The exon splicing silencer in human immunodeficiency virus type 1 Tat exon 3 is bipartite and acts early in spliceosome assembly.

Inefficient splicing of human immunodeficiency virus type 1 (HIV-1) RNA is necessary to preserve unspliced and singly spliced viral RNAs for transport to the cytoplasm by the Rev-dependent pathway. Signals within the HIV-1 genome that control the rate of splicing include weak 3' splice sites, exon splicing enhancers (ESE), and exon splicing silencers (ESS). We have previously shown that an ESS present within tat exon 2 (ESS2) and a suboptimal 3' splice site together act to inhibit splicing at the 3' splice site flanking tat exon 2. This occurs at an early step in spliceosome assembly. Splicing at the 3' splice site flanking tat exon 3 is regulated by a bipartite element composed of an ESE and an ESS (ESS3). Here we show that ESS3 is composed of two smaller elements (AGAUCC and UUAG) that can inhibit splicing independently. We also show that ESS3 is more active in the context of a heterologous suboptimal splice site than of an optimal 3' splice site. ESS3 inhibits splicing by blocking the formation of a functional spliceosome at an early step, since A complexes are not detected in the presence of ESS3. Competitor RNAs containing either ESS2 or ESS3 relieve inhibition of splicing of substrates containing ESS3 or ESS2. This suggests that a common cellular factor(s) may be required for the inhibition of tat mRNA splicing mediated by ESS2 and ESS3.

Presence of exon splicing silencers within human immunodeficiency virus type 1 tat exon 2 and tat-rev exon 3: evidence for inhibition mediated by cellular factors.

Human immunodeficiency virus type 1 (HIV-1) pre-mRNA splicing is regulated in order to maintain pools of unspliced and partially spliced viral RNAs as well as the appropriate levels of multiply spliced mRNAs during virus infection. We have previously described an element in tat exon 2 that negatively regulates splicing at the upstream tat 3' splice site 3 (B. A. Amendt, D. Hesslein, L.-J. Chang, and C. M. Stoltzfus, Mol. Cell. Biol. 14:3960-3970, 1994). In this study, we further defined the element to a 20-nucleotide (nt) region which spans the C-terminal vpr and N-terminal tat coding sequences. By analogy with exon splicing enhancer (ESE) elements, we have termed this element an exon splicing silencer (ESS). We show evidence for another negative cis-acting region within tat-rev exon 3 of HIV-1 RNA that has sequence motifs in common with a 20-nt ESS element in tat exon 2. This sequence is juxtaposed to a purine-rich ESE element to form a bipartite element regulating splicing at the upstream tat-rev 3' splice site. Inhibition of the splicing of substrates containing the ESS element in tat exon 2 occurs at an early stage of spliceosome assembly. The inhibition of splicing mediated by the ESS can be specifically abrogated by the addition of competitor RNA. Our results suggest that HIV-1 RNA splicing is regulated by cellular factors that bind to positive and negative cis elements in tat exon 2 and tat-rev exon 3.

[Studies on morphology and histology of medicinal part of Corydalis yanhusuo].

OBJECTIVE: To Study the morphology, microstructure and morphosis of medicinal part of Corydalis yanhusuo, and provide a basis for the knowledge of the type of modified stem of Corydalis yanhusuo and the identification of crude drug. METHOD: Sample collection, culture observation and microsectile observation. RESULT: Morphological and histological characters and morphogenetic and developmental regulation of medicinal part of Corydalis yanhusuo are described with pictures. CONCLUSION: The medicinal part of Corydalis yanhusuo is bulb, and the conception that the medicinal part of Corydalis yanhusuo is tuber is wrong.

A base-paired structure in the avian sarcoma virus 5' leader is required for efficient encapsidation of RNA.

Selective encapsidation of avian sarcoma-leukosis virus genomic RNA within virions requires recognition of a cis-acting signal (termed psi) located in the 5' leader of the RNA between the primer binding site and the splice donor site. Computer analyses indicate the potential for numerous secondary structure interactions within this region, including alternative conformations with similar free energy levels. We have constructed mutations designed to disrupt and restore potential secondary structure interactions within psi to investigate the role of these structures in RNA packaging. To test for the ability of psi mutants to package a heterologous reporter gene into virions, chimeric constructs bearing avian sarcoma virus 5' sequences fused to lacZ were transiently cotransfected with a nonpackageable helper construct into chicken embryo fibroblasts. lacZ virions produced from cotransfected cells were used to infect new cultures of chicken embryo fibroblasts, and then an in situ assay for individual cells expressing lacZ was done. Results obtained with this assay were confirmed in direct analyses of isolated virion RNA by RNase protection assays. Two mutations, predicted to disrupt a potential stem structure forming between elements located at nucleotides 160 to 167 and 227 to 234, severely inhibited packaging when either element was mutated. A construct in which these mutations were combined to restore potential base pairing between the two elements displayed a partially restored packaging phenotype. These results strongly suggest that the structure, referred to as the O3 stem, is required for efficient encapsidation of avian sarcoma virus RNA. Site-directed mutagenesis of additional sequence elements located in the O3 loop reduced packaging as measured by the indirect assay, suggesting that these sequences may also be components of the encapsidation signal. The possible implications of the O3 stem structure with regard to translation of avian sarcoma-leukosis virus short upstream open reading frames are discussed.

Association of structural changes in the V2 and V3 loops of the gp120 envelope glycoprotein with acquisition of neutralization resistance in a simian-human immunodeficiency virus passaged in vivo.

The in vivo passage of a neutralization-sensitive, laboratory-adapted simian-human immunodeficiency virus (SHIV-HXBc2) generated a pathogenic, neutralization-resistant virus, SHIV-HXBc2P 3.2. SHIV-HXBc2P 3.2 differs from SHIV-HXBc2 only in 13 amino acid residues of the viral envelope glycoproteins. Here we used antibody competition analysis to examine the structural changes that occurred in the SHIV-HXBc2P 3.2 gp120 exterior envelope glycoprotein. The relationships among the antibody epitopes on the conserved gp120 core of SHIV-HXBc2 and SHIV-HXBc2P 3.2 were similar. The third variable (V3) loop was more closely associated with the fourth conserved (C4) region and CD4-induced epitopes on the gp120 core in the HXBc2P 3.2 gp120 glycoprotein compared with the HXBc2 gp120 glycoprotein. Rearrangements of the second variable (V2) loop with respect to the CD4 binding site and associated epitopes were evident in comparisons of the two gp120 glycoproteins. Thus, the in vivo evolution of a neutralization-resistant virus involves conformational adjustments of the V2 and V3 variable loops with respect to the conserved receptor-binding regions of the gp120 core.