Attempts to combat HIV have been hampered due to the virus's ability to rapidly mutate and produce genetic variants that can circumvent the immune response and resist drug therapy. Most mutations are generated during genome replication. Recombination serves to reassort them, further amplifying genetic diversity. Two viral proteins, reverse transcriptase (RT) and nucleocapsid (NC) have been clearly implicated in these processes. The goal of this proposal is to answer key questions regarding the mechanism of recombination and to better understand how RT and NC have evolved to interact with replication intermediates. In addition, tight binding nucleic acid molecules (aptamers) that could potentially be used as viral inhibitors will be selected.
Three specific aims are listed: (1) To determine if a recombination hotspot in the gag-pol frameshift region discovered in vitro is also a hotspot in cellular infections and whether occurrence of hotspots is dependent on in vitro test conditions;(2) To determine how HIV nucleocapsid protein inhibits priming of DNA synthesis by random sequence RNAs but not polypurine tract RNA;(3): To isolate primer-template sequences that bind RTs (MuLV, AMV, and TY3) with high affinity and determine if like HIV-RT, other RTs also bind their cognate polypurine tract sequences (ppt) very tightly. A combination of in vitro and cell culture approaches will be used for these experiments. For example, in Aim 1 recombination assays will be conducted under both conditions and compared in order to verify conclusions.
Aim 2 builds on preliminary data from our lab showing that NC inhibits non-ppt RNA priming but does not affect the ppt usage. This could be important to preventing spurious priming 2nd strand synthesis and producing proviruses with multiple discontinuities.
Aim 3 is related to aim 2 and will address the possibility that RTs evolved to recognize and bind tightly to ppt sequences that are used for second strand DNA priming. This tight binding could potentially be used to design inhibitors that may be difficult for the virus to escape from.
This work is relevant to human health because it will help identify possible leads for nucleic acid-based drug therapies (aptamers). In addition, it will help define the potential of new specific targets (i.e. ppt, recombination) for drug intervention. This work is relevant to human health because it will help identify possible leads for nucleic acid-based drug therapies (aptamers). In addition, it will help define the potential of new specific targets (NC, ppt, recombination) for drug intervention.
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