Human immunodeficiency virus type 1 hnRNP A/B-dependent exonic splicing silencer ESSV antagonizes binding of U2AF65 to viral polypyrimidine tracts

Human immunodeficiency virus type 1 (HIV-1) exonic splicing silencers (ESSs) inhibit production of certain spliced viral RNAs by repressing alternative splicing of the viral precursor RNA. Several HIV-1 ESSs interfere with spliceosome assembly by binding cellular hnRNP A/B proteins. Here, we have further characterized the mechanism of splicing repression using a representative HIV-1 hnRNP A/B-dependent ESS, ESSV, which regulates splicing at the vpr 3' splice site. We show that hnRNP A/B proteins bound to ESSV are necessary to inhibit E complex assembly by competing with the binding of U2AF65 to the polypyrimidine tracts of repressed 3' splice sites. We further show evidence suggesting that U1 snRNP binds the 5' splice site despite an almost complete block of splicing by ESSV. Possible splicing-independent functions of U1 snRNP-5' splice site interactions during virus replication are discussed.     

Inhibition of Rous sarcoma virus replication by antisense RNA.

Previous results have indicated that Rous sarcoma virus env gene expression is specifically inhibited by antisense RNA (L.-J. Chang and C. M. Stoltzfus, Mol. Cell. Biol. 5:2341-2348, 1985). In this study, we compare the extents of inhibition by antisense RNA derived from different parts of the Rous sarcoma virus genome, and we show that antisense constructs containing the 3'-end noncoding region inhibit env expression to a similar extent as those containing the 5'-end noncoding region or coding region. Furthermore, we show that antisense RNA inhibits virus replication at other levels in addition to translation.     

Noncoding region between the env and src genes of Rous sarcoma virus influences splicing efficiency at the src gene 3' splice site.

Viral RNA and proteins in chicken embryo fibroblasts infected with different cloned variants of the Prague strain Rous sarcoma virus (RSV) were analyzed. The ratio of immunoprecipitated pp60src to the gag gene product p27 in Prague A (PrA) and Prague B (PrB) RSV-infected cells was two to three times that in Prague C (PrC) RSV-infected cells. A significant increase in the steady-state ratio of spliced 2.7-kilobase src gene mRNA to unspliced 9.3-kilobase genome-size RNA was observed in PrA- and PrB- compared with PrC-infected cells, consistent with the differences in the ratios of the gag to src gene protein products. Similar results were obtained when hybrid-selected RNA, which had been labeled for 3 h with [3H]uridine, was analyzed on formaldehyde-agarose gels, suggesting that the observed differences were due to splicing rather than RNA stability. Recombinant plasmids from infectious molecular clones of PrA and PrC were constructed to localize the regions responsible for the effects on src gene splicing. The substitution in place of the corresponding PrA region of the 262-base-pair region between the env gene and the src gene coding sequences from the PrC clone into the infectious PrA plasmid conferred the low src splicing efficiency of the PrC strain. The nucleotide sequence of this region of the PrA plasmid was determined and compared with the sequence of the PrC strain. Only four nucleotide differences were found; two changes were within the intron sequence, and two were in the exon sequence. The possible role of these differences in determining the extent of viral RNA splicing is discussed.     

Oral papillomas and papilliform lesions in rhesus macaques (Macaca mulatta)

Oral papillomas in two male rhesus macaques that were diagnosed morphologically as filiform and squamous types are described. Two additional macaques had oral papilliform lesions consistent histologically with papillary hyperplasia. Immunohistochemistry, along with electron microscopy and PCR assays, failed to demonstrate evidence of papillomavirus in any of the tumors; however, such results are often lacking when suspect oral lesions in humans and other species are assessed. Other potential causes of the papillary masses include chronic irritation and perhaps a genetic susceptibility. Benign tumors of the oral epithelium in macaques have not been reported previously; they appear to be rare and of variable clinical significance.     

Inflammation and Cancer. I. Rodent models of infectious gastrointestinal and liver cancer

Chronic gastrointestinal and liver infections account for a significant percentage of human cancer deaths. Rodent models help elucidate how infection can lead to malignancy. Helicobacter pylori, the leading cause of human gastric tumors, produces similar disease in Mongolian gerbils. H. pylori, H. felis, and H. hepaticus induce stomach, lower bowel, or liver tumors in susceptible wild-type and genetically engineered mice. Immune dysregulated mice recapitulate features of inflammatory bowel disease including colon carcinoma. Hepatitis B and C virus transgenic mice provide insights into viral hepatitis and hepatocellular carcinoma. Rodent models enhance our understanding of infectious cancer pathogenesis and suggest novel targets for intervention.     

Differential expression of the Sesbania rostrata leghemoglobin glb3 gene promoter in transgenic legume and non-legume plants

The involvement of the Sesbania rostrata glb3 gene promoter NICE (nodule-infected cell expression) element in root-enhanced expression of 5-Srglb3-uidA-3nos chimeric gene was investigated in transgenic Nicotiana tabacum plants. The full-length wild-type Srglb3 promoter directed root meristem-enhanced expression in transgenic tobacco plants. The expression pattern of nine selected Srglb3 promoter mutations in the NICE element was examined in transgenic tobacco plants and compared with the pattern observed in nodules of transgenic Lotus corniculatus plants. The results suggest that the highly conserved motifs in the NICE element play an important role in expression in roots of non-legume plants.     

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.     

Repair of a Rev-minus human immunodeficiency virus type 1 mutant by activation of a cryptic splice site

We isolated a revertant virus after prolonged culturing of a replication-impaired human immunodeficiency virus type 1 (HIV-1) mutant of which the Rev open reading frame was inactivated by mutation of the AUG translation initiation codon. Sequencing of the tat-rev region of this revertant virus identified a second-site mutation in tat that restored virus replication in the mutant background. This mutation activated a cryptic 5' splice site (ss) that, when used in conjunction with the regular HIV 3' ss #5, fuses the tat and rev reading frames to encode a novel T-Rev fusion protein that rescues Rev function. We also demonstrate an alternative route to indirectly activate this cryptic 5' ss by mutational inactivation of an adjacent exon splicing silencer element.     

Fine mapping of chromosome 22 breakpoints within the breakpoint cluster region (bcr) implies a role for bcr exon 3 in determining disease duration in chronic myeloid leukemia.

The chromosomal translocation that fuses the phl gene with the c-abl proto-oncogene appears to be a pivotal step in the pathogenesis of some leukemias. In chronic myeloid leukemia (CML) the breakage within the phl gene is largely confined to a 5.8-kb segment referred to as the breakpoint cluster region (bcr). To determine whether the presence of specific bcr exons on the Philadelphia chromosome has any clinical significance, we have analyzed the bcr breakpoints in 134 patients with CML. As many as five probes were used in this analysis, including a synthetic oligonucleotide probe homologous to the bcr exon 3 (phl exon 14) region. The distribution of breakpoints indicates that, in fact, breakage is largely confined to a 3.1-kb segment lying between bcr exon 2 and exon 4 (phl exons 13-15). In 61 CML patients analyzed within 1 year of diagnosis, the distribution of breakpoints appeared to be random within the 3.1-kb region. However, a significant excess of 5' breakpoints was observed in the total population studied, consistent with previous data showing that patients with 3' breakpoints have shorter disease durations. Analysis using the bcr exon 3 sequence probe indicated it was probably the presence or absence of bcr exon 3 on the Philadelphia chromosome that accounts for some of the variability in disease duration seen in CML. The data suggest that the phl/abl protein product may influence the timing of the onset of blast crisis and imply a continuing role for this protein during the evolution of the disease.