bViruses employ a variety of strategies to usurp and control cellular activities through the orchestrated recruitment of macromolecules to specific cytoplasmic or nuclear compartments. Formation of such specialized virus-induced cellular microenvironments, which have been termed viroplasms, virus factories, or virus replication centers, complexes, or compartments, depends on molecular interactions between viral and cellular factors that participate in viral genome expression and replication and are in some cases associated with sites of virion assembly. These virus-induced compartments function not only to recruit and concentrate factors required for essential steps of the viral replication cycle but also to control the cellular mechanisms of antiviral defense. In this review, we summarize characteristic features of viral replication compartments from different virus families and discuss similarities in the viral and cellular activities that are associated with their assembly and the functions they facilitate for viral replication.

Adenovirus type 5 (Ad5) inactivates the host cell DNA damage response by facilitating the degradation of Mre11, DNA ligase IV, and p53. In the case of p53, this is achieved through polyubiquitylation by Ad5E1B55K and Ad5E4orf6, which recruit a Cul5-based E3 ubiquitin ligase. Recent evidence indicates that this paradigm does not apply to other adenovirus serotypes, since Ad12, but not Ad5, causes the degradation of TOPBP1 through the action of E4orf6 alone and a Cul2-based E3 ubiquitin ligase. We now have extended these studies to adenovirus groups A to E. While infection by Ad4, Ad5, and Ad12 (groups E, C, and A, respectively) cause the degradation of Mre11, DNA ligase IV, and p53, infection with Ad3, Ad7, Ad9, and Ad11 (groups B1, B1, D, and B2, respectively) only affects DNA ligase IV levels. Indeed, Ad3, Ad7, and Ad11 cause the marked accumulation of p53. Despite this, MDM2 levels were very low following infection with all of the viruses examined here, regardless of whether they increase p53 expression. In addition, we found that only Ad12 causes the degradation of TOPBP1, and, like Ad5, Ad4 recruits a Cul5-based E3 ubiquitin ligase to degrade p53. Surprisingly, Mre11 and DNA ligase IV degradation do not appear to be significantly affected in Ad4-, Ad5-, or Ad12-infected cells depleted of Cul2 or Cul5, indicating that E1B55K and E4orf6 recruit multiple ubiquitin ligases to target cellular proteins. Finally, although Mre11 is not degraded by Ad3, Ad7, Ad9, and Ad11, no viral DNA concatemers could be detected. We suggest that group B and D adenoviruses have evolved mechanisms based on the loss of DNA ligase IV and perhaps other unknown molecules to disable the host cell DNA damage response to promote viral replication.

The biological relevance of extracellular vesicles (EV) in intercellular communication has been well established. Thus far, proteins and RNA were described as main cargo. Here, we show that EV released from human bone marrow derived mesenchymal stromal cells (BM-hMSC) also carry high-molecular DNA in addition. Extensive EV characterization revealed this DNA mainly associated with the outer EV membrane and to a smaller degree also inside the EV. Our EV purification protocol secured that DNA is not derived from apoptotic or necrotic cells. To analyze the relevance of EV-associated DNA we lentivirally transduced Arabidopsis thaliana-DNA (A.t.-DNA) as indicator into BM-hMSC and generated EV. Using quantitative polymerase chain reaction (qPCR) techniques we detected high copy numbers of A.t.-DNA in EV. In recipient hMSC incubated with tagged EV for two weeks we identified A.t.-DNA transferred to recipient cells. Investigation of recipient cell DNA using quantitative PCR and verification of PCR-products by sequencing suggested stable integration of A.t.-DNA. In conclusion, for the first time our proof-of-principle experiments point to horizontal DNA transfer into recipient cells via EV. Based on our results we assume that eukaryotic cells are able to exchange genetic information in form of DNA extending the known cargo of EV by genomic DNA. This mechanism might be of relevance in cancer but also during cell evolution and development.

