Truncation of the Human Adenovirus Type 5 L4 33-kDa Protein: Evidence for an Essential Role of the Carboxy-Terminus in the Viral Infectious Cycle

Finnen, Renée L.; Biddle, Jennifer F.; Flint, Jane

doi:10.1006/viro.2001.1130

Cited by 32 publications

(44 citation statements)

References 81 publications

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“…Alternatively, Finnen et al suggested that their truncation may have created a dominant-negative form of the protein (14). However, both studies point to a role for 33K in the assembly of particles, whereas data presented here show that 33K is required to turn on full late gene expression.…”

Section: Discussioncontrasting

confidence: 52%

“…Of the three studies, only the present one unambiguously examined the effect of a total lack of 33K; the other two retained substantial parts of the 33K reading frame. According to this reasoning, the central part of 33K, deleted here but retained and potentially expressed in the studies of Fessler and Young and of Finnen et al (13,14), would be necessary and sufficient for regulating the MLTU switch. Alternatively, the primary function of 33K may be in activating late gene expression, leading secondarily to defective assembly.…”

Section: Discussionmentioning

confidence: 99%

“…Both DNA replication and late protein production were normal, but assembly of particles was impaired. A second group attempted to isolate virus carrying a mutation that would remove 47 residues from the C terminus of 33K but were unable to recover virus from transfections of full-length cloned mutant genomes (14). Viral DNA replication could be detected, but onset posttransfection was delayed compared to the wild type.…”

Section: Discussionmentioning

confidence: 99%

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Activation of the Early-Late Switch in Adenovirus Type 5 Major Late Transcription Unit Expression by L4 Gene Products

Farley

Brown

Leppard

2004

J Virol

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The adenovirus major late transcription unit (MLTU) encodes multiple proteins from five regions, L1 to L5, through differential splicing and polyadenylation. MLTU expression is temporally regulated; only a single product from L1 (52/55K) is expressed prior to replication, but a subsequent switch, the mechanism of which has not been defined, leads to full expression that encompasses L1 IIIa and all L2 to L5 products. Transfection of a plasmid containing the complete MLTU gave a full array of proteins in proportions similar to those in a late infection, and in a time course, the temporal pattern of expression in a natural infection was reproduced. However, a plasmid truncated after the L3 poly(A) site exclusively expressed the L1 52/55K protein and was defective in the switch to full gene expression from L1 to L3. The L4 33K protein, supplied in trans, was sufficient to upregulate cytoplasmic mRNA for MLTU products characteristic of the late pattern of expression to levels comparable to those produced by the full-length MLTU. There was a corresponding increase in expression of the L1 IIIa, L2, and L3 proteins, except hexon. Hexon protein expression additionally required both the L4 100K protein in trans and sequences downstream of the L3 poly(A) site in cis. These results indicate that induction of L4 protein expression is a key event in the early-late switch in MLTU expression, which we propose is precipitated by small amounts of L4 expression in a feed-forward activation mechanism.Human adenoviruses, especially group C viruses such as serotype 5 (Ad5), have been studied extensively; their tropism, gene expression, host cell interactions, and transforming capabilities are well characterized. Despite this knowledge, however, there remain important aspects of adenovirus biology that are not understood. One of these is the control of protein expression from the major late transcription unit (MLTU) during the infectious cycle.Upon Ad5 infection, a temporally phased program of gene expression occurs. The E1A gene products are produced immediately and upregulate transcription from all of the early regions: E1A, E1B, E2, E3, and E4. The MLTU, which encodes the majority of the virus structural proteins within five subregions, L1 to L5, is also active at a low level during this early phase, but transcription proceeds only as far as the L3 region and mRNA production is focused on the L1 52/55K reading frame (33). After replication, the major late promoter (MLP) becomes fully activated and transcription is allowed to proceed through the full length of the MLTU to supply the proteins for the packaging of newly replicated genomes into virions. The MLP is activated by the E1A 289R protein (32) and also by the intermediate-late protein IVa2 (28). However, unreplicated genomes from virus allowed to superinfect cells already in the late phase of infection, and hence containing these activators, continue to display the early pattern of MLTU expression until they themselves have begun to replicate (44). This indicates a role for a ...

