Effect of Escherichia coli Chaperonin GroELS on Heterologously Expressed Human Immunodeficiency Virus Type 1 Reverse Transcriptase In Vivo and In Vitro

Abstract

The two subunits of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (HIV-1 RT), p66 and p51, were coexpressed in Escherichia coli along with the E. coli chaperonin system GroEL/GroES. Coexpression increases the yield of heterodimeric HIV-1 RT by a factor of 4 to 5 and improves the nucleic acid binding affinity of HIV-1 RT by a factor of 1.6. We have analyzed the reasons for the improvements. The total increase in yield of HIV-1 RT can be attributed to an accumulation of RT subunits in the cells (factor of about 2.8) and an increased growth of the E. coli cells (factor of about 1.4). One reason for the accumulation in the cells is an improved stability of HIV-1 RT subunits toward bacterial proteases. In vitro studies showed that the nucleic acid binding affinity of HIV-1 RT purified from cells that did not coexpress GroELS was stimulated by adding purified GroELS (approx 1.5-fold), whereas HIV-1 RT stemming from cells coexpressing GroELS was stimulated only marginally (approx 1.1-fold). The in vivo as well as the in vitro studies suggest that the chaperonin interacts with HIV-1 RT and therefore affects the folding process of HIV-1 RT.

Efficient Concerted Integration by Recombinant Human Immunodeficiency Virus Type 1 Integrase without Cellular or Viral Cofactors

“…The purification procedure involved lysis of bacteria without nonionic detergent, low-salt washes of "insoluble" IN-DNA pellet fractions followed by high-salt extraction of IN, and further purification by column chromatography (33). In the second approach, we examined the effect of coexpressing protein chaperones (GroEL and GroES) (16,23,30) with HIV-1 IN in bacteria to investigate if possible protein-folding defects in IN are corrected. With both approaches, highly purified recombinant HIV-1 IN was capable of efficiently performing both the half-site and full-site integration reactions nearly equivalently without the addition of purified cellular or viral proteins.…”

Diversity in heat shock protein families: functional implications in virus infection with a comprehensive insight of their role in the HIV-1 life cycle

“…The co-expression of two subunits of HIV-1 reverse transcriptase (HIV-1 RT) i.e. p66 and p55, with Escherichia coli chaperonin complex GroEL-GroES in E. coli , reportedly improved the yield and nucleic acid–binding activity of HIV-1 RT (Maier et al 2000 ). The human HSP60 has been demonstrated to interact with the catalytic core domain of HIV-1 integrase (IN) and is also able to stimulate the IN activity.…”