Nuclear Rotation and Cellular Reorganization During Cytomegalovirus Infection

Human Cytomegalovirus (HCMV) is a widespread herpesvirus that is the leading infectious cause of congenital birth defects and causes coronary and other health problems in adults, as well as serious complications in immunosuppressed transplant recipients or AIDS patients. Despite this, there is no vaccine or cure, and we continue to have a relatively limited understanding of several unique aspects of HCMV replication.  

Unlike most other viruses, HCMV has a protracted replication cycle that spans several days. During this time HCMV forms a unique cytoplasmic structure termed the Assembly Compartment (AC). While fixed imaging approaches provided insights into its organization, revealing that it comprises a remodeled Golgi surrounded by various host vesicles, in the prior award period we developed innovative multi-color live cell imaging approaches that revealed the dynamic behavior of the AC. In doing so, we revealed that the AC not only acts as a virion maturation site but also serves as a Golgi-derived microtubule organizing center (MTOC) that enables HCMV to generate acetylated microtubules (MTs). Our imaging approaches further revealed a temporal series of events whereby HCMV rotates the nucleus for several days, after which time infected cells become motile. Acetylation provides the mechanical strength for AC-derived MT filaments to rotate the nucleus, with connections to the nuclear membrane being formed by the dynein adaptor, Bicaudal D2 (BICD2) and SUN1, a component of the nuclear membrane-spanning Linker of Nucleoskeleton and Cytoskeleton (LINC) complex. Rotation of the nucleus is symptomatic of the extreme pulling forces that MTs and dynein exert on SUN1-LINC complexes, polarizing them towards the AC. This in turn polarizes the actin-regulatory protein, Emerin inside the nucleus and drives the formation of transient nuclear actin filaments.  

Using both RNAi-mediated targeting and dominant-negatives to each individual component in this newly identified chain connecting the AC all the way to nuclear actin, combined with the development of artificial intelligence-based neural networks for large scale, automated image analysis, we revealed that MT-based connections to the nuclear surface remodel the intranuclear environment in order to segregate viral genomic DNA from host heterochromatin. In additional preliminary data, approaches such as transient transcriptomic sequencing (TT- Seq) reveal that MT-based nuclear rotation serves to control specific host gene expression programs associated with actin regulation, cell attachment and motility. Furthermore, blocking MT-based nuclear rotation prevents HCMV from downregulating “actin caps”, which form through distinct SUN2-based LINC complexes and serve to anchor the nucleus in place. Finally, additional data reveals the remarkably deformable nature of the HCMV nucleus and supports a central hypothesis, tested here, that HCMV establishes a cytoskeletal mechano-transduction pathway that releases actin caps that anchor the nucleus place, allowing MTs to then remodel nuclear architecture and gene expression programs to promote cell motility and virus spread. 

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