Given the limited genetic coding capacity of HIV-1, it is reasonable to expect that the virus must interact with an extensive battery of cellular factors to complete its passage through the cell. Indeed, it is remarkable that the viral genome, comprising only about 0.0003% of the entire genetic capacity of the cell, commandeers the cellular environment to its own advantage. However, to date and despite being easily the most studied virus, only a relatively small group of cellular proteins have been shown essential for HIV-1 propagation. A genetic/proteomic approach has been developed that utilizes a random mutagenic protocol followed by a stringent step of biological selection in efforts to pinpoint those rare sites within the HIV-1 proteome that can accommodate the incorporation of a potent affinity tag without significant loss of those structure/function relationships that are required for viral replication-competency. In conjunction with a novel cryogenic methodology to capture and preserve transient viral-host interactions during immunoisolation, we have recovered several host proteins in association with the replicating virus but previously obscured from conventional scientific investigation. We are reassured that our method is sound since among the cellular proteins recovered and identified using our methodologies are those previously determined by other groups to interact with HIV-1. Furthermore, a straightforward mass spectrometric technique is presented that discriminates with high accuracy between specific and nonspecific proteins in immunoisolated protein complexes. The combined genetic and proteomic method described is a powerful tool set that can be utilized to identify cellular proteins in transient association with a given viral protein at points during the viral life cycle, a subset of which may be absolutely required by the virus for its livelihood but in part, dispensable by the host cell.

Public Health Relevance

Given the known complexity of biological processes within the cell as well as the rigid constraints imposed by the small coding size of the HIV-1 genome, it is reasonable to expect that the HIV-1 proteome must rely upon a battery of host factor arrays to complete its intracellular tasks. In an effort to recover and identify requisite host proteins that interact in complex with the viral machinery, we have developed a systematic method to select derivatives that can encode a small, but potent, foreign epitope tag yet remain fully replication-competent in culture. In conjunction with a novel cryogenic methodology to capture and preserve transient viral-host interactions, we have recovered and identified new sets of host proteins existing at the viral/host interface (in association with HIV-1 but previously obscured from conventional scientific investigation), a subset of which may have the potential to provide a new targets for small molecule intervention against this virus.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI081615-05
Application #
8415922
Study Section
AIDS Discovery and Development of Therapeutics Study Section (ADDT)
Program Officer
Stansell, Elizabeth H
Project Start
2009-02-15
Project End
2015-01-31
Budget Start
2013-02-01
Budget End
2015-01-31
Support Year
5
Fiscal Year
2013
Total Cost
$420,341
Indirect Cost
$190,017
Name
Aaron Diamond AIDS Research Center
Department
Type
DUNS #
786658872
City
New York
State
NY
Country
United States
Zip Code
10016
Luo, Yang; Muesing, Mark A (2014) Mass spectrometry-based proteomic approaches for discovery of HIV-host interactions. Future Virol 9:979-992
Trinité, Benjamin; Chan, Chi N; Lee, Caroline S et al. (2014) Suppression of Foxo1 activity and down-modulation of CD62L (L-selectin) in HIV-1 infected resting CD4 T cells. PLoS One 9:e110719