The nucleoplasm is not homogenous; it consists of many types of nuclear bodies, also known as nuclear domains or nuclear subcompartments. These self-organizing structures gather machinery involved in various nuclear activities. Nuclear speckles (NSs) or splicing speckles, also called interchromatin granule clusters, were discovered as sites for splicing factor storage and modification. Further studies on transcription and mRNA maturation and export revealed a more general role for splicing speckles in RNA metabolism. Here, we discuss the functional implications of the localization of numerous proteins crucial for epigenetic regulation, chromatin organization, DNA repair and RNA modification to nuclear speckles. We highlight recent advances suggesting that NSs facilitate integrated regulation of gene expression. In addition, we consider the influence of abundant regulatory and signaling proteins, i.e. protein kinases and proteins involved in protein ubiquitination, phosphoinositide signaling and nucleoskeletal organization, on pre-mRNA synthesis and maturation. While many of these regulatory proteins act within NSs, direct evidence for mRNA metabolism events occurring in NSs is still lacking. NSs contribute to numerous human diseases, including cancers and viral infections. In addition, recent data have demonstrated close relationships between these structures and the development of neurological disorders.

/pdf/nuclear-speckles-molecular-organization-biological-function-41s2d5i5e1.pdf

Nuclear speckles: molecular organization, biological function and role in disease.

Summary


Herpesvirus capsids are assembled in the nucleus of infected cells whereas final maturation occurs in the cytosol. To access the final maturation compartment, intranuclear capsids have to cross the nuclear envelope which represents a formidable barrier. They do so by budding at the inner nuclear membrane, thereby forming a primary enveloped particle residing in the perinuclear cleft. Formation of primary envelopes is driven by a heterodimeric complex of two conserved herpesviral proteins, designated in the herpes simplex virus nomenclature as pUL34, a tail-anchored transmembrane protein located in the nuclear envelope, and pUL31. This nuclear egress complex recruits viral and cellular kinases to soften the nuclear lamina and allowing access of capsids to the inner nuclear membrane. How capsids are recruited to the budding site and into the primary virus particle is still not completely understood, nor is the composition of the primary enveloped virion in the perinuclear cleft. Fusion of the primary envelope with the outer nuclear membrane then results in translocation of the capsid to the cytosol. This fusion event is clearly different from fusion during infectious entry of free virions into target cells in that it does not require the conserved essential core herpesvirus fusion machinery. Nuclear egress can thus be viewed as a vesicle (primary envelope)-mediated transport of cargo (capsids) through thenuclear envelope, a process which had been unique in cell biology. Only recently has a similar process been identified in Drosophila for nuclear egress of large ribonucleoprotein complexes. Thus, herpesviruses appear to subvert a hitherto cryptic cellular pathway for translocation of capsids from the nucleus to the cytosol.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/cmi.12044

The way out: what we know and do not know about herpesvirus nuclear egress

https://www.cell.com/trends/microbiology/pdf/S0966-842X(12)00081-9.pdf

Viral and host control of cytomegalovirus maturation

In eukaryotic cells the nuclear genome is enclosed by the nuclear envelope (NE). In metazoans, the NE breaks down in mitosis and it has been assumed that the physical barrier separating nucleoplasm and cytoplasm remains intact during the rest of the cell cycle and cell differentiation. However, recent studies suggest that nonmitotic NE remodeling plays a critical role in development, virus infection, laminopathies, and cancer. Although the mechanisms underlying these NE restructuring events are currently being defined, one common theme is activation of protein kinase C family members in the interphase nucleus to disrupt the nuclear lamina, demonstrating the importance of the lamina in maintaining nuclear integrity.

