The adenovirus E4-ORF3 protein stimulates SUMOylation of general transcription factor TFII-I to direct proteasomal degradation

Modulation of host cell transcription, translation, and posttranslational modification processes is critical for the ability of many viruses to replicate efficiently within host cells. The human adenovirus (Ad) early region 4 open reading frame 3 (E4-ORF3) protein forms unique inclusions throughout the nuclei of infected cells and inhibits the antiviral Mre11-Rad50-Nbs1 DNA repair complex through relocalization. E4-ORF3 also induces SUMOylation of Mre11 and Nbs1. We recently identified additional cellular targets of E4-ORF3 and found that E4-ORF3 stimulates ubiquitin-like modification of 41 cellular proteins involved in a wide variety of processes. Among the proteins most abundantly modified in an E4-ORF3-dependent manner was the general transcription factor II–I (TFII-I). Analysis of Ad-infected cells revealed that E4-ORF3 induces TFII-I relocalization and SUMOylation early during infection. In the present study, we explored the relationship between E4-ORF3 and TFII-I. We found that Ad infection or ectopic E4-ORF3 expression leads to SUMOylation of TFII-I that precedes a rapid decline in TFII-I protein levels. We also show that E4-ORF3 is required for ubiquitination of TFII-I and subsequent proteasomal degradation. This is the first evidence that E4-ORF3 regulates ubiquitination. Interestingly, we found that E4-ORF3 modulation of TFII-I occurs in diverse cell types but only E4-ORF3 of Ad species C regulates TFII-I, providing critical insight into the mechanism by which E4-ORF3 targets TFII-I. Finally, we show that E4-ORF3 stimulates the activity of a TFII-I-repressed viral promoter during infection. Our results characterize a novel mechanism of TFII-I regulation by Ad and highlight how a viral protein can modulate a critical cellular transcription factor during infection

Altering the Ad5 Packaging Domain Affects the Maturation of the Ad Particles

We have previously described a new family of mutant adenoviruses carrying different combinations of attB/attP sequences from bacteriophage PhiC31 flanking the Ad5 packaging domain. These novel helper viruses have a significantly delayed viral life cycle and a severe packaging impairment, regardless of the presence of PhiC31 recombinase. Their infectious viral titers are significantly lower (100–1000 fold) than those of control adenovirus at 36 hours post-infection, but allow for efficient packaging of helper-dependent adenovirus. In the present work, we have analyzed which steps of the adenovirus life cycle are altered in attB-helper adenoviruses and investigated whether these viruses can provide the necessary viral proteins in trans. The entry of attB-adenoviral genomes into the cell nucleus early at early timepoints post-infection was not impaired and viral protein expression levels were found to be similar to those of control adenovirus. However, electron microscopy and capsid protein composition analyses revealed that attB-adenoviruses remain at an intermediate state of maturation 36 hours post-infection in comparison to control adenovirus which were fully mature and infective at this time point. Therefore, an additional 20–24 hours were found to be required for the appearance of mature attB-adenovirus. Interestingly, attB-adenovirus assembly and infectivity was restored by inserting a second packaging signal close to the right-end ITR, thus discarding the possibility that the attB-adenovirus genome was retained in a nuclear compartment deleterious for virus assembly. The present study may have substantive implications for helper-dependent adenovirus technology since helper attB-adenovirus allows for preferential packaging of helper-dependent adenovirus genomes

Upper Ordovician chronostratigraphic correlation between the Appalachian and Midcontinent basins

