Searching for genes of Aujeszky's disease virus required for neurotropism and virulence

Aujeszky's Disease Virus (ADV) is an alphaherpesvirus with the remarkable ability to cause lethal encephalitis in a wide variety of animal species including the swine, its natural host. The molecular mechanisms that enable the virus to invade and spread in the nervous systems of so many different animals are not known, but it is well known that the virion envelope proteins gE and gI are required in almost every case studied for efficient cell-to-cell spread both in non-neuronal and neuronal cells. To determine other viral genes involved in pathogenesis, we have been testing selected ADV mutants for defects in neuroinvasion in a rodent model. Preliminary results indicate that the absence of the Us9 gene, not previously known to affect pathogenesis, has striking effects on virus spread. The Us9 gene is highly conserved among the alphaherpesviruses sequenced to date, yet its function remains unknown. Us9 is a novel type II membrane protein expressed as a highly phosphorylated protein late in ADV infection. Unlike the HSV-L Us9 homologue which was reported to be associated with nucleocapsids in the nuclei of infected cells, ADV Us9 localises to the secretory system (predominantly to the Golgi apparatus) and not to the nucleus. By fusing the jellyfish enhanced green fluorescent protein reporter molecule (EGFP) to the carboxy-terminus of Us9, we demonstrated that Us9 not only is capable of targeting a Us9-EGFP fusion protein to the Golgi compartment, it also is able to direct efficient incorporation of such chimeric molecules into infectious viral particles. The predominant localisation of Us9 to the Golgi apparatus may have important ramifications for models of herpesvirus envelopment. The steady-state residence of the Us9 protein is in a cellular compartment in or near the trans-Golgi network (TGN). Through internalization assays with an EGFP epitope-tagged Us9 protein, we demonstrate that the maintenance of Us9 to the TGN region is a dynamic process involving retrieval of molecules from the cell surface. Deletion analysis of the cytoplasmic tail reveals that an acidic cluster containing putative phosphorylation sites is necessary for the recycling of Us9 from the plasma membrane. The absence of this cluster results in the relocalization of Us9 to the plasma membrane due to a defect in endocytosis. The acidic motif, however, does not contain signals needed to direct the incorporation of Us9 into viral envelopes. We also have investigated the role of a dileucine endocytosis signal in the Us9 cytoplasmic tail in the recycling and retention of Us9 to the TGN region. Site-directed mutagenesis of the dileucine motif results in an increase in Us9 plasma membrane staining and a partial internalisation defect. While mutant viruses lacking the Us9 gene have no obvious growth or plaque size defects in tissue culture, these mutants are defective for anterograde directional spread in a subset of retinal ganglion neurons that make up visual circuitry after infection of the rat eye. Viral mutants lacking the highly conserved Us9 acidic motif required for endocytosis and trans-Golgi network targeting are defective for directional spread in the rat visual system. However, mutants lacking the Us9 dileucine motif required for efficient endocytosis from the plasma membrane have wild type spread and virulence in the rat eye infection model. We have constructed revertants, independent null mutations, and completed complementation analyses with gE, gI and Us9 mutants in this animal model. We conclude that at least three ADV genes (gE, gI and Us9) are all necessary, but each is not sufficient to sponsor anterograde directed spread of the virus in specific rodent neurons

Actin Is a Component of the Compensation Mechanism in Pseudorabies Virus Virions Lacking the Major Tegument Protein VP22

Despite being a major component of the pseudorabies virus tegument, VP22 is not required for PRV replication, virulence, or neuroinvasion (T. del Rio, H. C. Werner, and L. W. Enquist, J. Virol. 76:774-782, 2002). In the absence of VP22, tegument assembly compensates in a limited fashion with increased incorporation of cellular actin. Infection of epithelial cell lines expressing fluorescent actin fusion proteins resulted in the incorporation of filamentous and nonfilamentous actin into individual virions that were predominately light, noninfectious particles. We conclude that cellular actin is incorporated in the tegument of wild-type virions and is part of a compensation mechanism for VP22-null virions

What is the price of science?

The peer-reviewed scientific literature is the bedrock of science. However, scientific publishing is undergoing dramatic changes, which include the expansion of open access, an increased number of for-profit publication houses, and ready availabil-ity of preprint manuscripts that have not been peer reviewed. In this opinion article, we discuss the inequities and concerns that these changes have wrought. © 2021 Alwine et al.Open access journalThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]