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Human adenovirus (HAdV) E1B-55K is a multifunctional regulator of productive viral replication and oncogenic transformation in nonpermissive mammalian cells. These functions depend on E1B-55K's posttranslational modification with the SUMO protein and its binding to HAdV E4orf6. Both early viral proteins recruit specific host factors to form an E3 ubiquitin ligase complex that targets antiviral host substrates for proteasomal degradation. Recently, we reported that the PML-NB-associated factor Daxx represses efficient HAdV productive infection and is proteasomally degraded via a SUMO-E1B-55K-dependent, E4orf6-independent pathway, the details of which remained to be established. RNF4, a cellular SUMO-targeted ubiquitin ligase (STUbL), induces ubiquitinylation of specific SUMOylated proteins and plays an essential role during DNA repair. Here, we show that E1B-55K recruits RNF4 to the insoluble nuclear matrix fraction of the infected cell to support RNF4/Daxx association, promoting Daxx PTM and thus inhibiting this antiviral factor. Removing RNF4 from infected cells using RNA interference resulted in blocking the proper establishment of viral replication centers and significantly diminished viral gene expression. These results provide a model for how HAdV antagonize the antiviral host responses by exploiting the functional capacity of cellular STUbLs. Thus, RNF4 and its STUbL function represent a positive factor during lytic infection and a novel candidate for future therapeutic antiviral intervention strategies.IMPORTANCE Daxx is a PML-NB-associated transcription factor that was recently shown to repress efficient HAdV productive infection. To counteract this antiviral measurement during infection, Daxx is degraded via a novel pathway including viral E1B-55K and host proteasomes. This virus-mediated degradation is independent of the classical HAdV E3 ubiquitin ligase complex, which is essential during viral infection to target other host antiviral substrates. To maintain a productive viral life cycle, HAdV E1B-55K early viral protein inhibits the chromatin-remodeling factor Daxx in a SUMO-dependent manner. In addition, viral E1B-55K protein recruits the STUbL RNF4 and sequesters it into the insoluble fraction of the infected cell. E1B-55K promotes complex formation between RNF4- and E1B-55K-targeted Daxx protein, supporting Daxx posttranslational modification prior to functional inhibition. Hence, RNF4 represents a novel host factor that is beneficial for HAdV gene expression by supporting Daxx counteraction. In this regard, RNF4 and other STUbL proteins might represent novel targets for therapeutic intervention.

Human adenoviruses (HAdV) are nonenveloped viruses containing a linear, double-stranded DNA genome surrounded by an icosahedral capsid. To allow proper viral replication, the genome is imported through the nuclear pore complex associated with viral core proteins. Until now, the role of these incoming virion proteins during the early phase of infection was poorly understood. The core protein V is speculated to bridge the core and the surrounding capsid. It binds the genome in a sequenceindependent manner and localizes in the nucleus of infected cells, accumulating at nucleoli. Here, we show that protein V contains conserved SUMO conjugation motifs (SCMs). Mutation of these consensus motifs resulted in reduced SUMOylation of the protein; thus, protein V represents a novel target of the host SUMOylation machinery. To understand the role of protein V SUMO posttranslational modification during productive HAdV infection, we generated a replication-competent HAdV with SCM mutations within the protein V coding sequence. Phenotypic analyses revealed that these SCM mutations are beneficial for adenoviral replication. Blocking protein V SUMOylation at specific sites shifts the onset of viral DNA replication to earlier time points during infection and promotes viral gene expression. Simultaneously, the altered kinetics within the viral life cycle are accompanied by more efficient proteasomal degradation of host determinants and increased virus progeny production than that observed during wild-type infection. Taken together, our studies show that protein V SUMOylation reduces virus growth; hence, protein V SUMOylation represents an important novel aspect of the host antiviral strategy to limit virus replication and thereby points to potential intervention strategies. IMPORTANCE Many decades of research have revealed that HAdV structural proteins promote viral entry and mainly physical stability of the viral genome in the capsid. Our work over the last years showed that this concept needs expansion as the functions are more diverse. We showed that capsid protein VI regulates the antiviral response by modulation of the transcription factor Daxx during infection. Moreover, core protein VII interacts with SPOC1 restriction factor, which is beneficial for efficient viral gene expression. Here, we were able to show that core protein V also represents a novel substrate of the host SUMOylation machinery and contains several conserved SCMs; mutation of these consensus motifs reduced SUMOylation of the protein. Unexpectedly, we observed that introducing these mutations into HAdV promotes adenoviral replication. In conclusion, we offer novel insights into adenovirus core proteins and provide evidence that SUMOylation of HAdV factors regulates replication efficiency.