Study of a subsurface core (named F688) from northern Indiana provides integrated data sets linking Katian chronostratigraphic records of the Appalachian and Midcontinent basins. The F688 core shows a variety of shallow- and deep-water facies containing numerous, well-preserved and zonally significant fossil species and diagnostic chemostratigraphic patterns. The succession belonging to the Cincinnatian Regional Stage in the F688 core is 210 m thick. Detailed benchtop examination of the succession revealed several phosphatic intervals, rich brachiopod faunas, multiple graptolitic horizons, and at least two tephras. Elemental analysis was conducted at 60 cm spacing quantifying lithofacies composition. Based on these results, the succession was assigned to six previously defined lithostratigraphic units (Kope, Waynesville, Liberty, Whitewater, Elkhorn, and Fort Atkinson formations). This lithostratigraphic succession shares components with both the Appalachian and Midcontinent basins, suggesting deposition near their shared margin. Twenty samples yielded abundant, well-preserved, low-diversity conodont assemblages with long-ranging taxa that clearly demarcate the position of the OrdovicianâSilurian boundary at the top of the succession in the core. More than fifty palynologic samples, targeting graptolite-bearing intervals, were processed for chitinozoans and produced important new insights. The Kope Formation contains the chitinozoan species Belonechitina kjellstromi, Hercochitina downiei, and Clathrochitina sp. nov., co-occurring with a graptolite assemblage suggestive of the Geniculograptus pygmaeus Zone. Samples from the overlying Waynesville Formation produced graptolites indicative of the upper G. pygmaeus to Paraorthograptus manitoulinensis zones co-occurring with the long-ranging chitinozoan species Belonechitina micracantha and Plectochitina spongiosa as well as several new species of the genera Tanuchitina and Hercochitina. Higher in the core, the Liberty, Whitewater, Elkhorn, and Fort Atkinson formations yielded chitinozoan species characteristic of the upper Katian biozones of Anticosti Island and Nevada, such as Tanuchitina anticostiensis, Hercochitina longi, and Eisenackitina ripae. Results of δ13Ccarb analysis reveal partial preservation of the Kope, Waynesville, and Elkhorn excursions. A tephra in the rising limb of the Waynesville Excursion yielded needle-shaped clear zircons that will provide a high-precision U-Pb age. The Fort Atkinson Formation is overlain by the Brassfield Formation containing Silurian conodonts and δ13Ccarb values suggesting an Aeronian age. Chronostratigraphic data from our study of the F688 core resolves longstanding uncertainty about correlations between strata of Katian Age in the Appalachian and Midcontinent basins. Integration of core F688 with our other regional chronostratigraphic data in the Midcontinent Basin demonstrates that the Fort Atkinson Formation of the Indiana and Illinois subsurface is age equivalent to the Fernvale Formation of Tennessee, Arkansas, and Oklahoma. Across this area, the Fernvale is overlain by graptolitic shales of the uppermost P. manitoulinensis to basal Dicellograptus complanatus graptolite zones. By contrast, the type Fort Atkinson Formation of Iowa is interpreted to occur completely within the younger D. complanatus Zone. These regional correlations taken as a whole suggest that the uppermost Katian (all of Ka4) and all but the uppermost Hirnantian are missing throughout much of the Appalachian Basin. By contrast, the Midcontinent Basin contains a much more complete upper Katian and Hirnantian succession. Our comprehensive approach is correcting temporal miscorrelation and providing robust chronostratigraphic context for study of biogeochemical events, which will further enable us to disentangle proxy data and identify the processes that drove the Katian diversity peak and culminated in the Late Ordovician mass extinction

The adenovirus major core protein VII is dispensable for virion assembly but is essential for lytic infection

The Adenovirus (Ad) genome within the capsid is tightly associated with a virus-encoded, histone-like core protein—protein VII. Two other Ad core proteins, V and X/μ, also are located within the virion and are loosely associated with viral DNA. Core protein VII remains associated with the Ad genome during the early phase of infection. It is not known if naked Ad DNA is packaged into the capsid, as with dsDNA bacteriophage and herpesviruses, followed by the encapsidation of viral core proteins, or if a unique packaging mechanism exists with Ad where a DNA-protein complex is simultaneously packaged into the virion. The latter model would require an entirely new molecular mechanism for packaging compared to known viral packaging motors. We characterized a virus with a conditional knockout of core protein VII. Remarkably, virus particles were assembled efficiently in the absence of protein VII. No changes in protein composition were evident with VII−virus particles, including the abundance of core protein V, but changes in the proteolytic processing of some capsid proteins were evident. Virus particles that lack protein VII enter the cell, but incoming virions did not escape efficiently from endosomes. This greatly diminished all subsequent aspects of the infectious cycle. These results reveal that the Ad major core protein VII is not required to condense viral DNA within the capsid, but rather plays an unexpected role during virus maturation and the early stages of infection. These results establish a new paradigm pertaining to the Ad assembly mechanism and reveal a new and important role of protein VII in early stages of infection

Role for the L1-52/55K Protein in the Serotype Specificity of Adenovirus DNA Packaging▿

The packaging of adenovirus (Ad) DNA into virions is dependent upon cis-acting sequences and trans-acting proteins. We studied the involvement of Ad packaging proteins in the serotype specificity of packaging. Both Ad5 and Ad17 IVa2 and L4-22K proteins complemented the growth of Ad5 IVa2 and L4-22K mutant viruses, respectively. In contrast, the Ad5 L1-52/55K protein complemented an Ad5 L1-52/55K mutant virus, but the Ad17 L1-52/55K protein did not. The analysis of chimeric proteins demonstrated that the N-terminal half of the Ad5 L1-52/55K protein mediated this function. Finally, we demonstrate that the L4-33K and L4-22K proteins have distinct functions during infection

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

ABSTRACT 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.</jats:p