The Carboxyl Terminus of Tegument Protein pUL21 Contributes to Pseudorabies Virus Neuroinvasion

Abstract

Following its entry into cells, pseudorabies virus (PRV) utilizes microtubules to deliver its nucleocapsid to the nucleus. Previous studies have shown that PRV VP1/2 is an effector of dynein-mediated capsid transport. However, the mechanism of PRV for recruiting microtubule motor proteins for successful neuroinvasion and neurovirulence is not well understood. Here, we provide evidence that PRV pUL21 is an inner tegument protein. We tested its interaction with the cytoplasmic light chains using a bimolecular fluorescence complementation (BiFC) assay and observed that PRV pUL21 interacts with Roadblock-1. This interaction was confirmed by coimmunoprecipitation (co-IP) assays. We also determined the efficiency of retrograde and anterograde axonal transport of PRV strains in explanted neurons using a microfluidic chamber system and investigated pUL21's contribution to PRV neuroinvasion in vivo. Further data showed that the carboxyl terminus of pUL21 is essential for its interaction with Roadblock-1, and this domain contributes to PRV retrograde axonal transport in vitro and in vivo. Our findings suggest that the carboxyl terminus of pUL21 contributes to PRV neuroinvasion. IMPORTANCE Herpesviruses are a group of DNA viruses that infect both humans and animals. Alphaherpesviruses are distinguished by their ability to establish latent infection in peripheral neurons. After entering neurons, the herpesvirus capsid interacts with cellular motor proteins and undergoes retrograde transport on axon microtubules. This elaborate process is vital to the herpesvirus lifecycle, but the underlying mechanism remains poorly understood. Here, we determined that pUL21 is an inner tegument protein of pseudorabies virus (PRV) and that it interacts with the cytoplasmic dynein light chain Roadblock-1. We also observed that pUL21 promotes retrograde transport of PRV in neuronal cells. Furthermore, our findings confirm that pUL21 contributes to PRV neuroinvasion in vivo. Importantly, the carboxyl terminus of pUL21 is responsible for interaction with Roadblock-1, and this domain contributes to PRV neuroinvasion. This study offers fresh insights into alphaherpesvirus neuroinvasion and the interaction between virus and host during PRV infection.

An improved animal model for herpesvirus encephalitis in humans

“…pUL21 has been implicated in different steps of the viral replication cycle in non-neuronal and neuronal cells [39,[62][63][64][65][66][67]. In particular, mutations in or complete absence of pUL21 affect retrograde transport processes in neurons [68,69], and point mutations in the UL21 gene of live-attenuated vaccine strain PrV-Bartha contribute to its avirulence in pigs [42]. Repair of the UL21 locus in PrV-Bartha has been shown to enhance retrograde intraaxonal and transneuronal spread [69].…”

“…Repair of the UL21 locus in PrV-Bartha has been shown to enhance retrograde intraaxonal and transneuronal spread [69]. The delay in retrograde transport of UL21-null PrV has been linked to an interaction of the carboxyl terminus of pUL21 with Roadblock-1, a dynein light chain belonging to the dynein motor complex important for retrograde transport along microtubules [68]. Mice infected intraocularly or intranasally with a UL21-deleted PrV showed prolonged survival times compared to mice infected with wildtype virus [68] confirming previous results that pUL21 has an impact on neuroinvasion and spread [28].…”

“…However, all mice developed severe clinical signs and had to be euthanized, although the disease developed slower. In the light of decelerated axonal spread [68,69] and subsequent delayed infection of higher areas an immune response is beginning to develop in PrV-ΔUL21-infected mice, but not effective enough to eliminate the virus. In summary, these data confirm that pUL21 contributes to neuroinvasion, but elucidation of the molecular function requires more in-depth investigation.…”

pUL21 is a viral phosphatase adaptor that promotes herpes simplex virus replication and spread

“…Pseudorabies virus (PrV) pUL21 has been extensively studied as mutations in the gene encoding this protein contribute to the attenuation of the vaccine strain Bartha (Klupp et al, 1995). PrV pUL21 is required for neurovirulence and a recent study identified that pUL21 binds the dynein light chain protein Roadblock-1, promoting retrograde axonal transport (Yan et al, 2019).…”

Establishment of a Fosmid Library for Pseudorabies Virus SC Strain and Application in Viral Neuronal Tracing

“…UL21 is another protein associated with neuroinvasion. It can promote PRV retrograde transport through interaction with cytoplasmic dynein light chain Roadblock-1 (Yan et al, 2019); however, UL21 mutation causes a delay in the spread to presynaptic neurons and reduces infectious particle production (Curanovic et al, 2009).…”