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Joshua Madsen


Mentor: C. Martin Stoltzfus, Ph.D.
Lab Phone: 335-7819

Analysis of the dependency of HIV-1 replication upon the presence of Exonic Splicing Silencers with the viral genome.

Over 40 unique viral mRNAs are expressed in an HIV-1 infected T-cell by alternative splicing of a precursor viral mRNA. Individual viral mRNAs are not present at similar levels, suggesting that utilization of the nine acceptor splice sites within the viral genomic mRNA occurs with unequal efficiency. Our lab has identified three negative regulatory elements, known as Exonic Splicing Silencers (ESS), containing binding sites for members of the cellular hnRNPA/B family of proteins, inhibiting spliceosome assembly at the respective acceptor splice site.

We have previously shown that ESS2, which regulates splicing at aceptor splice site A3, is conserved in Group M HIV-1 but not Group O HIV-1. Analysis of HIV-1 mRNA by RT-PCR from Group O infected T-cells reveals that splicing at acceptor splice sites A2 and A3 does not occur with greater frequency. This observation suggests that Group O HIV-1 regulates acceptor splice site utilization in manner which differs from Group M HIV-1. We are currently investigating the mechanism by which Group O HIV-1 regulates acceptor splice site utilization at A2 and A3.

To determine if the regulation of acceptor splice site utilization is critical for viral replication we replaced either Group M HIV-1 ESSV or ESS2 with the respective Group O HIV-1 sequence within the infectious plasmid pNL4-3. Transient transfection into 293 cells and RT-PCR of viral mRNA revealed that the Group O HIV-1 sequences at acceptor splice sites A2 and A3 relieved the splicing inhibition at these splice sites, leading to an increase in the inclusion of viral exon 3 and exon 4, respectively. Relief of splicing inhibition at A2 resulted in the inability to produce replication competent viral particles. These results indicate that regulated alternative splicing is critical for efficient HIV-1 replication. We are currently investigating the ramifications of increased splicing at A2 on viral replication.

Abstracts:

Madsen, J.M. and Stoltzfus, C.M. An Exonic Splicing Silencer Downstream of HIV-1 3’ss A2 is Necessary for Viral Replication. All Iowa Virology Symposium, October 2004.

Madsen, J.M. and Stoltzfus, C.M. Negative regulation of alternative splicing at HIV-1 3’ss A2 by ESSV is necessary for productive viral replication. RNA 2004, May 2004.

Madsen, J.M. and Stoltzfus, C.M. An Exonic Splicing Silencer Downstream of HIV-1 3’ss A2 is Necessary for Viral Replication. Cold Spring Harbor Retrovirus Meeting, May 2004.

Madsen, J.M., Domsic, J.K., Vowels, C.R., and Stoltzfus, C.M. Group O HIV-1 strain MVP5180 Regulates 3’ Splice Site Utilization in the Absence of Exonic Splicing Silencers. Cold Spring Harbor Laboratory Retrovirus Meeting. Abstract 158. May 2003.

Publications:

Madsen JM, Stoltzfus CM. A suboptimal 5' splice site downstream of HIV-1 splice site A1 is required for unspliced viral mRNA accumulation and efficient virus replication. Retrovirology. 2006 Feb 3;3:10. PubMed PMID: 16457729; PubMed Central PMCID: PMC1403798.

Stoltzfus CM, Madsen JM. Role of viral splicing elements and cellular RNA binding proteins in regulation of HIV-1 alternative RNA splicing. Curr HIV Res. 2006 Jan;4(1):43-55. Review. PubMed PMID: 16454710.

Madsen JM, Stoltzfus CM. An exonic splicing silencer downstream of the 3' splice site A2 is required for efficient human immunodeficiency virus type 1 replication. J Virol. 2005 Aug;79(16):10478-86. PubMed PMID: 16051840; PubMed Central PMCID: PMC1182660.



Honors and Awards

  • Molecular Biology Retreat Travel Award, 2002. Predoctoral Training Fellowship in Virology, NIH 8/02
  • Teaching: Fall 2002, Animal Viruses Laboratory, Spring 2002, Microbiology for Nurses Laboratory, Fa
  • May 2004, RNA Society Meeting. May 2003, Cold Spring Harbor Laboratory Retrovirus Meeting. June 2