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Section: Discussioncontrasting

confidence: 52%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Activation of the Early-Late Switch in Adenovirus Type 5 Major Late Transcription Unit Expression by L4 Gene Products

Farley

Brown

Leppard

2004

J Virol

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“…7B). The second paper introduced two tandem translational stop codons in the conserved carboxy-terminal part of L4-33K (30). This mutation was lethal.…”

Section: Discussionmentioning

confidence: 99%

L4-33K, an Adenovirus-encoded Alternative RNA Splicing Factor

Törmänen¹,

Backström²,

Carlsson

et al. 2006

Journal of Biological Chemistry

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Splicing of the adenovirus IIIa mRNA is subjected to a strict temporal regulation during virus infection such that efficient IIIa 3 splice site usage is confined to the late phase of the infectious cycle. Here we show that the adenovirus L4-33K protein functions as a virus-encoded RNA splicing factor that preferentially activates splicing of transcripts with a weak 3 splice site sequence context, a sequence configuration that is shared by many of the late adenovirus 3 splice sites. Furthermore, we show that L4-33K activates IIIa splicing through the IIIa virus infection-dependent splicing enhancer element (3VDE). This element was previously shown to be the minimal element, both necessary and sufficient, for activation of IIIa splicing in the context of an adenovirus-infected cell. L4-33K stimulates an early step in spliceosome assembly and appears to be the only viral protein necessary to convert a nuclear extract prepared from uninfected HeLa cells to an extract with splicing properties very similar to a nuclear extract prepared from adenovirus lateinfected cells. Collectively, our results suggest that L4-33K is the key viral protein required to activate the early to late switch in adenovirus major late L1 alternative splicing.Most late adenovirus proteins are translated from mRNAs originating from the major late transcription unit (MLTU), 3 which extends from the major late promoter (MLP) at coordinate 16.8 to a termination signal close to the right-hand end of the genome (reviewed in Ref. 1). The ϳ28,000-nucleotide pre-mRNA expressed from the MLTU becomes polyadenylated at one of five possible sites, generating five families of mRNAs with co-terminal 3Ј-ends (L1-L5; Fig. 1A). Following selection of a poly(A) site, the primary transcript is spliced in such a way that each mature mRNA receives a common set of three short 5Ј-leader segments, the tripartite leader (Fig. 1A). This leader is then spliced to one of several alternative 3Ј splice sites, generating a total of more than 20 cytoplasmic mRNAs.The accumulation of mRNA from the MLTU is subjected to a temporal regulation at the levels of transcription elongation, poly(A) site choice and alternative 3Ј splice site selection (reviewed in Ref. 1). Thus, during the early phase of infection the MLP is active at a level comparable with the other early transcription units, whereas the same promoter accounts for most of the transcriptional activity at late times of infection (2, 3). However, at early times transcription initiated at the MLP decreases gradually over a large region beginning after the L1 unit, with few RNA polymerases extending beyond the L3 polyadenylation sequence (4). At late times, this block in elongation is alleviated and transcripts initiated at the MLP continue to the right hand end of the genome. The control of MLTU transcription is further regulated by events taking place at the level of poly(A) and alternative 3Ј splice site selection (2, 5, 6). Thus, although nuclear transcription proceeds across at least the L1, L2 and L3 poly(A) sites a...