/pdf/breaching-the-nuclear-envelope-in-development-and-disease-i5ufaa4ftf.pdf

Breaching the nuclear envelope in development and disease

Nuclear egress of herpesvirus capsids through the nuclear envelope is mediated by the multimeric nuclear egress complex (NEC). The human cytomegalovirus (HCMV) core NEC is defined by an interaction between the membrane-anchored pUL50 and its nuclear co-factor pUL53, tightly associated through heterodimeric corecruitment to the nuclear envelope. Cellular proteins, such as p32/gC1qR, emerin and protein kinase C (PKC), are recruited by direct interaction with pUL50 for the multimeric extension of the NEC. As a functionally important event, the recruitment of both viral and cellular protein kinases leads to site-specific lamin phosphorylation and nuclear lamina disassembly. In this study, interaction domains within pUL50 for its binding partners were defined by co-immunoprecipitation. The interaction domain for pUL53 is located within the pUL50 N-terminus (residues 10-169), interaction domains for p32/gC1qR (100-358) and PKC (100-280) overlap in the central part of pUL50, and the interaction domain for emerin is located in the C-terminus (265-397). Moreover, expression and formation of core NEC proteins at the nuclear rim were consistently detected in cells permissive for productive HCMV replication, including two trophoblast-cell lines. Importantly, regular nuclear-rim formation of the core NEC was blocked by inhibition of cyclin-dependent kinase (CDK) activity. In relation to the recently published crystal structure of the HCMV core NEC, our findings result in a refined view of NEC assembly. In particular, we suggest that CDKs may play an important regulatory role in NEC formation during HCMV replication.

Cytomegalovirus pUL50 is the multi-interacting determinant of the core nuclear egress complex (NEC) that recruits cellular accessory NEC components.

Viral replication is a complex process relying on a network of interacting viral and cellular proteins, in which particularly protein kinases play an important regulatory role. The specific phosphorylation of substrate proteins induces activation, inactivation, or other functional modification and thus determines virus-host cell interregulation. During herpesviral infections, both viral and cellular protein kinases are expressed and provide activities crucial for the efficiency of virus replication. The protein kinase pUL97 encoded by human cytomegalovirus (HCMV) is a multifunctional regulatory enzyme which exerts strong regulatory effects on early and late steps of the viral replication cycle. A number of interacting proteins and substrates of pUL97 have been described, including retinoblastoma (Rb) protein, nuclear lamins and viral pUL69. Recently, it was demonstrated that pUL97 has structural and functional resemblance to cyclin-dependent protein kinases (CDKs) and thus represents a CDK ortholog. pUL97 can phosphorylate and inactivate Rb, resulting in a stimulation of cell cycle progression. In addition, the association of pUL97 activity with nucleocytoplasmic export of viral capsids has been demonstrated by several investigators. We could show that pUL97 is able to phosphorylate nuclear lamins and to contribute to the HCMV-induced reorganization of the nuclear lamina. On the basis of very recent findings, it is becoming increasingly clear that pUL97 is a component of a multiprotein nuclear egress complex (NEC). The NEC contains a small number of egress proteins involved in the recruitment of protein kinases, such as pUL97 and cellular protein kinase C (PKC), to specific sites of the nuclear lamina. Current information about the composition, function, and regulatory complexity of the NEC leads to a mechanistic concept which may set the key features of HCMV nuclear egress in a new light.

Regulatory roles of protein kinases in cytomegalovirus replication.

Cyclin-dependent protein kinases (CDKs) are important regulators of cellular processes and are functionally integrated into the replication of human cytomegalovirus (HCMV). Recently, a regulatory impact of CDK activity on the viral mRNA export factor pUL69 was shown. Here, specific aspects of the mode of interaction between CDK9/cyclin T1 and pUL69 are described. Intracellular localization was studied in the presence of a novel selective CDK9 inhibitor, R22, which exerts anti-cytomegaloviral activity in vitro. A pronounced R22-induced formation of nuclear speckled aggregation of pUL69 was demonstrated. Multi-labelling confocal laser-scanning microscopy revealed that CDK9 and cyclin T1 co-localized perfectly with pUL69 in individual speckles. The effects were similar to those described recently for the broad CDK inhibitor roscovitine. Co-immunoprecipitation and yeast two-hybrid analyses showed that cyclin T1 interacted with both CDK9 and pUL69. The interaction region of pUL69 for cyclin T1 could be attributed to aa 269–487. Moreover, another component of CDK inhibitor-induced speckled aggregates was identified with RNA polymerase II, supporting earlier reports that strongly suggested an association of pUL69 with transcription complexes. Interestingly, when using a UL69-deleted recombinant HCMV, no speckled aggregates were formed by CDK inhibitor treatment. This indicated that pUL69 is the defining component of aggregates and generally may represent a crucial viral interactor of cyclin T1. In conclusion, these data emphasize that HCMV inter-regulation with CDK9/cyclin T1 is at least partly based on a pUL69–cylin T1 interaction, thus contributing to the importance of CDK9 for HCMV replication.

