Biomedical subjects

Alan Cochrane

Publications and source records attributed to Alan Cochrane.

6 recordsLinked to original sources

PubMed2004

A novel function for Sam68: enhancement of HIV-1 RNA 3' end processing.

Both cis elements and host cell proteins can significantly affect HIV-1 RNA processing and viral gene expression. Previously, we determined that the exon splicing silencer (ESS3) within the terminal exon of HIV-1 not only reduces use of the adjacent 3' splice site but also prevents Rev-induced export of the unspliced viral RNA to the cytoplasm. In this report, we demonstrate that loss of unspliced viral RNA export is correlated with the inhibition of 3' end processing by the ESS3. Furthermore, we find that the host factor Sam68, a stimulator of HIV-1 protein expression, is able to reverse the block to viral RNA export mediated by the ESS3. The reversal is associated with a stimulation of 3' end processing of the unspliced viral RNA. Our findings identify a novel activity for the ESS3 and Sam68 in regulating HIV-1 RNA polyadenylation. Furthermore, the observations provide an explanation for how Sam68, an exclusively nuclear protein, modulates cytoplasmic utilization of the affected RNAs. Our finding that Sam68 is also able to enhance 3' end processing of a heterologous RNA raises the possibility that it may play a similar role in regulating host gene expression.

3' Untranslated Regions↗

PubMed2004

Controlling HIV-1 Rev function.

Although current therapies used in the treatment of HIV-1 infection have proven effective in reducing mortality due to the infection, the increase in drug resistant strains of the virus call for increased effort to explore and develop alternative treatment modalities. In this review, the various strategies to control HIV-1 replication through the disruption of Rev function are outlined. A wide range of methods have been developed including antisense DNA, ribozymes, decoy RNAs, transdominant proteins and suicide vectors targeted at disrupting Rev function. Although many of these methods have proven effective alone, it is hoped that a more robust antiviral response can be attained through combination of these strategies. As the methods of delivering these therapeutic agents matures through the development of lentiviral-based vectors, it is hoped that they will eventually reach the clinic where they may not only supplement the current treatment strategies but also provide resistance to those at high risk of infection or failing therapy.

Animals↗

PubMed2003

Control of HIV-1 env RNA splicing and transport: investigating the role of hnRNP A1 in exon splicing silencer (ESS3a) function.

The control of HIV-1 viral RNA splicing and transport plays an important role in the successful replication of the virus. Previous studies have identified both an exon splicing enhancer (ESE) and a bipartite exon splicing silencer (ESS3a and ESS3b) within the terminal exon of HIV-1 that are involved in modulating both splicing and Rev-mediated export of viral RNA. To define the mechanism of ESS3a function, experiments were carried out to better define the cis and trans components required for ESS3a activity. Mutations throughout the 30-nt element resulted in partial loss of ESS function. Combining mutations was found to have an additive effect, suggesting the presence of multiple binding sites. Analysis of interacting factors identified hnRNP A1 as one component of the complex that modulates ESS3a activity. However, subsequent binding analyses determined that hnRNP A1 interacts with only one portion of ESS3a, suggesting the involvement of another host factor. Parallel analysis of the effect of the mutations on Rev-mediated export determined that there is not a direct correlation between the effect of the mutations on splicing and RNA transport. Consistent with this hypothesis, replacement of ESS3a with consensus hnRNP A1 binding sites was found to be insufficient to block Rev-mediated RNA export.

Base Sequence↗

PubMed2003

Mapping of determinants required for the function of the HIV-1 env nuclear retention sequence.

Control of HIV-1 RNA processing and transport are critical to the successful replication of the virus. In previous work, we identified a region within the HIV-1 env that is involved in mediating nuclear retention of unspliced viral RNA. To define this sequence further and identify elements required for function, deletion mutagenesis was carried out. Progressive 5' and 3' deletions map the nuclear retention sequence (NRS) within the intron between nts 8281 and 8381. While deletion of sequences comprising the 3'ss had no effect, removal of the 5'ss resulted in cytoplasmic accumulation of unspliced RNA. Sequence analysis determined that the region corresponding to the NRS is highly conserved among HIV-1 strains. To evaluate whether this NRS interacts with cellular factors, RNA electrophoretic mobility shift assays (REMSA) were performed. We show that the NRS specifically interacts with cellular factors present in HeLa nuclear extracts, and, by UV crosslinking, correlates with the binding of a 49-kDa protein. Immunoprecipitation of the UV crosslinked products determined that this 49-kDa protein corresponds to hnRNP C.

Cell Nucleus↗

PubMed2003

Induction of HIV-1 long terminal repeat-mediated transcription by Neisseria gonorrhoeae.

Gonorrhoea enhances the transmission of HIV through increased viral shedding and the increased probability of seroconversion among previously HIV-negative individuals. However, the mechanism(s) underlying these influences remain poorly understood. We demonstrated that exposure to Neisseria gonorrhoeae induces the nuclear factor kappa B-dependent transcription from the HIV-1 long terminal repeat in derivatives of the Jurkat CD4 T cell line. These data suggest that gonococcal infection directly impacts HIV-1 transmission through the localized stimulation of viral expression.

CD4-Positive T-Lymphocytes↗

PubMed2002

Positive and negative modulation of human immunodeficiency virus type 1 Rev function by cis and trans regulators of viral RNA splicing.

Expression of the entire complement of human immunodeficiency virus type 1 (HIV-1) viral proteins depends on the competing activities of viral RNA splicing and export into the cytoplasm by Rev. To investigate the possibility that modulation of viral RNA metabolism may alter Rev function, we analyzed the impact of multiple SR proteins on both processes. While overexpression of several of the SR factors altered splicing of HIV-1 env mRNA, they had disparate effects on Rev function that varied with the cell line used. Subsequent examination of exon splicing enhancer (ESE) and/or silencer (ESS) deletions suggests that the effects of the SR proteins on Rev function are not mediated through interaction with these elements. However, analysis of the deletions did indicate that the ESE and/or ESS does have significant effects on Rev function, with deletion of the ESS augmenting the magnitude of the response to Rev and deletion of the ESE significantly reducing it. In situ hybridization and reverse transcription-PCR indicated that the loss of Rev response upon deletion of the ESE was due to a failure of Rev to induce transport of the unspliced RNA into the cytoplasm. Together, the data indicate that cellular splicing factors and viral regulatory elements can have significant stimulatory and inhibitory effects on Rev function, raising the possibility that cells can be rendered permissive or nonpermissive for virus replication by modulation of splicing activities.

Animals↗