Once transported to the replication sites, HAdVs need to assure decondensation and transcriptional activation of their viral genomes to synthesize viral proteins and initiate steps to reprogram the host cell for viral replication. These early stages during adenoviral infection are poorly characterized, but represent a decisive moment in establishing a productive infection. Here, we identify a novel host viral restriction factor, KAP1. This heterochromatin associated transcription factor regulates the dynamic organization of host chromatin structure via its ability to influence epigenetic marks and chromatin compaction. In response to DNA damage, KAP1 is phosphorylated and functionally inactive, resulting in chromatin relaxation. We discovered that KAP1 posttranslational modification is dramatically altered during HAdV infection to limit the antiviral capacity of this host restriction factor, which represents an essential step required for efficient viral replication. Conversely, we also observed an HAdV-mediated decrease of KAP1 SUMO moieties during infection, known to promote chromatin-decondensation events. Based on our findings, we provide evidence that HAdV induces KAP1 deSUMOylation to minimize epigenetic gene silencing and to promote SUMO modification of E1B-55K by a so far unknown mechanism. IMPORTANCE: Here we describe a novel cellular restriction factor for Human Adenovirus (HAdV) that sheds light on very early modulation processes in viral infection. We reported that chromatin formation and cellular SWI/SNF chromatin remodeling play a key role in HAdV transcriptional regulation (1-4). We observed that the cellular chromatin-associated factor, and epigenetic reader SPOC1 represses HAdV infection and gene expression. Here, we illustrate the role of the SPOC1 interacting factor KAP1 during productive HAdV growth. KAP1 binds to the viral E1B-55K protein, promoting its SUMO modification, therefore illustrating a crucial step for efficient viral replication. Simultaneously, KAP1 posttranslational modification is dramatically altered during infection. We observed an HAdV-mediated decrease in KAP1 SUMOylation, known to promote chromatin-decondensation events. These findings indicate that HAdV induces loss of KAP1 SUMOylation to minimize epigenetic gene silencing and to promote SUMO modification of E1B-55K by a so far unknown mechanism.

Аденовирусы являются причиной большого числа инфекционных заболеваний человека. Офтальмологические инфекции аденовирусный конъюнктивит и эпидемический кератоконъюнктивит вызывают, в основном, аденовирусы человека, относящиеся к группе D. Недостаточная эффективность современных методов лечения аденовирусной инфекции обусловливает необходимость поиска и разработки лекарственных средств, избирательно воздействующих на возбудителей данного заболевания. Потенциальными мишенями для создания терапевтических препаратов служат ранние гены аденовирусов человека, такие, как ген ДНК-полимеразы Е2В и ген Е1А, играющие важную роль в репликации вирусной ДНК. В настоящей работе предложен подход к эффективному подавлению репликации аденовирусов группы D человека с помощью РНК-интерференции. Созданы модельные линии клеток, которые позволяют быстро определять эффективность действия интерферирующих РНК, комплементарных различным участкам мРНК гена Е1А. Нами показано значительное подавление экспрессии Е1А с помощью интерференции РНК в таких клетках. Также показана высокая эффективность малых шпилечных РНК, специфичных в отношении ранних генов E1A и E2B аденовирусов группы D человека, при подавлении репликации аденовирусов типа D8 и D37 в первичных клетках лимба роговицы глаза человека. Мы надеемся, что результаты нашего исследования смогут послужить основой для разработки современных лекарственных средств для борьбы с заболеваниями человека, вызываемыми аденовирусами.

The heterogeneous nuclear ribonucleoprotein (hnRNP) family includes predominantly nuclear proteins acting at different stages of mRNA metabolism. A characteristic feature of hnRNPs is to undergo post-translational asymmetric arginine methylation catalysed by different type 1 protein arginine methyltransferases (PRMTs). A novel mammalian hnRNP, E1B-AP5, recently identified by its interaction with adenovirus early protein E1B-55kDa, has been proposed to have a regulatory role in adenoviral and host-cell mRNA processing/nuclear export [Gabler, Schutt, Groitl, Wolf, Shenk and Dobner (1998) J. Virol. 72, 7960–7971]. Here we report that E1B-AP5 is methylated in vivo in its Arg-Gly-Gly (RGG)-box domain, known to mediate protein–RNA interactions. The activity responsible for E1B-AP5 methylation forms a complex with E1B-AP5 in vivo. The predominant mammalian arginine methyltransferase HRMT1L2 (hPRMT1) did not detectably methylate endogenous E1B-AP5 despite efficiently methylating a recombinant RGG-box domain of E1B-AP5. Using yeast two-hybrid screening we identified HRMT1L1 (PRMT2) as one of the proteins interacting with E1B-AP5. By in situ immunofluorescence we demonstrated that E1B-AP5 co-localizes with the nuclear fraction of HRMT1L1. The Src homology 3 (SH3) domain of HRMT1L1 was essential for its interaction with E1B-AP5 in vivo. We suggest that HRMT1L1 is responsible for specific E1B-AP5 methylation in vivo.

We propose that human adenoviruses (HAdVs) be identified, characterized, and typed on the basis of complete genome sequence analyses rather than serological approaches. This idea has recently percolated through the community of adenovirologists. As a result, an open-floor discussion took place at