During the adenovirus infectious cycle, the early proteins E4orf6 and E1B55K are known to perform several functions. These include nuclear export of late viral mRNAs, a block of nuclear export of the bulk of cellular mRNAs, and the ubiquitin-mediated degradation of selected proteins, including p53 and Mre11. Degradation of these proteins occurs via a cellular E3 ubiquitin ligase complex that is assembled through interactions between elongins B and C and BC boxes present in E4orf6 to form a cullin 5-based ligase complex. E1B55K, which has been known for some time to associate with the E4orf6 protein, is thought to bind to specific substrate proteins to bring them to the complex for ubiquitination. Earlier studies with E4orf6 mutants indicated that the interaction between the E4orf6 and E1B55K proteins is optimal only when E4orf6 is able to form the ligase complex. These and other observations suggested that most if not all of the functions ascribed to E4orf6 and E1B55K during infection, including the control of mRNA export, are achieved through the degradation of specific substrates by the E4orf6 ubiquitin ligase activity. We have tested this hypothesis through the generation of a virus mutant in which the E4orf6 product is unable to form a ligase complex and indeed have found that this mutant behaves identically to an E4orf6 ؊ virus in production of late viral proteins, growth, and export of the late viral L5 mRNA.The late phase of an adenoviral infection, typified by human adenovirus type 5 (Ad5), is characterized by a massive production of progeny virions. To support this production, the virus takes control of the cellular machinery to produce abundant amounts of its own late proteins to the detriment of the synthesis of cellular proteins. This host cell shutoff is achieved via several mechanisms. The translation of cellular mRNAs is blocked by the action of the L4-100K protein on the ribosomal machinery (15, 16) so that only the late viral mRNAs containing the tripartite leader sequence can be translated (26). At the same time, export of cellular mRNAs to the cytoplasm is blocked by the action of the early viral proteins E4orf6 and E1B55K (3, 6, 23, 42). These same proteins were also shown to be required for the export of the late viral mRNAs (4,20,23,42,53). The E4orf6 and E1B55K proteins were shown to interact during infection (48) and to function in the same pathway, as a viral mutant with defects in both of these products was seen to have essentially the same phenotype as mutants affecting only one of these species (3,17,23,42). The role that E4orf6 and E1B55K proteins play in the control of mRNA export has yet to be defined.It has been known for some time that the E4orf6 and E1B55K proteins play a role in the degradation of the p53 tumor suppressor, and recently the mechanism for such degradation has been elucidated by our group. Expression of the virus E1A protein results in increased levels of p53 (10, 34); however, during infection, in the presence of both E4orf6 and E1B55K products, p53 is degraded ...

Adenoviral replication depends on viral as well as cellular proteins. However, little is known about cellular proteins promoting adenoviral replication. In our screens to identify such proteins, we discovered a cellular component of the ubiquitin proteasome pathway interacting with the central regulator of adenoviral replication. Our binding assays mapped a specific interaction between the N-terminal domains of both viral E1B-55K and USP7, a deubiquitinating enzyme. RNA interference-mediated downregulation of USP7 severely reduced E1B-55K protein levels, but more importantly negatively affected adenoviral replication. We also succeeded in resynthesizing an inhibitor of USP7, which like the knockdown background reduced adenoviral replication. Further assays revealed that not only adenoviral growth, but also adenoviral oncogene-driven cellular transformation relies on the functions of USP7. Our data provide insights into an intricate mechanistic pathway usurped by an adenovirus to promote its replication and oncogenic functions, and at the same time open up possibilities for new antiviral strategies.

The therapeutic effect of mesenchymal stromal cells (MSC) in tissue regeneration is based mainly on the secretion of bioactive molecules. Here, we report that the radioprotective effect of mouse bone marrow derived mesenchymal stromal cells (mMSC) can be attributed to extracellular vesicles (EV) released from mMSC. The transplantation of mMSC-derived EV into lethally irradiated mice resulted in long-term survival but no improvement in short-term reconstitution of the recipients. Importantly, the radiation rescue was efficient without additional hematopoietic support. In vitro we show a protection by EV of irradiated hematopoietic stem cells but not progenitor cells using stroma-cell cultures and colony-forming assays. After systemic infusion into lethally irradiated recipients, labeled EV traveled freely through the body reaching the bone marrow within 2 hours. We further show that long-term repopulating Sca-1 positive and c-kit low-positive stem cells were directly targeted by EV leading to long-term survival. Collectively, our data suggest EV as an effective first-line treatment to combat radiation-induced hematopoietic failure which might also be helpful in alleviating myelosuppression due to chemotherapy and toxic drug reaction. We suggest the infusion of MSC-derived EV as efficient and immediate treatment option after irradiation injuries. STEM CELLS 2017;35:2379-2389 SIGNIFICANCE STATEMENTRadiation as used in conditioning of patients prior to hematopoietic stem cell transplantation is associated with severe side effects. Previous research has shown survival-promoting properties of mouse mesenchymal stromal cells in a model of lethal irradiation without support of hematopoietic stem cells. This phenomenon formerly was attributed to "paracrine" effects. In recent years, extracellular vesicles (EVs) have gained increasing interest as mediators of intercellular communication based on their capability to transfer new information to sites of action without loss at physiological barriers. This novel data demonstrate that EV derived from mouse bone marrow-derived mesenchymal stromal cells are the active component of these cells providing a radiation-protective effect. Results show that hematopoietic, long-term repopulating stem cells are targeted rapidly by the EV and thereby rescued from radiation damage. These results open a new line of treatment options for patients suffering from radiotherapy induced myelosuppression without the potential side effects of cell therapies.

