Evaluating the role of CRM1-mediated export for adenovirus gene expression

Carter, Christoph C; Izadpanah, Reza; Bridge, Eileen

doi:10.1016/s0042-6822(03)00526-9

Cited by 13 publications

(21 citation statements)

References 39 publications

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“…Consistent with previous observations (11,25,62), our findings demonstrated that inhibition of CRM1-mediated export of E1B-55K or E4orf6 has no effect on viral late mRNA export. Rather, the NES present in E1B-55K and E4orf6 are differentially required for the export of p53 and Mre11, two important cellular targets of the adenovirus-dependent cell-specific E3 ubiquitin ligase, indicating that although dispensable for viral late mRNA export, CRM1 is required for other activities that depend on E1B-55K/E4orf6 shuttling (44,69).…”

supporting

confidence: 93%

“…To address the role of CRM1 in E1B-55K and E4orf6-mediated viral mRNA transport, the CRM1 inhibitor LMB was used, which associates covalently to cysteine 528 in the NES binding region of CRM1 and thereby irreversibly blocks its interaction with NES containing proteins (26,47). These experiments showed that the functional inhibition of CRM1 by LMB had only a minor effect on viral late protein synthesis (an indirect measurement of viral late mRNA export), leading to the conclusion that LMB treatment during the late phase of adenoviral replication does not abrogate viral late mRNA accumulation in the cytoplasm (11,62). A different experimental approach, in which a specific peptide inhibitor of CRM1 was used to make direct measurements of viral late mRNA export, supported these findings.…”

mentioning

confidence: 99%

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CRM1-Dependent Transport Supports Cytoplasmic Accumulation of Adenoviral Early Transcripts

Schmid¹,

González²,

Dobner³

2012

J Virol

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The life cycle of adenoviruses is divided by convention into early and late phases, separated by the onset of viral genome replication. Early events include virus adsorption, transport of the genome into the nucleus, and the expression of early genes. After the onset of viral DNA replication, transcription of the major late transcription unit (MLTU) and thereby synthesis of late proteins is induced. These steps are controlled by an orchestra of regulatory processes and require import of the genome and numerous viral proteins into the nucleus, as well as active transport of viral transcripts and proteins from the nucleus to the cytoplasm. The latter is achieved by exploiting the shuttling functions of cellular transport receptors, which normally stimulate the nuclear export of cellular mRNA and protein cargos. A set of adenoviral early and late proteins contains a leucine-rich nuclear export signal of the HIV-1 Rev type, known to be recognized by the cellular export receptor CRM1. However, a role for CRM1-dependent export in supporting adenoviral replication has not been established. To address this issue in detail, we investigated the impact of two different CRM1 inhibitors on several steps of the adenoviral life cycle. Inhibition of CRM1 led to a reduction in viral early and late gene expression, viral genome replication, and progeny virus production. For the first time, our findings indicate that CRM1-dependent shuttling is required for the efficient export of adenoviral early mRNA. The human subgroup C adenoviruses, such as adenovirus type 5 (Ad5), encode several gene products that are required for efficient synthesis of viral macromolecules and progeny production. During the early phase, the E1A gene products interact with a variety of cellular proteins to induce an optimal cellular environment that is conducive to viral gene expression and replication (reviewed in references 23, 28, and 66). The late phase is characterized by the production of large quantities of viral macromolecules and a severe inhibition of cellular protein synthesis (2,53,83). This highly efficient expression of viral late genes is achieved by the preferential accumulation of viral late transcripts in the cytoplasm and inhibition of nuclear export of most cellular mRNA induced by the E1B-55K and E4orf6 proteins (1,10,37,49,60). Furthermore, the selective translation of viral late mRNA is induced by VA-RNA1 and the L4-100K protein (64,75,79,80). In addition, the L4-100K and pVI structural proteins are known to participate in trimerization and nuclear import of Hexon, as well as capsid assembly (14,15,39,78).As infection progresses the early E1A, E1B-55K, E4orf6, and late L4-100K and pVI proteins localize in both the cytoplasm and nucleus at different times of viral replication. However, the impact of their precise intracellular distribution on their activities is not well understood. These proteins all possess nuclear export signals (NES) of the HIV-1 Rev type, and it is clearly established that the nucleocytoplasmic export of E1A, E1B-5...

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supporting

confidence: 93%

mentioning

confidence: 99%

CRM1-Dependent Transport Supports Cytoplasmic Accumulation of Adenoviral Early Transcripts

Schmid¹,

González²,

Dobner³

2012

J Virol

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“…Adenovirus E1B-55K products also form aggregates in the nucleus, in particular under conditions that inactivate nucleocytoplasmic transport of Ad2/5 E1B-55K by the CRM1-mediated export pathway (Kra¨tzer et al, 2000;Endter et al, 2001Endter et al, , 2005Carter et al, 2003;Flint et al, 2005). Similar to cytoplasmic inclusion bodies, formation of the spherical nuclear aggregates is accompanied by the relocalization of p53, PML (Endter et al, 2005) and Mre11 ( Figures 7A and B).…”

Section: Discussionmentioning

confidence: 96%

Adenovirus type 5 early region 1B 55-kDa oncoprotein can promote cell transformation by a mechanism independent from blocking p53-activated transcription

