Some 30-40 million people are living with Human immunodeficiency virus, HIV. Other retroviruses account for many more infections, and emerging infectious diseases are on the rise. Unfortunately, antiretroviral resistance develops in the presence of the selective pressure of drug exposure. Vaccine development has been problematic. A novel, validated target of intervention for retrovirus infection is the virus'dependence on the recruitment of a specific host cell transfer RNA (tRNA). The host tRNA is recruited by viral proteins into the new viral particles. When the virus infects the very next cell, this tRNA becomes the primer for replication of its RNA genome through reverse transcription. Recently, small proteins, peptides of 15 and 16 amino acids, have been selected from vast libraries of peptides for their abilities to bind specifically the one human tRNA, tRNALys3, that is recruited by HIV, and other lentiviruses for priming reverse transcription. Thus, the peptides mimic HIV viral proteins in their specificity for tRNALys3. The long-term objectives of this program are to understand and exploit the dedicated recruitment of human tRNALys3 by HIV, and to develop peptides as tools for designing new small molecule therapeutics that will inhibit the recruitment, and the peptides themselves as putative therapeutics. The experimental specific aims of the revised project are: 1) The peptides bind the anticodon domain of human tRNALys3 (ASLLys3) with high affinity and specificity characteristic of HIV proteins that recruit the tRNA in vivo. The RNA binding properties of those peptides from 20 that have the highest affinities and specificities will be characterized in detail. 2) Peptide affinity and specificity will compete with one or more of the HIV proteins involved in the recruitment of tRNALys3. Using electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry, gel mobility shifts, and fluorescence the abilities of the peptides to mimic HIV proteins involved in recruiting the tRNALys3 will be determined. As a result of the two Specific Aims, the HIV protein/htRNALys3 interaction will be elucidated, and peptides will have been developed as proven tools for investigating viral protein/host cell RNA interactions that are critical to virus replication. The discovered peptides could be progenitors of therapeutics and/or tools in the development of small candidate drugs.
Globally, over 30 million adults and children were living with HIV/AIDS in 2006. Existing antivirals target HIV protein function such as replication (reverse transcriptase) and HIV polyprotein hydrolysis (protease). Almost all experimental therapeutics target the function of an HIV protein. Antiretroviral resistance develops when viral replication continues in the presence of the selective pressure of drug exposure, especially when the drugs are directed against the same or similar targets. Unfortunately to date, vaccine development has been problematic. New paradigms are required that reduce the probability of antiviral resistance. Novel targets for intervention in retrovirus infection are sought in which the development of resistance will be difficult. All retroviruses use a host cell tRNA species as the primer for reverse transcription (RT) of their single stranded RNA genomes into double stranded DNA. HIV is among the lentiviruses that use the host tRNALys3 species as the primer of RT. The anticodon stem and loop domain (ASLLys3) and the posttranscriptional modifications of mammalian tRNALys3 are the most chemically interesting and distinct parts of the tRNA. The extensive modification chemistry of ASLLys3 distinguishes the tRNA from the other two lysine isoacceptors and from other tRNAs, in general. These chemistries and the anticodon domain conformation they generate are probable identity elements for the recognition of the tRNA by viral proteins. Lentiviruses are so strongly dedicated to the use of tRNALys3 that no single mutation at the primer binding site (PBS) is sufficient to produce a lasting substitution of another human tRNA as a primer. Thus, the viral recruitment and preparation of human tRNALys3 as primer for reverse transcription are persistent characteristics that are unique to HIV and other lentivirus infections and replication. HIV's dedication to this single human tRNA species validates the viral protein Interaction with the human tRNA as a possible target of intervention (1). Because of the central role of the tRNALys3 in cellular protein synthesis, as well as in viral replication and because of the cell viability required of the virus, it would be quite difficult for the virus to develop resistance to a therapeutic intervention that targets this HIV protein/human tRNA interaction. The recruitment, packaging and function of tRNALys3 in the initiation of reverse transcriptase and genome replication have been reviewed quite recently (1). However, there are very significant gaps in our understanding of the chemical identities and structural interactions involved in the recruitment, denaturation and annealing of tRNALys3 to the HIV genome by HIV proteins. Thus, it is imperative to understand the molecular and structural biology of novel targets and to develop other approaches to drug design for HIV specifically, and for retroviruses, in general. We are interested in the unique molecular and structural biology of HIV's protein recognition of tRNALys3 as a novel target of intervention
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