PROJECT SUMMARY/ABSTRACT
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 SUN2-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.