Chromatin structure of adenovirus DNA throughout infection

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...

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

“…How viruses resist cellular chromatinization and silencing is known for only a few viruses (9,12,13). These include herpes simplex viruses (HSVs) (14)(15)(16), polyomaviruses (17,18), adenoviruses (19), and cytomegaloviruses (20,21), all of which encode proteins that promote transcriptionally activating histone modifications on chromatinized viral genomes during lytic infection.…”

Section: Viral Dna Introduced Into Cell Nuclei Is Exposed To Cellularmentioning

confidence: 99%

Promoter-Targeted Histone Acetylation of Chromatinized Parvoviral Genome Is Essential for the Progress of Infection

Mäntylä

Salokas

Oittinen

et al. 2016

The association of host histones with parvoviral DNA is poorly understood. We analyzed the chromatinization and histone acetylation of canine parvovirus DNA during infection by confocal imaging and in situ proximity ligation assay combined with chromatin immunoprecipitation and high-throughput sequencing. We found that during late infection, parvovirus replication bodies were rich in histones bearing modifications characteristic of transcriptionally active chromatin, i.e., histone H3 lysine 27 acetylation (H3K27ac). H3K27ac, in particular, was located in close proximity to the viral DNA-binding protein NS1. Importantly, our results show for the first time that in the chromatinized parvoviral genome, the two viral promoters in particular were rich in H3K27ac. Histone acetyltransferase (HAT) inhibitors efficiently interfered with the expression of viral proteins and infection progress. Altogether, our data suggest that the acetylation of histones on parvoviral DNA is essential for viral gene expression and the completion of the viral life cycle. IMPORTANCEViral DNA introduced into cell nuclei is exposed to cellular responses to foreign DNA, including chromatinization and epigenetic silencing, both of which determine the outcome of infection. How the incoming parvovirus resists cellular epigenetic downregulation of its genes is not understood. Here, the critical role of epigenetic modifications in the regulation of parvovirus infection was demonstrated. We showed for the first time that a successful parvovirus infection is characterized by the deposition of nucleosomes with active histone acetylation on the viral promoter areas. The results provide new insights into the regulation of parvoviral gene expression, which is an important aspect of the development of parvovirus-based virotherapy. N uclear chromatin is composed of DNA and histone proteins (1). The histone proteins assemble DNA into nucleosomes, the composition and spacing of which contribute to higher-order chromatin packing. The chromatin is organized into regions of less-condensed actively transcribed chromatin (euchromatin) and highly condensed transcriptionally repressed chromatin (heterochromatin). Epigenetic modifications of histone proteins have been shown to correlate with the spatial distribution of active and repressed chromatin (2, 3). The acetylation of lysine 9 or 27 of histone H3 (H3K9ac and H3K27ac, respectively) and trimethylation of lysine 4 (H3K4me3) correlate with transcriptional activity, while repressed chromatin is characterized by, e.g., trimethylation or dimethylation of the same H3 lysine residues (H3K9me3 and H3K27me3 as well as H3K9me2 and H3K27me2) (4-8).Foreign DNA introduced into mammalian cells can be recognized as a threat by the host cell. The cellular responses to the foreign DNA, such as viral DNA, include the chromatinization of the entering DNA, leading to its transcriptional silencing (9-11). How viruses resist cellular chromatinization and silencing is known for only a few viruses (9, 12, 13). These include h...

“…Pre-viously, we identified host cell-derived remodeling factors for Ad chromatin by biochemical analyses (19,22,24,26) and demonstrated that TAF-I, one of these host factors, plays an important role in the regulation of viral early gene expression in infected cells through interactions with protein VII (15,17,18,20,27). Thus, it was indicated that the remodeling of Ad chromatin is a crucial process for its genome functions (13), as is the case for the cellular genome. In addition, using chromatin immunoprecipitation (ChIP) assays, we recently reported that in early phases of infection, cellular histones are incorporated into viral DNA-protein VII complexes and that a histone modification occurs depending upon the transcription states on viral chromatin, suggesting that cellular histones could be functional components of viral chromatin in infected cells (20).…”

mentioning

confidence: 97%

Replication-Uncoupled Histone Deposition during Adenovirus DNA Replication

Komatsu

Nagata

2012

In infected cells, the chromatin structure of the adenovirus genome DNA plays critical roles in its genome functions. Previously, we reported that in early phases of infection, incoming viral DNA is associated with both viral core protein VII and cellular histones. Here we show that in late phases of infection, newly synthesized viral DNA is also associated with histones. We also found that the knockdown of CAF-1, a histone chaperone that functions in the replication-coupled deposition of histones, does not affect the level of histone H3 bound on viral chromatin, although CAF-1 is accumulated at viral DNA replication foci together with PCNA. Chromatin immunoprecipitation assays using epitope-tagged histone H3 demonstrated that histone variant H3.3, which is deposited onto the cellular genome in a replication-independent manner, is selectively associated with both incoming and newly synthesized viral DNAs. Microscopic analyses indicated that histones but not USF1, a transcription factor that regulates viral late gene expression, are excluded from viral DNA replication foci and that this is achieved by the oligomerization of the DNA binding protein (DBP). Taken together, these results suggest that histone deposition onto newly synthesized viral DNA is most likely uncoupled with viral DNA replication, and a possible role of DBP oligomerization in this replication-uncoupled histone deposition is discussed.