Mapping of determinants involved in the stimulation of HIV-1 expression by Sam68

McLaren, Meredith; Cochrane, Alan

doi:10.1016/j.virol.2008.10.049

Cited by 6 publications

(4 citation statements)

References 49 publications

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“…This may in part explain why mutations in ESE3 were found to influence Rev activity. 59 Like nucleolin, and sometimes together with nucleolin, hnRNP K increases the stability of cellular mRNAs. 29 It may therefore stabilize HIV-1 mRNAs containing the SLS2-A7 sequence.…”

Section: Methodsmentioning

confidence: 99%

Identification of protein partners of the human immunodeficiency virus 1tat/revexon 3 leads to the discovery of a new HIV-1 splicing regulator, protein hnRNP K

Marchand¹,

Santerre²,

Aigueperse³

et al. 2011

RNA Biology

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HIV-1 pre-mRNA splicing depends upon 4 donor and 8 acceptor sites, which are used in combination to produce more than 40 different mRNAs. The acceptor site A7 plays an essential role for tat and rev mRNA production. The SLS2-A7 stem-loop structure containing site A7 was also proposed to modulate HIV-1 RNA export by the Rev protein. To further characterize nuclear factors involved in these processes, we purified RNP complexes formed by incubation of SLS2-A7 RNA transcripts in HeLa cell nuclear extracts by affinity chromatography and identified 33 associated proteins by nanoLC-MS/MS. By UV cross-linking, immunoselection and EMSA, we showed that, in addition to the well-known hnRNP A1 inhibitor of site A7, nucleolin, hnRNP H and hnRNP K interact directly with SLS2-A7 RNA. Nucleolin binds to a cluster of successive canonical NRE motifs in SLS2-A7 RNA, which is unique in HIV-1 RNA. Proteins hnRNP A1 and hnRNP K bind synergistically to SLS2-A7 RNA and both have a negative effect on site A7 activity. By the use of a plasmid expressing a truncated version of HIV-1 RNA, we showed a strong effect of the overexpression of hnRNP K in HeLa cells on HIV-1 alternative splicing. As a consequence, production of the Nef protein was strongly reduced. Interestingly also, many proteins identified in our proteomic analysis are known to modulate either the Rev activity or other mechanisms required for HIV-1 multiplication and several of them seem to be recruited by hnRNP K, suggesting that hnRNP K plays an important role for HIV-1 biology.

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Section: Methodsmentioning

confidence: 99%

Identification of protein partners of the human immunodeficiency virus 1tat/revexon 3 leads to the discovery of a new HIV-1 splicing regulator, protein hnRNP K

Marchand¹,

Santerre²,

Aigueperse³

et al. 2011

RNA Biology

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“…Sam68 enhances protein expression from the intron-containing HIV-1 RNA to a much greater extent than the RNA nuclear export itself in a Gag-Pol reporter system (Coyle et al 2003). On the other hand, Δ410, a NLSdeleted Sam68 mutant, inhibits HIV-1 RNA translation through sequestering the unspliced HIV-1 RNA at the perinulcear bundles from the translation machinery (Soros et al 2001) or altering the viral ribonucleoprotein (RNP) complex (Marsh et al 2008;McLaren and Cochrane 2009). Our recent studies have also shown that Sam68 enhances and Δ410 inhibits HIV-1 nef mRNA translation through stress granule formation ).…”

Section: Sam68 Function In Hiv-1 Rna Translationmentioning

confidence: 99%

Translational Regulation of HIV-1 Replication by HIV-1 Rev Cellular Cofactors Sam68, eIF5A, hRIP, and DDX3

