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

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

Adenovirus major core protein condenses DNA in clusters and bundles, modulating genome release and capsid internal pressure

Some viruses package dsDNA together with large amounts of positively charged proteins, thought to help condense the genome inside the capsid with no evidence. Further, this role is not clear because these viruses have typically lower packing fractions than viruses encapsidating naked dsDNA. In addition, it has recently been shown that the major adenovirus condensing protein (polypeptide VII) is dispensable for genome encapsidation. Here, we study the morphology and mechanics of adenovirus particles with (Ad5-wt) and without (Ad5-VII-) protein VII. Ad5-VII- particles are stiffer than Ad5-wt, but DNA-counterions revert this difference, indicating that VII screens repulsive DNA-DNA interactions. Consequently, its absence results in increased internal pressure. The core is slightly more ordered in the absence of VII and diffuses faster out of Ad5-VII- than Ad5-wt fractured particles. In Ad5-wt unpacked cores, dsDNA associates in bundles interspersed with VII-DNA clusters. These results indicate that protein VII condenses the adenovirus genome by combining direct clustering and promotion of bridging by other core proteins. This condensation modulates the virion internal pressure and DNA release from disrupted particles, which could be crucial to keep the genome protected inside the semi-disrupted capsid while traveling to the nuclear pore

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

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

Relocalization of the Mre11-Rad50-Nbs1 Complex by the Adenovirus E4 ORF3 Protein Is Required for Viral Replication

Adenovirus replication is controlled by the relocalization or modification of nuclear protein complexes, including promyelocytic leukemia protein (PML) nuclear domains and the Mre11-Rad50-Nbs1 (MRN) DNA damage machinery. In this study, we demonstrated that the E4 ORF3 protein effects the relocalization of both PML and MRN proteins to similar structures within the nucleus at early times after infection. These proteins colocalize with E4 ORF3. Through the analysis of specific viral mutants, we found a direct correlation between MRN reorganization at early times after infection and the establishment of viral DNA replication domains. Further, the reorganization of MRN components may be uncoupled from the ability of E4 ORF3 to rearrange PML. At later stages of infection, components of the MRN complex disperse within the nucleus, Nbs1 is found within viral replication centers, Rad50 remains localized with E4 ORF3, and Mre11 is degraded. The importance of viral regulation of the MRN complex is underscored by the complementation of E4 mutant viruses in cells that lack Mre11 or Nbs1 activity. These results illustrate the importance of nuclear organization in virus growth and suggest that E4 ORF3 regulates activities in both PML nuclear bodies and the MRN complex to stimulate the viral replication program

Distinct Roles of the Adenovirus E4 ORF3 Protein in Viral DNA Replication and Inhibition of Genome Concatenation

Adenovirus early proteins E4 ORF3 and E4 ORF6 have complementary functions during viral infection. Both proteins facilitate efficient viral DNA replication, late protein expression, and prevention of concatenation of viral genomes. Additionally, E4 ORF6 is involved in the shutoff of the host cell protein synthesis through its interaction with the E1B 55K protein. This complex also leads to the degradation of p53. A unique function of E4 ORF3 is the reorganization of nuclear structures known as PML oncogenic domains (PODs). The function of these domains is unclear, but PODs have been implicated in a number of important cellular processes, including transcriptional regulation, apoptosis, transformation, and response to interferon. The goal of this study was to determine the functional significance of the reorganization of PODs by E4 ORF3. Point mutations were made in the E4 ORF3 gene. These mutants were recombined into a virus lacking E4 ORF6 and expressed under the control of the natural virus E4 promoter. The panel of mutant viruses was used to investigate the role of E4 ORF3 during the course of the viral infection program. One of the mutant viruses exhibited aberrant reorganization of PODs and had a severe defect in viral DNA replication, thus leading to a dramatic decrease in virus production. A number of mutants accumulated viral DNA and infectious virus particles to wild-type levels but showed significant viral genome concatenation. These data show that E4 ORF3 is a multifunctional protein and that a specific rearrangement of nuclear PML domains is coupled to efficient viral DNA replication. This function is distinct from the role of E4 ORF3 in the regulation of virus genome concatenation via inhibition of cellular double-strand break repair

Mechanism of Adenovirus E4-ORF3-Mediated SUMO Modifications

Viruses interplay with the host SUMOylation system to manipulate diverse cellular responses. The Ad E4-ORF3 protein forms a dynamic nuclear network to interfere with and exploit different host processes, including the DNA damage and interferon responses. We previously reported that E4-ORF3 is a SUMO E3 ligase. Here, we demonstrate that this activity is a conserved function of evolutionarily diverse human Ad E4-ORF3 proteins and that E4-ORF3 functions directly to promote SUMO conjugations to multiple cellular proteins. Recruitment of cellular substrates into E4-ORF3 nuclear inclusions is required for SUMO conjugation to occur in vivo. We probed the mechanism by which E4-ORF3 functions as a SUMO E3 ligase. Only multimeric, but not dimeric, E4-ORF3 binds to the SUMO E2 conjugation enzyme UBC9 in vitro only in a trimeric complex with SUMO. These results reveal a novel mechanism by which a conserved viral protein usurps the cellular SUMO conjugation machinery.Regulation of a variety of different cellular processes, including posttranslational modifications, is critical for the ability of many viruses to replicate efficiently within host cells. The adenovirus (Ad) E4-ORF3 protein assembles into polymers and forms a unique nuclear scaffold that leads to the relocalization and sequestration of cellular proteins, including small ubiquitin-like modifiers (SUMOs). Previously, we showed that E4-ORF3 functions as a SUMO E3 ligase of transcriptional intermediary factor-1 gamma (TIF-1γ) and promotes poly-SUMO chain formation. Here, we present cellular and biochemical data to further understand E4-ORF3 SUMO ligase activity. E4-ORF3 proteins from five different Ad species were found to possess SUMO E3 ligase activities in vitro. In infected cells, SUMO modifications of target proteins occurred only when the proteins were recruited into E4-ORF3 polymeric structures. By analyzing SUMO-deficient TIF-1γ, we demonstrated that SUMO conjugations are not required for E4-ORF3-mediated relocalization of target proteins in infected cells, implying that sequestration is followed by SUMO modification. In vitro SUMO conjugation assays revealed the Ad E1B-55K oncoprotein as a new viral target of E4-ORF3-mediated SUMOylation. We also verified a direct function of E4-ORF3 as a SUMO ligase for multiple cellular proteins, including transcription factor II-I (TFII-I), Nbs1, and Mre11. Moreover, we discovered that E4-ORF3 associates with SUMO-bound UBC9, and E4-ORF3 polymerization is crucial for this ternary interaction. Together, our findings characterize E4-ORF3 as a novel polymer-type SUMO E3 ligase and provide mechanistic insights into the role of E4-ORF3 in SUMO conjugation