Adenovirus DNA Packaging: Construction and Analysis of Viral Mutants

Schmid, Susanne I.; Hearing, Patrick

doi:10.1385/0-89603-551-4:47

Cited by 11 publications

(8 citation statements)

References 11 publications

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“…Infected cells were then identified by immunostaining with an antibody against the early E2A protein (B6-8). Viral DNA was isolated as described previously (49) from viral particles purified by cesium chloride equilibrium density centrifugation (22,49) and analyzed by HindIII restriction endonuclease digestion. In addition, the viral DNA was partially sequenced to verify the presence of the E4orf6 mutations.…”

Section: Methodsmentioning

confidence: 99%

Control of mRNA Export by Adenovirus E4orf6 and E1B55K Proteins during Productive Infection Requires E4orf6 Ubiquitin Ligase Activity

Blanchette¹,

Kindsmüller

Groitl

et al. 2008

J Virol

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During the adenovirus infectious cycle, the early proteins E4orf6 and E1B55K are known to perform several functions. These include nuclear export of late viral mRNAs, a block of nuclear export of the bulk of cellular mRNAs, and the ubiquitin-mediated degradation of selected proteins, including p53 and Mre11. Degradation of these proteins occurs via a cellular E3 ubiquitin ligase complex that is assembled through interactions between elongins B and C and BC boxes present in E4orf6 to form a cullin 5-based ligase complex. E1B55K, which has been known for some time to associate with the E4orf6 protein, is thought to bind to specific substrate proteins to bring them to the complex for ubiquitination. Earlier studies with E4orf6 mutants indicated that the interaction between the E4orf6 and E1B55K proteins is optimal only when E4orf6 is able to form the ligase complex. These and other observations suggested that most if not all of the functions ascribed to E4orf6 and E1B55K during infection, including the control of mRNA export, are achieved through the degradation of specific substrates by the E4orf6 ubiquitin ligase activity. We have tested this hypothesis through the generation of a virus mutant in which the E4orf6 product is unable to form a ligase complex and indeed have found that this mutant behaves identically to an E4orf6 ؊ virus in production of late viral proteins, growth, and export of the late viral L5 mRNA.The late phase of an adenoviral infection, typified by human adenovirus type 5 (Ad5), is characterized by a massive production of progeny virions. To support this production, the virus takes control of the cellular machinery to produce abundant amounts of its own late proteins to the detriment of the synthesis of cellular proteins. This host cell shutoff is achieved via several mechanisms. The translation of cellular mRNAs is blocked by the action of the L4-100K protein on the ribosomal machinery (15, 16) so that only the late viral mRNAs containing the tripartite leader sequence can be translated (26). At the same time, export of cellular mRNAs to the cytoplasm is blocked by the action of the early viral proteins E4orf6 and E1B55K (3, 6, 23, 42). These same proteins were also shown to be required for the export of the late viral mRNAs (4,20,23,42,53). The E4orf6 and E1B55K proteins were shown to interact during infection (48) and to function in the same pathway, as a viral mutant with defects in both of these products was seen to have essentially the same phenotype as mutants affecting only one of these species (3,17,23,42). The role that E4orf6 and E1B55K proteins play in the control of mRNA export has yet to be defined.It has been known for some time that the E4orf6 and E1B55K proteins play a role in the degradation of the p53 tumor suppressor, and recently the mechanism for such degradation has been elucidated by our group. Expression of the virus E1A protein results in increased levels of p53 (10, 34); however, during infection, in the presence of both E4orf6 and E1B55K products, p53 is degraded ...

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Section: Methodsmentioning

confidence: 99%

Control of mRNA Export by Adenovirus E4orf6 and E1B55K Proteins during Productive Infection Requires E4orf6 Ubiquitin Ligase Activity

Blanchette¹,

Kindsmüller

Groitl

et al. 2008

J Virol

View full text Add to dashboard Cite

show abstract

“…Infected cells were then identified by immunostaining with an antibody against the early E2A protein (antibody B6-8). Viral DNA was isolated as described previously (50) from viral particles purified by cesium chloride equilibrium density centrifugation (18,50) and analyzed by HindIII restriction endonuclease digestion. In addition, the viral DNA was partially sequenced to verify the presence of the E4orf4 mutations/insertion.…”

Section: Methodsmentioning

confidence: 99%

Localization and Importance of the Adenovirus E4orf4 Protein during Lytic Infection

