the presence of Rev, many of the SC-35-containing granules were colocalized on their edges or completely colocalized with HIV-1 unspliced RNA speckles. In the absence of Rev, however, little colocalization of the unspliced HIV-1 RNA speckles and the SC-35-containing granules was observed. Quantitative RT-PCR was utilized to examine the effect of Rev on the level of fully spliced HIV-1 RNA. In the presence of Rev, a decrease in the level of fully spliced HIV-1 RNA was observed. Thus both the intranuclear localization and posttranscriptional processing of HIV-1 unspliced RNA are affected by Rev.

Involvement of human immunodeficiency virus type-1 splice sites in the cytoplasmic accumulation of viral RNA.

To define the role of human immunodeficiency virus type 1 splice sites in the cytoplasmic accumulation of viral RNAs, sequential deletion mutagenesis on an infectious proviral clone of HIV-1 was performed. Deletion of the majority of intron sequences, containing previously identified CRS, did not attenuate CRS activity. Retention of either the first or second tat intron preserved CRS activity. RNAs containing splice donor sequences, in the absence of known downstream splice acceptor sequences, retained CRS activity. Unexpectedly, these splice donors were still utilized for splicing. These results indicate that the major HIV-1 splice donors can function as CRS and function to negatively regulate the cytoplasmic accumulation of HIV-1 RNAs in COS cells.

Expression of metalloproteases by cardiac myocytes and fibroblasts in vitro.

Regulation of the turnover of extracellular matrix (ECM) components has been attributed in part to matrix metalloproteases (MMP). Isolated cardiac myocytes and fibroblasts from different developmental stages express different patterns of MMPs in vitro. Zymography of media and cell extracts of fibroblasts and myocytes indicated several apparent molecular weights (Mr) with gelatinolytic activity with prominent bands at 92 and 72 kDa. No caseinolytic activity was detected. These MMPs were characteristic of known MMP-2 and MMP-9. Fibroblasts predominantly expressed the latent 72-kDa MMP, whereas myocytes expressed a latent 92-kDa MMP. Expression of these MMPs was not affected by density of culture or the type of ECM substrate on which the cells were grown. Sodium dodecyl sulfate (SDS)-activated MMP-2 showed specific cleavage patterns on collagen types I and III but not on fibronectin, collagen type IV, or laminin. The reaction of SDS-activated MMP-2 produced a 140-kDa fragment from collagen types I and III. No specific substrate patterns were observed with activated MMP-9. MMP-2 from fibroblasts could also be activated by mechanical tension developed by fibroblasts within collagen gels or by cyclically stretching Silastic membranes on which the fibroblasts were grown. When mechanical tension was inhibited in collagen gels by antibodies against the ß1 integrin, the 72-kDa MMP, or cytochalasin D, the activated band at 62 kDa was not detected. Immunocytochemical localization with antibodies against MMP-2 showed a weak reaction on cardiac myocytes, but intense staining around the focal adhesions of migrating fibroblasts. In collagen gels, staining was localized to the leading pseudopodia of the fibroblasts. Together, these data indicate that the rat MMP-2 is a collagenase primarily associated with cardiac fibroblasts, activated by mechanical tension, and may be important in cellular ECM interactions.

Differential effects of intronic and exonic locations of the human immunodeficiency virus type 1 Rev-responsive element.

The influence of the location of the Rev-response element (RRE) on human immunodeficiency virus type 1 (HIV-1) protein and RNA expression in COS cells was assessed. The RRE was placed into nef where it would be present in all HIV-1 RNAs. At this location, Gag and Env proteins were produced and the unspliced gag/pol and partially spliced env/vpu RNAs were able to accumulate in the cytoplasm. The RRE was also relocated from its normal location in the env exon to the env intron. In this way, the RRE would be present in the nuclear env pre-mRNA, but not in the spliced env mRNA. Gag, but not Env protein production was detected. Th presence of the RRE in the env pre-mRNA allowed the cytoplasmic accumulation of the spliced env mRNA, which lacked the RRE. However, this mRNA accumulated at a reduced level relative to that produced by constructs containing the RRE within the env mRNA. The cytoplasmic accumulation of this mRNA was dependent on the presence of Rev and the RRE. These results demonstrate that the location of the RRE can have differential effects on the fate of HIV-1 RNAs.

Human immunodeficiency virus type 1 Rev is required in vivo for binding of poly(A)-binding protein to Rev-dependent RNAs.

In the absence of Rev or the Rev-responsive element, the Rev-dependent human immunodeficiency virus type 1 (HIV-1) RNAs do not behave as mRNAs; rather, they exhibit nuclear defects in splicing and/or nuclear export and cytoplasmic defects in stability and translation. A translational initiation factor, eIF-5A, has recently been shown to bind specifically to the Rev activation domain. As the binding of poly(A)-binding protein 1 (PAB1) to the poly(A) tail of mRNAs is involved in both the stability and translation of cytoplasmic mRNAs, we investigated whether Rev might influence the association of PAB1 with cytoplasmic HIV-1 RNAs. Antibodies were generated against PAB1. We used these antibodies in an immunoprecipitation assay to detect specific binding of PAB1 to cytoplasmic mRNAs. We found that in the presence of Rev, PAB1 was associated with Rev-dependent and Rev-independent RNAs in the cytoplasm of transfected cells. However, in the absence of functional Rev, we found little or no PAB1 associated with Rev-dependent RNAs. These RNAs were capable of binding PAB1 in vitro. These results demonstrate that HIV-1 RNAs are defective in PAB1 association in the absence of Rev.