Molecular Features Contributing to Virus-Independent Intracellular Localization and Dynamic Behavior of the Herpesvirus Transport Protein US9

Abstract

Reaching the right destination is of vital importance for molecules, proteins, organelles, and cargoes. Thus, intracellular traffic is continuously controlled and regulated by several proteins taking part in the process. Viruses exploit this machinery, and viral proteins regulating intracellular transport have been identified as they represent valuable tools to understand and possibly direct molecules targeting and delivery. Deciphering the molecular features of viral proteins contributing to (or determining) this dynamic phenotype can eventually lead to a virus-independent approach to control cellular transport and delivery. From this virus-independent perspective we looked at US9, a virion component of Herpes Simplex Virus involved in anterograde transport of the virus inside neurons of the infected host. As the natural cargo of US9-related vesicles is the virus (or its parts), defining its autonomous, virus-independent role in vesicles transport represents a prerequisite to make US9 a valuable molecular tool to study and possibly direct cellular transport. To assess the extent of this autonomous role in vesicles transport, we analyzed US9 behavior in the absence of viral infection. Based on our studies, Us9 behavior appears similar in different cell types; however, as expected, the data we obtained in neurons best represent the virus-independent properties of US9. In these primary cells, transfected US9 mostly recapitulates the behavior of US9 expressed from the viral genome. Additionally, ablation of two major phosphorylation sites (i.e. Y32Y33 and S34ES36) have no effect on protein incorporation on vesicles and on its localization on both proximal and distal regions of the cells. These results support the idea that, while US9 post-translational modification may be important to regulate cargo loading and, consequently, virion export and delivery, no additional viral functions are required for US9 role in intracellular transport.

The Basic Domain of Herpes Simplex Virus 1 pUS9 Recruits Kinesin-1 To Facilitate Egress from Neurons

“…Our identified interaction of kinesin-1 and the pUS9 basic domain most likely plays a functional role in the context of the motor-adaptor scenario to directly mediate axonal anterograde transport. In support of this, a recent study has shown that HSV-1 pUS9 still undergoes microtubule-dependent axonal transport in neurons in a process independent of other viral proteins (41). This axonal transport also did not depend on the acidic domain of pUS9 (41).…”

“…In support of this, a recent study has shown that HSV-1 pUS9 still undergoes microtubule-dependent axonal transport in neurons in a process independent of other viral proteins (41). This axonal transport also did not depend on the acidic domain of pUS9 (41). It is also of note that the pUS9 basic domain contains a consensus recognition sequence for HSV-1 pUS3 kinase (42).…”

The lipid raft-dwelling protein US9 can be manipulated to target APP compartmentalization, APP processing, and neurodegenerative disease pathogenesis

“…US9 is a small type 2 membrane ‘carrier’ protein essential for anterograde spread of viral particles in neurons 28 – 31 . As we recently demonstrated 32 , the cellular distribution and functional properties of US9 do not require additional viral factors, are solely dependent on US9 sequence, and are not affected by attachment of a reporter/cargo. Furthermore, these US9 behaviors are maintained in multiple cell types, both primary and cultured lines, from different species 32 .…”

“…As we recently demonstrated 32 , the cellular distribution and functional properties of US9 do not require additional viral factors, are solely dependent on US9 sequence, and are not affected by attachment of a reporter/cargo. Furthermore, these US9 behaviors are maintained in multiple cell types, both primary and cultured lines, from different species 32 . In the context of lipid rafts, there are additional features unique to US9 that are important to our long-term goal.…”

“…Previous studies have shown that modification of the US9 N-terminus by the attachment of green fluorescent protein (gfp) does not alter its function in the context of viral infection 42 . The gfp-US9 fusion protein also retains its properties in the absence of other viral proteins and its natural cargo, the virion 32 . Furthermore, US9 from pseudorabies viruses (PRV) was found to associate with lipid rafts 43 .…”