Host Protein Ku70 Binds and Protects HIV-1 Integrase from Proteasomal Degradation and Is Required for HIV Replication

Zheng, Yingfeng; Ao, Zhujun; Wang, Binchen; Jayappa, Kallesh D.; Yao, Xiaojian

doi:10.1074/jbc.m110.184739

Cited by 70 publications

(117 citation statements)

References 70 publications

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“…In addition to Gemin2, several other host proteins have been shown to interact with HIV-1 IN (for a review, see reference 78). Some of these host proteins, such as Ku70 and dynein, can bind to the IN CTD, and their interactions with IN appear to play a role during reverse transcription (79,80). We do not know at present if these INbinding host proteins can affect positively the RT-IN interaction or compete with RT for IN binding.…”

Section: Characterization Of Mutant In Viruses To Determine Functionamentioning

confidence: 93%

Interaction between Reverse Transcriptase and Integrase Is Required for Reverse Transcription during HIV-1 Replication

Tekeste

Wilkinson

Weiner

et al. 2015

J Virol

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Human immunodeficiency virus type 1 (HIV-1T o establish an infection after entry into a susceptible cell, human immunodeficiency virus type 1 (HIV-1) has to reverse transcribe its RNA genome to double-stranded DNA, followed by integration into the host genome. Reverse transcriptase (RT) and integrase (IN) are the viral enzymes responsible for catalyzing the essential steps of reverse transcription and integration, respectively. Both enzymes are synthesized as part of the Gag-Pol polyprotein, which is later processed by the viral protease to produce active RT and IN during HIV-1 maturation (1, 2). RT is a heterodimeric enzyme consisting of 66-and 51-kDa subunits and catalyzes the RNA-and DNA-dependent reverse transcription of the viral RNA genome into double-stranded cDNA through a complex cascade of events (3, 4). The 32-kDa IN has three domains: an N-terminal zinc-binding domain, a catalytic core domain, and a C-terminal domain (CTD) that binds DNA nonspecifically. IN catalyzes the integration of the viral cDNA into the host genome in two steps: an initial 3=-end processing step that removes two nucleotides at each 3= end and exposes a highly conserved CA 5= overhang, followed by a strand transfer step that inserts both processed viral DNA ends into the host cell genome (5, 6). In vitro, IN can also catalyze a reverse reaction, termed disintegration, resolving a DNA mimic of the viral-host DNA intermediate to products corresponding to a 3= processed viral DNA end and a target duplex DNA (7). IN can multimerize and forms a complex with viral DNA ends, termed the intasome (8-10). Structural studies of the prototype foamy virus (PFV) intasome found the tetramer to be the active IN configuration (9,11,12). HIV-1 IN has also been proposed to function as a tetramer (10,(13)(14)(15)(16)(17).Mutations in

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Section: Characterization Of Mutant In Viruses To Determine Functionamentioning

confidence: 93%

Interaction between Reverse Transcriptase and Integrase Is Required for Reverse Transcription during HIV-1 Replication

Tekeste

Wilkinson

Weiner

et al. 2015

J Virol

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“…Similarly, the ATP-dependent RNA helicase A (DHX9) has been hypothesized to play a role in HIV-1 reverse transcription (31) by facilitating tRNA annealing onto the viral genomic RNA (32). Also detected was XRCC6 (also known as Ku70), a subunit of the ATPdependent complex that protects HIV-1 integrase from the host ubiquitin-proteasome system and, together with PRKDC, associates with HIV-1 preintegration complexes (33,34). Finally, our mass spectrometry analysis may have missed some important factors, as proteins smaller than 30 kDa were not included due to poor protein resolution following SDS/PAGE.…”

Section: Discussionmentioning

confidence: 99%

Eukaryotic elongation factor 1 complex subunits are critical HIV-1 reverse transcription cofactors