Liu

Henao‐Mejia

Líu

et al. 2011

J Neuroimmune Pharmacol

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Nuclear export and translation of HIV-1 RNA are two important posttranscriptional events for HIV-1 gene expression and replication. HIV-1 Rev functions to export unspliced and incompletely spliced HIV-1 RNA from the nucleus to the cytoplasm; it requires interaction with several cellular cofactors such as Sam68, eIF5A, hRIP, and DDX3. Meanwhile, some studies have also implicated Rev and some of its cofactors such as Sam68 in HIV-1 RNA translation. Thus, in this study, we aimed to characterize the potential function of all these four Rev cofactors in HIV-1 RNA translation. Ectopic expression, siRNA knockdown, and trans-complementation assays confirmed that all these cofactors were very important for HIV-1 gene expression and production through Rev and, accordingly, Rev-dependent reporter gene expression. Importantly, these studies revealed for the first time that each of these cofactors also regulated Rev-independent reporter gene expression. To directly determine the roles of these cofactors in HIV-1 RNA translation, we designed and synthesized a full-length capped HIV-1 RNA in vitro, transfected it into cells to bypass the RNA nuclear export step, and determined HIV-1 Gag expression from the cytoplasmic RNA in the cells that had ectopically expressed or siRNA knocked down cofactors. Gag expression was found to closely correlate with the expression levels of all these cofactors. Furthermore, we took advantage of a HIV-1 internal ribosomal entry site (IRES)-based bicistronic reporter gene assay and determined the effects of these cofactors on cap-independent IRES-mediated HIV-1 translation. The results showed that DDX3, eIF5A, and hRIP enhanced HIV-1 IRES-mediated translation, whereas Sam68 did not. Taken together, these results show that HIV-1 Rev cofactors Sam68, eIF5A, hRIP, and DDX3 also function in the translation of HIV-1 RNA and suggest that the regulatory mechanisms of HIV-1 RNA translation are likely different among these cofactors.

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“…In contrast, the MPMV CTE has been shown to require host proteins, Tap (Nxf1), p15 (Nxt1), Sam68 [15], [16], [17] and the RNA helicase Dbp5 [14]. Sam68 may be involved in other aspects of HIV-1 gene expression including the Rev-RRE-Crm1 pathway [18], [19], [20], [21]. An HIV-1 packaging system based on MPMV CTE would not only allow one to target Rev function using dominant negative proteins or RNA based approaches, but also host proteins unique to the Rev-RRE-Crm1 RNA transport pathway [22].…”

Section: Introductionmentioning

confidence: 99%

Rev-Free HIV-1 Gene Delivery System for Targeting Rev-RRE-Crm1 Nucleocytoplasmic RNA Transport Pathway

Srinivasakumar

2011

PLoS ONE

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The use of RNA transport elements from different viruses can provide novel attributes to HIV-1-based gene delivery systems such as improved safety or Rev independence. We previously described an HIV-1 based gene delivery system that utilized the simian immunodeficiency virus Rev-response element (RRE) in place of the HIV-1 RRE. Despite the use of Rev for the production of vector stocks, we showed the utility of this system for delivery of Rev M10, a dominant-negative mutant of HIV-1 Rev, into T-cells. Here, we investigated the use of RNA transport elements from Mason-Pfizer monkey virus or MPMV for the creation of high-titered Rev-free HIV-1-based packaging systems. The HIV-1 gag/pol expression constructs containing one or more copies of MPMV constitutive RNA transport element (CTE) were used to package similarly modified gene-transfer vectors in the presence or absence of Rev. An inverse correlation between the number of CTE modules and Rev dependency was noted for vector stock production. While packaging systems containing multiple CTEs were resistant to exogenously expressed Rev M10, the titers of vectors encoding Rev M10 were nevertheless reduced in comparison to vectors encoding only green fluorescent protein (GFP). In contrast, a gene transfer vector encoding the Rev M10 transgene and containing both RNA transport elements exhibited almost no loss in titer in comparison to a corresponding vector encoding only GFP. The optimized Rev-independent gene delivery system was used for delivery of Rev M10 transgene into T-lymphocytes. Upon challenge in single round infection assays with HIV-1, the modified T-cells produced fewer virus particles than control cells expressing GFP. This Rev-free packaging system may prove useful for targeting the Rev-RRE-Crm1 nucleocytoplasmic RNA transport pathway for inhibiting HIV replication.

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Mapping of determinants involved in the stimulation of HIV-1 expression by Sam68

Cited by 6 publications

References 49 publications

Identification of protein partners of the human immunodeficiency virus 1tat/revexon 3 leads to the discovery of a new HIV-1 splicing regulator, protein hnRNP K

Identification of protein partners of the human immunodeficiency virus 1tat/revexon 3 leads to the discovery of a new HIV-1 splicing regulator, protein hnRNP K

Translational Regulation of HIV-1 Replication by HIV-1 Rev Cellular Cofactors Sam68, eIF5A, hRIP, and DDX3

Rev-Free HIV-1 Gene Delivery System for Targeting Rev-RRE-Crm1 Nucleocytoplasmic RNA Transport Pathway

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