The ability of adenovirus early region proteins, E1B-55K and E4orf6, to usurp control of cellular ubiquitin ligases and target proteins for proteasome-dependent degradation during infection is well established. Here we show that the E4 gene product, E4orf3 can, independently of E1B-55K and E4orf6, target the transcriptional corepressor transcriptional intermediary factor 1␥ (TIF1␥) for proteasome-mediated degradation during infection. Initial mass spectrometric studies identified TIF1 family members-TIF1␣, TIF1␤, and TIF1␥-as E1B-55K-binding proteins in both transformed and infected cells, but analyses revealed that, akin to TIF1␣, TIF1␥ is reorganized in an E4orf3-dependent manner to promyelocytic leukemia protein-containing nuclear tracks during infection. The use of a number of different adenovirus early region mutants identified the specific and sole requirement for E4orf3 in mediating TIF1␥ degradation. Further analyses revealed that TIF1␥ is targeted for degradation by a number of divergent human adenoviruses, suggesting that the ability of E4orf3 to regulate TIF1␥ expression is evolutionarily conserved. We also determined that E4orf3 does not utilize the Cullin-based ubiquitin ligases, CRL2 and CRL5, or the TIF1␣ ubiquitin ligase in order to promote TIF1␥ degradation. Further studies suggested that TIF1␥ possesses antiviral activity and limits adenovirus early and late gene product expression during infection. Indeed, TIF1␥ knockdown accelerates the adenovirus-mediated degradation of MRE11, while TIF1␥ overexpression delays the adenovirus-mediated degradation of MRE11. Taken together, these studies have identified novel adenovirus targets and have established a new role for the E4orf3 protein during infection.H uman adenoviruses (Ad) are small, nonenveloped viruses with a linear double-stranded DNA genome and are classified into species A to F according to various criteria (7). Since the observation that Ad12 could induce tumors in newborn rodents, Ad has served as a reliable model for dissecting the molecular basis of the key cellular signaling pathways that underlie the transformation process (28,33,69,70). Studies investigating the roles of the Ad early region proteins in both Ad-transformed and Adinfected cells have led to key advances in the understanding of basic cellular processes and how Ad usurps control of these pathways in order to promote viral replication (8,33,67).The Ad early region proteins E1B-55K, E4orf3, and E4orf6 have a complex inter-relationship and serve together to regulate RNA processing, late viral mRNA nuclear export, the shutoff of host-cell protein synthesis, and neutralization of the host cell DNA damage response during infection (4,29,57,61,67,73). They can also function synergistically and cooperate with E1A to promote Ad-induced cellular transformation (47-49). It is perhaps not surprising, therefore, that they share many common functions. For instance, E1B-55K interacts directly with p53 to repress transcriptional activity and also promotes p53 sumoylation and targeting t...

Congenital Zika virus (ZIKV) syndrome may cause fetal microcephaly in ~1% of affected newborns. Here, we investigate whether the majority of clinically inapparent newborns might suffer from long-term health impairments not readily visible at birth. Infection of immunocompetent pregnant mice with high-dose ZIKV caused severe offspring phenotypes, such as fetal death, as expected. By contrast, low-dose (LD) maternal ZIKV infection resulted in reduced fetal birth weight but no other obvious phenotypes. Male offspring born to LD ZIKV-infected mothers had increased testosterone (TST) levels and were less likely to survive in utero infection compared to their female littermates. Males also presented an increased number of immature neurons in apical and basal hippocampal dendrites, while female offspring had immature neurons in basal dendrites only. Moreover, male offspring with high but not very high (storm) TST levels were more likely to suffer from learning and memory impairments compared to females. Future studies are required to understand the impact of TST on neuropathological and neurocognitive impairments in later life. In summary, increased sex-specific vigilance is required in countries with high ZIKV prevalence, where impaired neurodevelopment may be camouflaged by a healthy appearance at birth.