The L4 22-Kilodalton Protein Plays a Role in Packaging of the Adenovirus Genome

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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“…The amino terminus may theoretically function in other aspects of the viral life cycle (for an example, see Ref. 25). …”

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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“…Expression plasmids were generated by using pcDNA3 (Invitrogen Life Technologies). Expression plasmids pcDNA3-22K and pcDNA3-33K were previously described (11). Plasmid pcDNA3-22K-C137S/C141S was constructed by introducing double serine substitution mutations into the L4-22K gene by site-directed mutagenesis (QuikChange mutagenesis; Stratagene).…”

Section: Methodsmentioning

confidence: 99%

“…Both studies presented evidence indicating that L4-22K acts at a posttranscriptional level, affecting late gene mRNA production and/or stability, and that this effect was on specific Ad late RNAs, not on a global scale. Prior to being linked to a regulatory function in viral gene expression, the L4-22K protein was first attributed to a role in packaging of the Ad genome into the empty capsid (11). Ad packaging is dependent on a cis-acting region located at the left end of the genome.…”

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confidence: 99%

“…Efficient packaging is required for the formation of mature virus particles, and a complex of DNA and proteins at the packaging domain plays a key role. The IVa2 and L4-22K proteins bind to the CG and TTTG motifs of the packaging repeats, respectively (11,20,21) and play an important role in viral DNA encapsidation. IVa2 and L4-22K mutant viruses produce empty particles containing no viral DNA (12,22).…”

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confidence: 99%

“…The L4 region encodes four proteins: L4-100K, a translation enhancer protein that targets specific late mRNAs (8,9); protein pVIII, a structural protein of the viral capsid; L4-33K, a viral splicing factor (10); and L4-22K. The L4-22K protein has been shown to bind to the packaging domain in vitro and in vivo (11) and is required for viral DNA packaging into the empty capsid (12). L4-22K also is associated with other functions pertaining to the regulation of Ad late gene expression (12,13).…”

mentioning

confidence: 99%

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The Adenovirus L4-22K Protein Has Distinct Functions in the Posttranscriptional Regulation of Gene Expression and Encapsidation of the Viral Genome

Guimet

Hearing

2013

J Virol

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The adenovirus L4-22K protein is multifunctional and critical for different aspects of viral infection. Packaging of the viral genome into an empty capsid absolutely requires the L4-22K protein to bind to packaging sequences in cooperation with other viral proteins. Additionally, the L4-22K protein is important for the temporal switch from the early to late phase of infection by regulating both early and late gene expression. To better understand the molecular mechanisms of these key functions of the L4-22K protein, we focused our studies on the role of conserved pairs of cysteine and histidine residues in the C-terminal region of L4-22K. We found that mutation of the cysteine residues affected the production of infectious progeny virus but did not interfere with the ability of the L4-22K protein to regulate viral gene expression. These results demonstrate that these two functions of L4-22K may be uncoupled. Mutation of the histidine residues resulted in a mutant with a similar phenotype as a virus deficient in the L4-22K protein, where both viral genome packaging and viral gene expression patterns were disrupted. Interestingly, both mutant L4-22K proteins bound to adenovirus packaging sequences, indicating that the paired cysteine and histidine residues do not function as a zinc finger DNA binding motif. Our results reveal that the L4-22K protein controls viral gene expression at the posttranscriptional level and regulates the accumulation of the L4-33K protein, another critical viral regulator, at the level of alternative pre-mRNA splicing.H uman adenoviruses (Ad) consist of a nonenveloped icosahedral capsid with a linear double-stranded viral genome of ϳ36,000 bp. Many questions remain about the basic biology of Ad infection. In particular, virus assembly for complex eukaryotic viruses is relatively poorly understood. Understanding the molecular mechanisms of the viral life cycle is critical for development of strategies for treatment of Ad infections and for the use of Ad as a gene therapy vector. The Ad5 genome contains five early transcription units (E1A, E1B, E2, E3, and E4), which encode ϳ25 proteins that are expressed before viral DNA replication, and a set of delayed mRNAs, which encode proteins IX and IVa2, synthesized at the onset of DNA replication. These early and intermediate transcripts encode proteins with various roles during infection, including transcriptional regulation, viral DNA replication, inhibition of cellular antiviral responses, and inhibition of immune responses (1-4). Following DNA replication, a single major late transcription unit (MLTU) is transcribed and includes five different groups of mRNAs (L1 to L5) that encode capsid structural proteins and proteins that promote virus assembly, direct Ad genome packaging, and serve regulatory functions (2). The Ad major late promoter (MLP) drives transcription from the MLTU regions L1 to L5, producing all late mRNAs by alternative splicing and polyadenylation of a primary transcript. Prior to DNA replication, the MLP is active at low leve...

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Imaging the adenovirus infection cycle

Pied

Wodrich

2019

FEBS Letters

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Edited by Urs GreberIncoming adenoviruses seize control of cytosolic transport mechanisms to relocate their genome from the cell periphery to specialized sites in the nucleoplasm. The nucleus is the site for viral gene expression, genome replication, and the production of progeny for the next round of infection. By taking control of the cell, adenoviruses also suppress cell-autonomous immunity responses. To succeed in their production cycle, adenoviruses rely on wellcoordinated steps, facilitated by interactions between viral proteins and cellular factors. Interactions between virus and host can impose remarkable morphological changes in the infected cell. Imaging adenoviruses has tremendously influenced how we delineate individual steps in the viral life cycle, because it allowed the development of specific optical markers to label these morphological changes in space and time. As technology advances, innovative imaging techniques and novel tools for specimen labeling keep uncovering previously unseen facets of adenovirus biology emphasizing why imaging adenoviruses is as attractive today as it was in the past. This review will summarize past achievements and present developments in adenovirus imaging centered on fluorescence microscopy approaches.Adenoviruses (Ads) are nonenveloped icosahedral viruses with a diameter of~90 nm with slight structural differences between genotypes. The majority of the Ad capsid shell is composed of 240 hexon trimers, and each of the 12 vertices of the icosahedron is occupied by a penton. Pentons are involved in cell attachment and receptor recognition and are composed of the pentameric penton base from which the trimeric fiber molecule extends. Minor capsid proteins IIIa, VI, VIII, and IX are embedded in the capsid and contribute to capsid stability. Protein VI is located at the inner surface of the capsid, and biochemical data suggest that it may connect the capsid to the core containing the viral genome via protein V. The genome is ã 36-kb double-stranded linear DNA molecule with the terminal protein (TP) covalently bound to each 5 0end. Adenovirus genomes are highly condensed and organized into chromatin by several hundred copies of Abbreviations ADP, adenovirus death protein

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The L4 22-Kilodalton Protein Plays a Role in Packaging of the Adenovirus Genome

Cited by 63 publications

References 40 publications

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

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

The Adenovirus L4-22K Protein Has Distinct Functions in the Posttranscriptional Regulation of Gene Expression and Encapsidation of the Viral Genome

Imaging the adenovirus infection cycle

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