Härtl

Zeller²,

Blanchette

et al. 2008

Oncogene

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Inhibition of p53-activated transcription is an integral part of the mechanism by which early region 1B 55K oncoprotein (E1B-55K) from adenovirus type 5 (Ad5) contributes to complete cell transformation in combination with Ad E1A. In addition, more recent data suggest that the mode of action of the Ad protein during transformation may involve additional functions and other protein interactions. In the present study, we performed a comprehensive mutational analysis to assign further transforming functions of Ad5 E1B-55K to distinct domains within the viral polypeptide. Results from these studies show that the functions required for transformation are encoded within several patches of the 55K primary sequence, including several clustered cysteine and histidine residues, some of which match the consensus for zinc fingers. In addition, two amino-acid substitutions (C454S/C456S) created a 55K mutant protein, which had substantially reduced transforming activity. Interestingly, the same mutations neither affected binding to p53 nor inhibition of p53-mediated transactivation. Therefore, an activity necessary for efficient transformation of primary rat cells can be separated from functions required for inhibition of p53-stimulated transcription. Our data indicate that this activity is linked to the ability of the Ad5 protein to bind to components of the Mre11/Rad50/NBS1 DNA doublestrand break repair complex, and/or its ability to assemble multiprotein aggregates in the cytoplasm and nucleus of transformed rat cells. These results introduce a new function for Ad5 E1B-55K and suggest that the viral protein contributes to cell transformation through p53 transcription-dependent and -independent pathways.

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“…On the other hand, it has also been reported that the E4 Orf 6 NES failed to direct efficient export of a heterologous protein and that export of this E4 protein is insensitive to leptomycin B (26,58). Regardless of whether the E4 Orf 6 protein leaves the nucleus via the exportin 1 pathway, there is clear evidence that this export receptor does not mediate export of viral late mRNAs: the inhibition of exportin 1 by leptomycin B or an NES containing peptide had no effect on synthesis of viral late proteins (15,78) or viral late mRNA export (34).…”

mentioning

confidence: 97%

Adenovirus E1B 55-Kilodalton Protein Is Required for both Regulation of mRNA Export and Efficient Entry into the Late Phase of Infection in Normal Human Fibroblasts

González

Wen-ying

Finnen

et al. 2006

J Virol

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The human adenovirus type 5 (Ad5) E1B 55-kDa protein is required for selective nuclear export of viral late mRNAs from the nucleus and concomitant inhibition of export of cellular mRNAs in HeLa cells and some other human cell lines, but its contributions(s) to replication in normal human cells is not well understood. We have therefore examined the phenotypes exhibited by viruses carrying mutations in the E1B 55-kDa protein coding sequence in normal human fibroblast (HFFs). Ad5 replicated significantly more slowly in HFFs than it does in tumor cells, a difference that is the result of delayed entry into the late phase of infection. The A143 mutation, which specifically impaired export of viral late mRNAs from the nucleus in infected HeLa cells (R. During the late phase of human adenovirus type 5 (Ad5) infection, cellular protein synthesis is severely inhibited, while viral late proteins are made in large quantities (3, 6). Such preferential expression of viral genes is the result of posttranscriptional regulatory mechanisms: several viral gene products, including VA-RNA1 and the L4 100-kDa protein, contribute to selective translation of viral late mRNAs (see references 18, 64, and 85 for reviews), while the E1B 55-kDa and E4 Orf 6 proteins induce selective export of these mRNAs from the nucleus (reviewed in references 25, 33, and 39). In infected cells, these last two early proteins form a complex (84) that has been implicated in regulation of mRNA export (12, 19). Indeed, the E1B 55-kDa and E4 Orf 6 proteins colocalize to specific sites within infected cell nuclei, the peripheral zones of replication centers (70). Transcription of viral late genes and at least initial processing of viral pre-mRNAs take place at these same sites (4,14,74,75). Mutations that prevent synthesis of the E4 Orf 6 protein or reduce binding of this to the E1B 55-kDa protein (83) result in both mislocalization of the E1B 55-kDa protein and defects in export of viral late mRNAs (39, 70). These properties indicate that E4 Orf 6 protein-dependent recruitment of the E1B protein to the specialized nuclear sites at which viral late pre-mRNAs are synthesized promotes selective export of the processed mRNAs. The observation that the accumulation of viral mRNAs at enlarged interchromatin granules, which form in infected cells as the late phase progresses, correlates with efficient late mRNA export (4, 13) provides further support for the view that efficient mRNA export is intimately coupled to the organization of infected cell nuclei. However, the molecular basis of such coupling remains unknown, nor has the cellular export pathway by which viral late mRNAs are transported from the nucleus to the cytoplasm been identified.AThe E1B 55-kDa protein contains a leucine-rich nuclear export signal (NES) that is recognized by the cellular exportin 1 export receptor and mediates shuttling of the viral protein when it is synthesized either alone or in Ad5-infected cells (26,58). It has also been reported that the E4 Orf 6 protein contains a similar NES nece...

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Evaluating the role of CRM1-mediated export for adenovirus gene expression

Cited by 13 publications

References 39 publications

CRM1-Dependent Transport Supports Cytoplasmic Accumulation of Adenoviral Early Transcripts

CRM1-Dependent Transport Supports Cytoplasmic Accumulation of Adenoviral Early Transcripts

Adenovirus type 5 early region 1B 55-kDa oncoprotein can promote cell transformation by a mechanism independent from blocking p53-activated transcription

Adenovirus E1B 55-Kilodalton Protein Is Required for both Regulation of mRNA Export and Efficient Entry into the Late Phase of Infection in Normal Human Fibroblasts

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