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“…Following digestion with PacI (New England BioLabs), the released viral genome was isolated using the Qiaex II kit (Qiagen) following electrophoresis in 0.75% low-meltingpoint agarose (International Biotechnologies Inc.). To isolate dl341 DNA, 293 cells at 90% confluence were infected with 5 PFU/cell of the mutant for 44 h, and virus particles were isolated by sequential sedimentation in CsCl step and continuous gradients as described previously (87). Viral DNA was purified from the particles following dialysis into 0.01 M TrisHCl, pH 7.5, containing 1 mM EDTA as described previously (88).…”

Section: Cells and Virusesmentioning

confidence: 99%

Impact of the Adenoviral E4 Orf3 Protein on the Activity and Posttranslational Modification of p53

DeHart

Perlman

Flint

2015

J Virol

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Our previous studies have established that the p53 populations that accumulate in normal human cells exposed to etoposide or infected by an E1B 55-kDa protein-null mutant of human adenovirus type 5 carry a large number of posttranslational modifications at numerous residues (C. J. DeHart, J. S. Chahal, S. J. Flint, and D. H. Perlman, Mol Cell Proteomics 13:1-17, 2014, http: //dx.doi.org/10.1074/mcp.M113.030254). In the absence of this E1B protein, the p53 transcriptional program is not induced, and it has been reported that the viral E4 Orf3 protein inactivates p53 (C. Soria, F. E. Estermann, K. C. Espantman, and C. C. O'Shea, Nature 466:1076 -1081, 2010, http://dx.doi.org/10.1038/nature09307). As the latter protein disrupts nuclear Pml bodies, sites at which p53 is modified, we used mass spectrometry to catalogue the posttranscriptional modifications of the p53 population that accumulates when neither the E1B 55-kDa nor the E4 Orf3 protein is made in infected cells. Eighty-five residues carrying 163 modifications were identified. The overall patterns of posttranslational modification of this population and p53 present in cells infected by an E1B 55-kDa-null mutant were similar. The efficiencies with which the two forms of p53 bound to a consensus DNA recognition sequence could not be distinguished and were lower than that of transcriptionally active p53. The absence of the E4 Orf3 protein increased expression of several p53-responsive genes when the E1B protein was also absent from infected cells. However, expression of these genes did not attain the levels observed when p53 was activated in response to etoposide treatment and remained lower than those measured in mock-infected cells. T he cellular p53 protein was discovered by virtue of its interaction with the major product of the simian virus 40 oncogene, large T antigen (1, 2). The p53 tumor suppressor is a master regulator of cellular responses to internal and external stresses, when it can induce inhibition of cell cycle progression, apoptosis, or other responses, such as changes in metabolism. Under normal conditions, the human p53 protein is maintained at low concentrations, for example, as a result of its targeting for proteasomal degradation by the E3 ubiquitin ligase Hdm2 (3-5). Once stabilized and activated in response to genotoxic and other forms of stress, p53 binds to specific promoter sequences to activate or repress the transcription of numerous target genes (6-10) and can also operate in the cytoplasm to induce apoptosis by transcription-independent mechanisms (reviewed in references 11 to 14).One of the first interactions between human adenovirus type 5 (Ad5) and cellular proteins to be identified was the association of the viral E1B 55-kDa protein with p53 (15). In view of its crucial roles in regulating cell survival and other aspects of cellular physiology, considerable effort has since been devoted to elucidation of the impacts of adenoviral gene products on the activities and properties of p53. The viral immediate-early E1A prote...

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Truncation of the Human Adenovirus Type 5 L4 33-kDa Protein: Evidence for an Essential Role of the Carboxy-Terminus in the Viral Infectious Cycle

Cited by 32 publications

References 81 publications

Activation of the Early-Late Switch in Adenovirus Type 5 Major Late Transcription Unit Expression by L4 Gene Products

Activation of the Early-Late Switch in Adenovirus Type 5 Major Late Transcription Unit Expression by L4 Gene Products

L4-33K, an Adenovirus-encoded Alternative RNA Splicing Factor

Impact of the Adenoviral E4 Orf3 Protein on the Activity and Posttranslational Modification of p53

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