Adenovirus Structural Protein IIIa Is Involved in the Serotype Specificity of Viral DNA Packaging

Ma, Hongyang; Hearing, Patrick

doi:10.1128/jvi.00467-11

Cited by 34 publications

(46 citation statements)

References 33 publications

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“…All washes and secondary antibody incubations were performed with 5% nonfat milk-TBS containing 0.05% Tween 20. Proteins were detected using the following antibodies: rabbit polyclonal antibodies to fiber and penton (gifts from Carl Anderson, Brookhaven National Laboratory); rabbit polyclonal antibody to VII (Daniel Engel, University of Virginia); rabbit polyclonal antibody to hexon (GeneTex, Inc., Irvine, CA); rabbit polyclonal antibodies to L4-22K and L4-33K (11); rabbit polyclonal antibody to VIII (William Wold, St. Louis University); rabbit polyclonal antibodies to IVa2 and L1-52/55K (19); rabbit polyclonal antibody to IIIa (24); rabbit polyclonal antibody to V (David Matthews, University of Bristol); rabbit polyclonal antibody to L4-100K (37); rabbit polyclonal antibody to VI (Christopher Wiethoff, Loyola University Chicago); rabbit polyclonal antibody to E1A (Santa Cruz Biotechnology); rabbit polyclonal antibody to ␥-tubulin (Sigma). Additional information on Western blot analyses and antibodies was provided previously (12).…”

Section: Methodsmentioning

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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Section: Methodsmentioning

confidence: 99%

“…In vivo studies have shown that the L4-22K and IVa2 proteins are dependent on each other for binding to the packaging domain (12). In addition to IVa2 and L4-22K, the L1-52/55K and IIIa proteins also play roles in the packaging process (23,24).…”

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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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“…Although the molecular mass of L1 52/55k calculated from its sequence is 47 kDa, the protein was named by its electrophoretic mobility; it moved as a doublet due to two different phosphorylation states (9). L1 52/55k is part of the genome packaging machinery, together with polypeptides IIIa, IVa2, L4 33k, and L4 22k (10)(11)(12)(13)(14)(15). An L1 52/55k deletion construct produces only empty capsids (10), and a thermosensitive mutation in the L1 52/55k C-terminal region (ts369; 333-EL-336 to 333-GP-336) causes partial packaging (16).…”

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“…L1 52/55k binds to the viral packaging sequence in vivo and to the putative packaging ATPase IVa2 in vitro (17)(18)(19). L1 52/55k contributes to the specificity of packaging, possibly via an interaction with capsid protein IIIa (12,20). Furthermore, L1 52/55k has been reported to bind nonspecifically to DNA and to interact with pVII and its mature form VII in infected cells (21).…”

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“…Both the full-length and 40-kDa bands disappear and are replaced by higher-molecular-mass species in electrophoresis performed in the absence of ␤-mercaptoethanol, suggesting the formation of disulfide-linked homodimers mediated by the only Cys residue (Cys24) in L1 52/55k (9,22). The 34-kDa band has been proposed to originate by an additional cleavage at the N terminus, because it does not react with antibodies against either residues 9 to 22 (9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22) or 402-415 in L1 52/55k, and its electrophoretic mobility is not sensitive to nonreducing conditions, a finding consistent with the absence of Cys24 (9,22).…”

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Processing of the L1 52/55k Protein by the Adenovirus Protease: a New Substrate and New Insights into Virion Maturation

Pérez-Berná

Mangel

McGrath

et al. 2014

J Virol

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Late in adenovirus assembly, the viral protease (AVP) becomes activated and cleaves multiple copies of three capsid and three core proteins. Proteolytic maturation is an absolute requirement to render the viral particle infectious. We show here that the L1 52/55k protein, which is present in empty capsids but not in mature virions and is required for genome packaging, is the seventh substrate for AVP. A new estimate on its copy number indicates that there are about 50 molecules of the L1 52/55k protein in the immature virus particle. Using a quasi-in vivo situation, i.e., the addition of recombinant AVP to mildly disrupted immature virus particles, we show that cleavage of L1 52/55k is DNA dependent, as is the cleavage of the other viral precursor proteins, and occurs at multiple sites, many not conforming to AVP consensus cleavage sites. Proteolytic processing of L1 52/55k disrupts its interactions with other capsid and core proteins, providing a mechanism for its removal during viral maturation. Our results support a model in which the role of L1 52/55k protein during assembly consists in tethering the viral core to the icosahedral shell and in which maturation proceeds simultaneously with packaging, before the viral particle is sealed.A denovirus morphogenesis ends with a maturation step comprising proteolytic cleavage of several capsid and core precursor proteins. Without these cleavages, the immature particle lacks infectivity because of its inability to uncoat (1-3). Maturation primes the viral particle for stepwise uncoating by facilitating penton release and by loosening the condensed genome and its attachment to the icosahedral shell (4, 5). Proteolytic processing is carried out by the adenovirus protease (AVP; or L3 23K protein) (6). In human adenovirus type 2 (HAdV-2), AVP recognizes (M/I/ L)XGX-G and (M/I/L)XGG-X sequence motifs (7,8). These sequences are present in the precursor proteins pIIIa, pVI, and pVIII in the icosahedral shell, as well as in the DNA-binding polypeptides pVII, pre-, and the terminal protein. These six precursor proteins have been shown to be substrates for AVP. Another potential substrate, because it contains an AVP consensus sequence motif, is polypeptide L1 52/55k.The L1 52/55k protein in HAdV-2 is 415 residues in length, with an AVP consensus cleavage site at the 351-352 position (LAGT-G). Although the molecular mass of L1 52/55k calculated from its sequence is 47 kDa, the protein was named by its electrophoretic mobility; it moved as a doublet due to two different phosphorylation states (9). L1 52/55k is part of the genome packaging machinery, together with polypeptides IIIa, IVa2, L4 33k, and L4 22k (10-15). An L1 52/55k deletion construct produces only empty capsids (10), and a thermosensitive mutation in the L1 52/55k C-terminal region (ts369; 333-EL-336 to 333-GP-336) causes partial packaging (16). L1 52/55k binds to the viral packaging sequence in vivo and to the putative packaging ATPase IVa2 in vitro (17-19). L1 52/55k contributes to the specificity of packaging, po...

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The Use of Chromatin Immunoprecipitation (ChIP) to Study the Binding of Viral Proteins to the Adenovirus Genome In Vivo

Zheng

Hearing

2013

Adenovirus

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The encapsidation of adenovirus (Ad) DNA into virus particles depends on cis-acting sequences located at the left end of the viral genome. Repeated DNA sequences in the packaging domain contribute to viral DNA encapsidation and several viral proteins bind to these repeats when analyzed using in vitro DNA-protein binding assays. In this chapter, we describe a chromatin immunoprecipitation (ChIP) approach to study the binding of viral proteins to packaging sequences in vivo. This assay permits accurate quantification over a wide range of DNA concentrations. The use of formaldehyde cross-linking to stabilize DNA-protein and protein-protein complexes formed in vivo allows the identification of macromolecular complexes found in living cells.

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Adenovirus Structural Protein IIIa Is Involved in the Serotype Specificity of Viral DNA Packaging

Cited by 34 publications

References 33 publications

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

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

Processing of the L1 52/55k Protein by the Adenovirus Protease: a New Substrate and New Insights into Virion Maturation

The Use of Chromatin Immunoprecipitation (ChIP) to Study the Binding of Viral Proteins to the Adenovirus Genome In Vivo

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