|
Human immunodeficiency virus (HIV-1) is a resilient pathogen causing the depletion of CD4+ T cells and ultimately AIDS disease in infected humans. Recent findings on HIV-1 replication cycle demonstrate that some host factors are necessary for the successful virus replication while other host factors inhibit or restrict virus replication. In this study, we explored the possibility that the heat shock protein 70 (Hsp70) and Hsp90 may play a role in HIV-1 replication. Hsp70 and Hsp90 are members of a large group of heat shock proteins that function as molecular chaperones that have a variety of functions ranging from protein folding to intracellular transportation. Hsp70 has ATPase domain and substrate-binding domain, and promotes proper folding and prevents protein aggregation. Hsp90 is similar in structure to Hsp70 but has a distinct function in extreme environments. To determine the role of Hsp70 and Hsp90 in HIV-1 replication, we knock-downed the levels of Hsp70 and Hsp90 or over-expressed either the wild-type or mutant Hsp70 in human HEK293T cells or HeLa cells, and then examined the infectivity of HIV-1 or the production of HIV-1 capsid protein (p24). Our findings suggest that Hsp70 enhances the production of HIV-1 p24 protein and in contrast Hsp90 has an inhibitory effect on HIV-1 p24 expression.
Human immunodeficiency virus (HIV-1) is a resilient pathogen causing the depletion of CD4+ T cells and ultimately AIDS disease in infected humans. Recent findings on HIV-1 replication cycle demonstrate that some host factors are necessary for the successful virus replication while other host factors inhibit or restrict virus replication. In this study, we explored the possibility that the heat shock protein 70 (Hsp70) and Hsp90 may play a role in HIV-1 replication. Hsp70 and Hsp90 are members of a large group of heat shock proteins that function as molecular chaperones that have a variety of functions ranging from protein folding to intracellular transportation. Hsp70 has ATPase domain and substrate-binding domain, and promotes proper folding and prevents protein aggregation. Hsp90 is similar in structure to Hsp70 but has a distinct function in extreme environments. To determine the role of Hsp70 and Hsp90 in HIV-1 replication, we knock-downed the levels of Hsp70 and Hsp90 or over-expressed either the wild-type or mutant Hsp70 in human HEK293T cells or HeLa cells, and then examined the infectivity of HIV-1 or the production of HIV-1 capsid protein (p24). Our findings suggest that Hsp70 enhances the production of HIV-1 p24 protein and in contrast Hsp90 has an inhibitory effect on HIV-1 p24 expression.
Heat shock proteins are extremely important molecular chaperones and they rescue active proteins from aggregates, securing their functions in a cell. Recent studies show that Hsp70 facilitates the nuclear import of HIV-1 preintegration complexes. Hsp70 appears to have an activity similar to the Viral Protein R (Vpr) of HIV-1 and rescues nuclear import of a Vpr-defective HIV-1 strain. In addition to Hsp70, Hsp60 and Hsc70 were also associated with virions and were incorporated within the virion membrane. The possibility of other Hsp’s involvement in facilitation of HIV replication cycle subsists.
-shRNA-mediated knockdown of Hsp70 and Hsp90
-Transfection
-Infection with HIV-1-GFP and FACS analysis
-Western blot and ELISA
Out of the four 293T stable cell lines, 293T-213 was selected to be used for subsequent experiments since it expressed the least amount of Hsp70 as shown in Figure 3 (b). For Hsp90 knockdown candidates, HeLa 221/222 and 293T 222/223 were selected according to Figures 3 (c) and (d).
The results shown here indicate that the knockdown of Hsp70 or Hsp90 does not affect the infectivity of HIV-1 GFP.
Over-expression of the wild-type or mutant Hsp70 did not affect HIV-1-GFP infectivity compared to the control.
The knockdown of Hsp90 in 293T cells significantly increased the level of HIV-1 p24 protein in the released virus (medium) and in the cell lysates measured by ELISA (Figure 6a, 6b) and by Western blot (Figure 6c)
Overexpression of WT Hsp70 caused a significant increase in the level of HIV-1 p24 protein in the released virus (media) and in the cell lysates, whereas overexpression of Hsp70 mutants (K71E and K71M) did not affect the level of HIV-1 p24.
Overexpression of Hsp70 in 293T cells caused an increase in the level of HIV-1 capsid protein (p24). On the contrary, knock-down of Hsp90 in 293T cells caused an increase in the level of HIV-1 p24. These experimental data suggest that heat shock protein 70 may have a positive role in HIV-1 replication whereas heat shock protein 90 may have a negative role. Consequently, Hsp70’s potentially positive influence on HIV-1 replication might be associated with Hsp70’s chaperone function, which could have facilitated viral protein assembly. How Hsp90 exerts an inhibitory role in HIV-1 p24 production is unknown. Further studies on the mechanisms of how Hsp70 and Hsp90 differentially regulate HIV-1 replication is necessary to fully understand the results from this research.
This work was supported by Ruth L. Kirschstein National Research Service Award and Emory SURE program. I would like to thank Chae Y. Hwang for providing Hsp70 KD stable cell lines and Younglang Lee for instructing ELISA and helping with the gag protein experiment. I would also like to thank Devi Rajan (mentor) and Dr. Song (P.I.) for mentoring the research.
Isabelle, Serguei, and Jianhua Li 2000. Heat shock protein 70 can
replace viral protein R of HIV-1 during nuclear import of the viral
preintegration complex. Experimental Cell Research 259: 398-402.
Goff S. 2007. Host factors exploited by retroviruses. Nature
Reviews 5: 253-263.
Goldberg, Alfred L. 2003. Protein degradaion and protection
against misfolded or damaged proteins. Nature 426: 895-899.
Gurer, Cimarelli, and Jeremy Luban 2002. Specific incorporation of
heat shock protein 70 family members into primate lentiviral
virions. Journal of Virology 76: 4666-4670.
Liberek K, Lewandowska A, and Zietkiewicz S. 2008. Chaperones in
control of protein disaggregation. The EMBO Journal 27: 328-335.
Wolf D, Goff P. 2008. Host restriction factors blocking retroviral
replication. Annual Review of Genetics 42: 142-163.
|
