Identification and characterization of a DNA binding domain on the adenovirus IVa2 protein

Christensen, Joan B.; Ewing, Sean G.; Imperiale, Michael J.

doi:10.1016/j.virol.2012.07.013

Cited by 9 publications

(8 citation statements)

References 53 publications

(78 reference statements)

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“…The MS signal intensities of the labeled peptides are compared to those of their unlabeled partners, thus serving as internal controls for estimation of the abundance of the corresponding unlabeled peptide in the virus. This methodology has also been used for AdV, where it helped to determine the copy number of the low-abundance polypeptide IVa2 (52).…”

Section: Discussionmentioning

confidence: 99%

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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Section: Discussionmentioning

confidence: 99%

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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“…The chromatographic profile and a partial digestion with chymotrypsin and trypsin suggested that the deletion does not affect the tertiary structure of IVa2. However, the mutant failed to interact with the AdV PD, and therefore, could not support genome packaging (Christensen et al, 2012 ).…”

Section: Proteins Involved In Adv Dna Packagingmentioning

confidence: 99%

Components of Adenovirus Genome Packaging

Ahi

Mittal

2016

Front. Microbiol.

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Adenoviruses (AdVs) are icosahedral viruses with double-stranded DNA (dsDNA) genomes. Genome packaging in AdV is thought to be similar to that seen in dsDNA containing icosahedral bacteriophages and herpesviruses. Specific recognition of the AdV genome is mediated by a packaging domain located close to the left end of the viral genome and is mediated by the viral packaging machinery. Our understanding of the role of various components of the viral packaging machinery in AdV genome packaging has greatly advanced in recent years. Characterization of empty capsids assembled in the absence of one or more components involved in packaging, identification of the unique vertex, and demonstration of the role of IVa2, the putative packaging ATPase, in genome packaging have provided compelling evidence that AdVs follow a sequential assembly pathway. This review provides a detailed discussion on the functions of the various viral and cellular factors involved in AdV genome packaging. We conclude by briefly discussing the roles of the empty capsids, assembly intermediates, scaffolding proteins, portal vertex and DNA encapsidating enzymes in AdV assembly and packaging.

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“…pIX and IVa2 expression requires the synthesis of a new DNA template and begins immediately after the onset of viral DNA replication (30,31). IVa2 is critical for Ad genome recognition and packaging through its interaction with the viral packaging signal and subsequent recruitment and interaction with critical packaging proteins, including L4-22K, L4-33K, L1-52/55K, and E2 DBP (33,34). IVa2 has also been implicated as a transcriptional Table 2 for quantification methods and base pair coordinates for defined regions).…”

Section: Early Gene Expression (I) E1mentioning

confidence: 99%

Comparison of the Life Cycles of Genetically Distant Species C and Species D Human Adenoviruses Ad6 and Ad26 in Human Cells

Turner

Middha

Hofherr

et al. 2015

J Virol

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Our understanding of adenovirus (Ad) biology is largely extrapolated from human species C Ad5. Most humans are immune to Ad5, so lower-seroprevalence viruses like human Ad6 and Ad26 are being tested as therapeutic vectors. Ad6 and Ad26 differ at the DNA level by 34%. To better understand how this might impact their biology, we examined the life cycle of the two viruses in human lung cells in vitro. Both viruses infected A549 cells with similar efficiencies, executed DNA replication with identical kinetics within 12 h, and began killing cells within 72 h. While Ad6-infected cells remained adherent until death, Ad26-infected cells detached within 12 h of infection but remained viable. Next-generation sequencing (NGS) of mRNA from infected cells demonstrated that viral transcripts constituted 1% of cellular mRNAs within 6 h and 8 to 16% within 12 h. Quantitative PCR and NGS revealed the activation of key early genes at 6 h and transition to late gene activation by 12 h by both viruses. There were marked differences in the balance of E1A and E1B activation by the two viruses and in the expression of E3 immune evasion mRNAs. Ad6 was markedly more effective at suppressing major histocompatibility complex class I (MHC I) display on the cell surface and in evading TRAIL-mediated apoptosis than was Ad26. These data demonstrate shared as well as divergent life cycles in these genetically distant human adenoviruses. An understanding of these differences expands the knowledge of alternative Ad species and may inform the selection of related Ads for therapeutic development. IMPORTANCEA burgeoning number of adenoviruses (Ads) are being harnessed as therapeutics, yet the biology of these viruses is generally extrapolated from Ad2 and Ad5. Here, we are the first to compare the transcriptional programs of two genetically distant Ads by mRNA next-generation sequencing (NGS). Species C Ad6 and Ad26 are being pursued as lower-seroprevalence Ad vectors but differ at the DNA level by 34%. Head-to-head comparison in human lung cells by NGS revealed that the two viruses generally conform to our general understanding of the Ad transcriptional program. However, fine mapping revealed subtle and strong differences in how these two viruses execute these programs, including differences in the balance of E1A and E1B mRNAs and in E3 immune evasion genes. This suggests that not all adenoviruses behave like Ad2 and Ad5 and that they may have unique strategies to infect cells and evade the immune system. T here are currently nearly 60 genotypes of adenoviruses (Ads) that infect humans, causing an array of ocular, respiratory, digestive, and systemic infections (reviewed in reference 1). Human mastadenoviruses (HAdVs) fall into genetically grouped species A (HAdV-A) through species G (HAdV-G), with the level of sequence diversity across the virome being as high as 40% (2). Despite this drastic genetic diversity, the biology of most Ads is often extrapolated from the study of just two respiratory serotypes of HAdV-C, Ad2 and Ad5.Transcripti...

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Identification and characterization of a DNA binding domain on the adenovirus IVa2 protein

Cited by 9 publications

References 53 publications

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

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

Components of Adenovirus Genome Packaging

Comparison of the Life Cycles of Genetically Distant Species C and Species D Human Adenoviruses Ad6 and Ad26 in Human Cells

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