Warren

Wei

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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Cellular proteins have been implicated as important for HIV-1 reverse transcription, but whether any are reverse transcription complex (RTC) cofactors or affect reverse transcription indirectly is unclear. Here we used protein fractionation combined with an endogenous reverse transcription assay to identify cellular proteins that stimulated late steps of reverse transcription in vitro. We identified 25 cellular proteins in an active protein fraction, and here we show that the eEF1A and eEF1G subunits of eukaryotic elongation factor 1 (eEF1) are important components of the HIV-1 RTC. eEF1A and eEF1G were identified in fractionated human T-cell lysates as reverse transcription cofactors, as their removal ablated the ability of active protein fractions to stimulate late reverse transcription in vitro. We observed that the p51 subunit of reverse transcriptase and integrase, two subunits of the RTC, coimmunoprecipitated with eEF1A and eEF1G. Moreover eEF1A and eEF1G associated with purified RTCs and colocalized with reverse transcriptase following infection of cells. Reverse transcription in cells was sharply down-regulated when eEF1A or eEF1G levels were reduced by siRNA treatment as a result of reduced levels of RTCs in treated cells. The combined evidence indicates that these eEF1 subunits are critical RTC stability cofactors required for efficient completion of reverse transcription. The identification of eEF1 subunits as unique RTC components provides a basis for further investigations of reverse transcription and trafficking of the RTC to the nucleus. purification | mass spectrometry | cellular factor | siRNA knock down | virus replication T he HIV type-1 (HIV-1) reverse transcriptase (RT) enzyme carries out reverse transcription through DNA polymerase and RNase H activities, which mediate a sequential series of steps that include the initiation of DNA synthesis producing negative strand strong stop DNA (−ssDNA), full-length negative-strand DNA, positive-strand DNA, and intact preintegrative doublestrand DNA (reviewed elsewhere 1). The ability of HIV-1 to efficiently produce early reverse transcription products in vitro has been described (2). However, under in vitro conditions, the production of late reverse transcription products is less efficient than that observed in HIV-1-infected cells (3, 4), suggesting that host cellular factors are required. After entry into the host cell cytoplasm, the HIV-1 core, containing the viral genomic RNA, RT, and integrase (IN), reorganizes to form the reverse transcription complex (RTC). The RTC requires both IN and RT to be active (5), and they are believed to recruit cellular factors to facilitate DNA synthesis (6). Although two-hybrid library and genomewide RNAi screening methods have implicated more than 50 cellular proteins as important for reverse transcription (6, 7), whether any of these cellular proteins are RTC components, or whether they direct the RTC in other ways, is unclear.Attempts to purify and to identify cellular RTC cofactors by more conventional methods ...

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“…In contrast, it is well known that HIV-1 IN is a metabolically unstable protein and subject to proteasome-mediated degradation via ubiquitination (36). Although IN is likely to escape from the degradation through interaction with other cellular proteins, such as Ku70 (51,54), the E3 ubiquitin ligase that leads IN into proteasomal degradation remains undetermined (51). This study presented a pilot screening using a limited number of E3 ubiquitin ligases to document the feasibility of proteomic screening for the PIC modulators.…”

Section: Discussionmentioning

confidence: 99%

Identification of RFPL3 Protein as a Novel E3 Ubiquitin Ligase Modulating the Integration Activity of Human Immunodeficiency Virus, Type 1 Preintegration Complex Using a Microtiter Plate-based Assay

Tan

Suzuki

Takahashi

et al. 2014

Journal of Biological Chemistry

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Background:The retroviral preintegration complex (PIC) exhibits full fidelity of integration in vitro. Results: Application of a protein library to the microplate-based integration assay identified RFPL3 as an enhancer of HIV-1 PIC activity. Conclusion: This screening platform permits efficient identification of cellular factors modulating PIC. Significance: This approach provides an advantage in advancing our understanding of virus-host interactions in retrovirus integration.

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Host Protein Ku70 Binds and Protects HIV-1 Integrase from Proteasomal Degradation and Is Required for HIV Replication

Abstract: HIV-1 integrase (IN) is

Cited by 70 publications

References 70 publications

Interaction between Reverse Transcriptase and Integrase Is Required for Reverse Transcription during HIV-1 Replication

Interaction between Reverse Transcriptase and Integrase Is Required for Reverse Transcription during HIV-1 Replication

Eukaryotic elongation factor 1 complex subunits are critical HIV-1 reverse transcription cofactors

Identification of RFPL3 Protein as a Novel E3 Ubiquitin Ligase Modulating the Integration Activity of Human Immunodeficiency Virus, Type 1 Preintegration Complex Using a Microtiter Plate-based Assay

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