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Principal Investigator: LLOYD M SMITH
Organization: UNIVERSITY OF WISCONSIN-MADISON
Fiscal Year: 2024
Award: $532,733
Funding agency: National Institute of Allergy and Infectious Diseases
Project Summary/Abstract
The complexity of the HIV-1 transcriptome has been progressively revealed throughout recent
decades using increasingly advanced RNA sequencing technologies. However, knowledge of the
RNA primary sequence alone has not been sufficient to determine the importance or function of
each of the over 40 highly conserved HIV-1 splice variants, which code for nine known proteins and
polyproteins. RNA-protein interactions are fundamental to RNA fate and function. From
transcription to cellular localization to translation of the gene product, and many steps in between,
proteins interact with RNA to regulate gene expression and, in the case of HIV-1, viral replication.
Notwithstanding the high significance of these splice variants in the HIV-1 life cycle, technologies
for interrogating the functions, interactions, and cellular localizations of individual splice variants
are woefully lacking.
We propose to develop and validate a suite of powerful new tools to interrogate the functions,
interactions, and cellular localizations of individual splice variants of HIV-1. We will develop sensitive
and multiplexed assays (HyPR-MS) to elucidate the protein interactomes of up to 20 conserved HIV-
1 mRNA splice variants. We will develop a branched DNA fluorescence in situ hybridization
technique (SV-FISH) to examine how changes in the expression levels of individual splice variant
protein interactors alter their localization. Finally, we will determine which of the splice variants and
protein interactors are critical to HIV replication and infectivity using siRNA and protein knock-down
and overexpression studies.
These powerful new tools will be used here to elucidate previously unobtainable information about
HIV-1 replication. Once developed, these same novel technologies will comprise a powerful new
toolset that can be applied to splice variant investigations in other viral and cellular systems.
Terms: <0-11 years old><3' Splice Site><AIDS><AIDS Virus><Acquired Immune Deficiency><Acquired Immune Deficiency Syndrome><Acquired Immune Deficiency Syndrome Virus><Acquired Immunodeficiency Syndrome><Acquired Immunodeficiency Syndrome Virus><Alternate Splicing><Alternative RNA Splicing><Alternative Splicing><Artifacts><Cell Body><Cells><Child><Child Youth><Children (0-21)><Classification><Code><Coding System><Complex><DNA><Deoxyribonucleic Acid><Detection><Development><Exons><FISH Technic><FISH Technique><FISH analysis><FISH assay><Fluorescence In Situ Hybridization><Fluorescence Light Microscopy><Fluorescence Microscopy><Fluorescent in Situ Hybridization><Gene Expression><Gene Splicing><Gene Transcription><Genes><Genetic Transcription><Glycoproteins><HIV><HIV Genome><HIV-1><HIV-1 genome><HIV-I><HIV1><HIV1 genome><Host Factor><Host Factor Protein><Human Immunodeficiency Virus Type 1><Human Immunodeficiency Viruses><Human immunodeficiency virus 1><Immune Evasion><Immunoblotting><Immunomodulation><Individual><Integration Host Factors><Intervening Sequences><Introns><Investigation><Knowledge><LAV-HTLV-III><Life Cycle><Life Cycle Stages><Lymphadenopathy-Associated Virus><Maps><Mass Photometry/Spectrum Analysis><Mass Spectrometry><Mass Spectroscopy><Mass Spectrum><Mass Spectrum Analyses><Mass Spectrum Analysis><Messenger RNA><Microscopy><Morphologic artifacts><Network Analysis><Non-Polyadenylated RNA><ORFs><Open Reading Frames><Organism><Pathway Analysis><Persons><Polyproteins><Post-Translational Modification Protein/Amino Acid Biochemistry><Post-Translational Modifications><Post-Translational Protein Modification><Post-Translational Protein Processing><Posttranslational Modifications><Posttranslational Protein Processing><Pre-mRNA><Process><Production><Protein Coding Region><Protein Modification><Proteins><Proteomics><RNA><RNA Expression><RNA Gene Products><RNA Seq><RNA Sequences><RNA Splicing><RNA Stability><RNA and protein interaction><RNA sequencing><RNA, Messenger, Precursors><RNA-Protein Interaction><RNAseq><Research><Resolution><Ribonucleic Acid><Short interfering RNA><Site><Small Interfering RNA><Splice Acceptor Sites><Spliced Genes><Splicing><System><Systematics><Techniques><Technology><Therapeutic><Transcript><Transcription><Translations><Variant><Variation><Viral><Viral Diseases><Viral Gene Products><Viral Gene Proteins><Viral Proteins><Virion><Virus><Virus Diseases><Virus Particle><Virus Replication><Virus-HIV><Western Blotting><Western Immunoblotting><Work><develop a vaccine><develop vaccines><development of a vaccine><developmental><gene product><global gene expression><global transcription profile><immune evasive><immune modulation><immune regulation><immunologic reactivity control><immunomodulatory><immunoregulation><immunoregulatory><improved><in vivo><interest><kids><knock-down><knockdown><life course><living system><mRNA><mRNA Precursor><multiplex assay><new technology><novel technologies><novel virus><overexpress><overexpression><protein blotting><protein expression><resolutions><siRNA><tool><transcriptome><transcriptome sequencing><transcriptomic sequencing><translation><vaccine development><viral infection><viral multiplication><viral replication><virus host interaction><virus infection><virus multiplication><virus protein><virus-induced disease><youngster>