Recruitment of cyclin-dependent kinase 9 to nuclear compartments during cytomegalovirus late replication: importance of an interaction between viral pUL69 and cyclin T1.

Cyclin-dependent kinases (CDKs) are multifaceted regulators involved in the replication of human cytomegalovirus. Recently, we demonstrated an interaction of CDK9–cyclin T1 as well as viral CDK orthologue pUL97 with the viral regulator pUL69, thereby leading to pUL69-activating phosphorylation. Here, we demonstrate that colocalization and direct pUL69–cyclin T1 interaction is independent of viral strains and host cell types. In vitro phosphorylation of pUL69 by CDK9 or pUL97 did not occur in a single site-specific manner, but at multiple sites. The previously described fine-speckled nuclear aggregation of pUL69 was assigned to the late phase of viral replication. CDK inhibitors, including a novel inhibitor of the CDK-activating kinase CDK7, massively intensified this fine-speckled accumulation. Interestingly, we also observed spontaneous pUL69 accumulation in the absence of inhibitors at a lower frequency. These findings provide new insight into pUL69 kinase interregulation and emphasize the importance of pUL69 phosphorylation for correct intranuclear localization.

New insight into the phosphorylation-regulated intranuclear localization of human cytomegalovirus pUL69 mediated by cyclin-dependent kinases (CDKs) and viral CDK orthologue pUL97.

Autophagy is a catabolic process contributing to intrinsic cellular defense by degrading viral particles or proteins; however, several viruses hijack this pathway for their own benefit. The role of autophagy during human cytomegalovirus (HCMV) replication has not been definitely clarified yet. Utilizing small interfering RNA (siRNA)-based screening, we observed that depletion of many autophagy-related proteins resulted in reduced virus release, suggesting a requirement of autophagy-related factors for efficient HCMV replication. Additionally, we could show that the autophagy-initiating serine/threonine protein kinase ULK1 as well as other constituents of the ULK1 complex were upregulated at early times of infection and stayed upregulated throughout the replication cycle. We demonstrate that indirect interference with ULK1 through inhibition of the upstream regulator AMP-activated protein kinase (AMPK) impaired virus release. Furthermore, this result was verified by direct abrogation of ULK1 kinase activity utilizing the ULK1-specific kinase inhibitors SBI-0206965 and ULK-101. Analysis of viral protein expression in the presence of ULK-101 revealed a connection between the cellular kinase ULK1 and the viral tegument protein pp28 (pUL99), and we identified pp28 as a novel viral substrate of ULK1 by in vitro kinase assays. In the absence of ULK1 kinase activity, large pp28- and pp65-positive structures could be detected in the cytoplasm at late time points of infection. Transmission electron microscopy demonstrated that these structures represent large perinuclear protein accumulations presumably representing aggresomes. Our results indicate that HCMV manipulates ULK1 and further components of the autophagic machinery to ensure the efficient release of viral particles.IMPORTANCE The catabolic program of autophagy represents a powerful immune defense against viruses that is, however, counteracted by antagonizing viral factors. Understanding the exact interplay between autophagy and HCMV infection is of major importance since autophagy-related proteins emerged as promising targets for pharmacologic intervention. Our study provides evidence for a proviral role of several autophagy-related proteins suggesting that HCMV has developed strategies to usurp components of the autophagic machinery for its own benefit. In particular, we observed strong upregulation of the autophagy-initiating protein kinase ULK1 and further components of the ULK1 complex during HCMV replication. In addition, both siRNA-mediated depletion of ULK1 and interference with ULK1 protein kinase activity by two chemically different inhibitors resulted in impaired viral particle release. Thus, we propose that ULK1 kinase activity is required for efficient HCMV replication and thus represents a promising novel target for future antiviral drug development.

Sabine Feichtinger

Papers

Regulatory roles of protein kinases in cytomegalovirus replication.

Recruitment of cyclin-dependent kinase 9 to nuclear compartments during cytomegalovirus late replication: importance of an interaction between viral pUL69 and cyclin T1.

New insight into the phosphorylation-regulated intranuclear localization of human cytomegalovirus pUL69 mediated by cyclin-dependent kinases (CDKs) and viral CDK orthologue pUL97.

The Autophagy-Initiating Protein Kinase ULK1 Phosphorylates Human Cytomegalovirus Tegument Protein pp28 and Regulates Efficient Virus Release.