Abstract
Purpose: Like all viruses, human immunodeficiency virus type 1 (HIV-1) requires host cellular factors for productive replication. Identification of these factors may lead to the development of novel cell-based inhibitors. Experimental design: A Strep-tag was inserted into the C-terminus of the matrix (MA) region of the HIV-1 gag gene. The resultant virus was replication competent and used to infect Jurkat T-cells. MA complexes were affinity purified with Strep-Tactin agarose. Protein quantification was performed using sequential window acquisition of all theoretical fragment ion spectra (SWATH) MS, data were log2-transformed, and Student t-tests with Bonferroni correction used to determine statistical significance. Several candidate proteins were validated by immunoblot and investigated for their role in virus infection by siRNA knockdown assays. Results: A total of 17 proteins were found to be statistically different between the infected versus uninfected and untagged control samples. X-ray repair cross-complementing protein 6 (Ku70), X-ray repair cross-complementing protein 5 (Ku80), and Y-box binding protein 1 (YB-1) were confirmed to interact with MA by immunoblot. Knockdown of two candidates, EZRIN and Y-box binding protein 1, enhanced HIV infection in vitro. Conclusions and clinical relevance: The Strep-tag allowed for the capture of viral protein complexes in the context of virus replication. Several previously described factors were identified and at least two candidate proteins were found to play a role in HIV-1 infection. These data further increase our understanding of HIV host -cell interactions.
| Original language | English |
|---|---|
| Pages (from-to) | 156-163 |
| Number of pages | 8 |
| Journal | Proteomics - Clinical Applications |
| Volume | 10 |
| Issue number | 2 |
| DOIs | |
| State | Published - Feb 1 2016 |
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All Science Journal Classification (ASJC) codes
- Clinical Biochemistry
Cite this
Investigation of the HIV-1 matrix interactome during virus replication. / Li, Yan; Frederick, Kristin M.; Haverland, Nicole A.; Ciborowski, Pawel; Belshan, Michael A.
In: Proteomics - Clinical Applications, Vol. 10, No. 2, 01.02.2016, p. 156-163.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Investigation of the HIV-1 matrix interactome during virus replication
AU - Li, Yan
AU - Frederick, Kristin M.
AU - Haverland, Nicole A.
AU - Ciborowski, Pawel
AU - Belshan, Michael A.
PY - 2016/2/1
Y1 - 2016/2/1
N2 - Purpose: Like all viruses, human immunodeficiency virus type 1 (HIV-1) requires host cellular factors for productive replication. Identification of these factors may lead to the development of novel cell-based inhibitors. Experimental design: A Strep-tag was inserted into the C-terminus of the matrix (MA) region of the HIV-1 gag gene. The resultant virus was replication competent and used to infect Jurkat T-cells. MA complexes were affinity purified with Strep-Tactin agarose. Protein quantification was performed using sequential window acquisition of all theoretical fragment ion spectra (SWATH) MS, data were log2-transformed, and Student t-tests with Bonferroni correction used to determine statistical significance. Several candidate proteins were validated by immunoblot and investigated for their role in virus infection by siRNA knockdown assays. Results: A total of 17 proteins were found to be statistically different between the infected versus uninfected and untagged control samples. X-ray repair cross-complementing protein 6 (Ku70), X-ray repair cross-complementing protein 5 (Ku80), and Y-box binding protein 1 (YB-1) were confirmed to interact with MA by immunoblot. Knockdown of two candidates, EZRIN and Y-box binding protein 1, enhanced HIV infection in vitro. Conclusions and clinical relevance: The Strep-tag allowed for the capture of viral protein complexes in the context of virus replication. Several previously described factors were identified and at least two candidate proteins were found to play a role in HIV-1 infection. These data further increase our understanding of HIV host -cell interactions.
AB - Purpose: Like all viruses, human immunodeficiency virus type 1 (HIV-1) requires host cellular factors for productive replication. Identification of these factors may lead to the development of novel cell-based inhibitors. Experimental design: A Strep-tag was inserted into the C-terminus of the matrix (MA) region of the HIV-1 gag gene. The resultant virus was replication competent and used to infect Jurkat T-cells. MA complexes were affinity purified with Strep-Tactin agarose. Protein quantification was performed using sequential window acquisition of all theoretical fragment ion spectra (SWATH) MS, data were log2-transformed, and Student t-tests with Bonferroni correction used to determine statistical significance. Several candidate proteins were validated by immunoblot and investigated for their role in virus infection by siRNA knockdown assays. Results: A total of 17 proteins were found to be statistically different between the infected versus uninfected and untagged control samples. X-ray repair cross-complementing protein 6 (Ku70), X-ray repair cross-complementing protein 5 (Ku80), and Y-box binding protein 1 (YB-1) were confirmed to interact with MA by immunoblot. Knockdown of two candidates, EZRIN and Y-box binding protein 1, enhanced HIV infection in vitro. Conclusions and clinical relevance: The Strep-tag allowed for the capture of viral protein complexes in the context of virus replication. Several previously described factors were identified and at least two candidate proteins were found to play a role in HIV-1 infection. These data further increase our understanding of HIV host -cell interactions.
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U2 - 10.1002/prca.201400189
DO - 10.1002/prca.201400189
M3 - Article
VL - 10
SP - 156
EP - 163
JO - Proteomics - Clinical Applications
JF - Proteomics - Clinical Applications
SN - 1862-8346
IS - 2
ER -