Miron¹,

Blanchette²,

Groitl

et al. 2009

J Virol

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The human adenovirus type 5 (Ad5) E4orf4 product has been studied extensively although in most cases as expressed from vectors in the absence of other viral products. Thus, relatively little is known about its role in the context of an adenovirus infection. Although considerable earlier work had indicated that the E4orf4 protein is not essential for replication, a recent study using dl359, an Ad5 mutant believed to produce a nonfunctional E4orf4 protein, suggested that E4orf4 is essential for virus growth in primary small-airway epithelial cells (C. O'Shea, et al., EMBO J. 24:1211-1221, 2005). Hence, to examine further the role of E4orf4 during virus infection, we generated for the first time a set of E4orf4 virus mutants in a common Ad5 genetic background. Such mutant viruses included those that express E4orf4 proteins containing various individual point mutations, those defective entirely in E4orf4 expression, and a mutant expressing wild-type E4orf4 fused to the green fluorescent protein. E4orf4 protein was found to localize primarily in nuclear structures shown to be viral replication centers, in nucleoli, and in perinuclear bodies. Importantly, E4orf4 was shown not to be essential for virus growth in either human tumor or primary cells, at least in tissue culture. Unlike E4orf4-null virus, mutant dl359 appeared to exhibit a gain-of-function phenotype that impairs virus growth. The dl359 E4orf4 protein, which contains a large in-frame internal deletion, clustered in aggregates enriched in Hsp70 and proteasome components. In addition, the late viral mRNAs produced by dl359 accumulated abnormally in a nuclear punctate pattern. Altogether, our results indicate that E4orf4 protein is not essential for virus growth in culture and that expression of the dl359 E4orf4 product interferes with viral replication, presumably through interactions with structures in the nucleus.

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“…For the generation of virus mutants, the viral genomes were released from the recombinant plasmids by PacI digestion, and viruses were generated exactly as described previously (61). Viral DNA was isolated from viral particles (62) and analyzed by HindIII restriction endonuclease digestion. In addition, the viral DNA was partially sequenced to verify the presence of the E1B-19K -and E3-PM-14.7K mutations.…”

Section: Methodsmentioning

confidence: 99%

Inhibition of TNF receptor 1 internalization by adenovirus 14.7K as a novel immune escape mechanism

Schneider-Brachert¹,

Tchikov²,

Merkel³

et al. 2006

J. Clin. Invest.

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The adenoviral protein E3-14.7K (14.7K) is an inhibitor of TNF-induced apoptosis, but the molecular mechanism underlying this protective effect has not yet been explained exhaustively. TNF-mediated apoptosis is initiated by ligand-induced recruitment of TNF receptor-associated death domain (TRADD), Fas-associated death domain (FADD), and caspase-8 to the death domain of TNF receptor 1 (TNFR1), thereby establishing the death-inducing signaling complex (DISC). Here we report that adenovirus 14.7K protein inhibits ligandinduced TNFR1 internalization. Analysis of purified magnetically labeled TNFR1 complexes from murine and human cells stably transduced with 14.7K revealed that prevention of TNFR1 internalization resulted in inhibition of DISC formation. In contrast, 14.7K did not affect TNF-induced NF-κB activation via recruitment of receptor-interacting protein 1 (RIP-1) and TNF receptor-associated factor 2 (TRAF-2). Inhibition of endocytosis by 14.7K was effected by failure of coordinated temporal and spatial assembly of essential components of the endocytic machinery such as Rab5 and dynamin 2 at the site of the activated TNFR1. Furthermore, we found that the same TNF defense mechanisms were instrumental in protecting wild-type adenovirus-infected human cells expressing 14.7K. This study describes a new molecular mechanism implemented by a virus to escape immunosurveillance by selectively targeting TNFR1 endocytosis to prevent TNF-induced DISC formation.

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Adenovirus DNA Packaging: Construction and Analysis of Viral Mutants

Cited by 11 publications

References 11 publications

Control of mRNA Export by Adenovirus E4orf6 and E1B55K Proteins during Productive Infection Requires E4orf6 Ubiquitin Ligase Activity

Control of mRNA Export by Adenovirus E4orf6 and E1B55K Proteins during Productive Infection Requires E4orf6 Ubiquitin Ligase Activity

Localization and Importance of the Adenovirus E4orf4 Protein during Lytic Infection

Inhibition of TNF receptor 1 internalization by adenovirus 14.7K as a novel immune escape mechanism

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