Role of Pseudorabies Virus Us9, a Type II Membrane Protein, in Infection of Tissue Culture Cells and the Rat Nervous System

Abstract

The protein product of the pseudorabies virus (PRV) Us9 gene is a phosphorylated, type II membrane protein that is inserted into virion envelopes and accumulates in the trans-Golgi network. It is among a linked group of three envelope protein genes in the unique short region of the PRV genome which are absent from the attenuated Bartha strain. We found that two different Us9 null mutants exhibited no obvious phenotype after infection of PK15 cells in culture. Unlike those of gE and gI null mutants, the plaque size of Us9 null mutants on Madin-Darby bovine kidney cells was indistinguishable from that of wild-type virus. However, both of the Us9 null mutants exhibited a defect in anterograde spread in the visual and cortical circuitry of the rat. The visual system defect was characterized by restricted infection of a functionally distinct subset of visual projections involved in the temporal organization of behavior, whereas decreased anterograde spread of virus to the cortical projection targets was characteristic of animals receiving direct injections of virus into the cortex. Spread of virus through retrograde pathways in the brain was not compromised by a Us9 deletion. The virulence of the Us9 null mutants, as measured by time to death and appearance of symptoms of infection, also was reduced after their injection into the eye, but not after cortical injection. Through sequence analysis, construction of revertants, measurement of gE and gI protein synthesis in the Us9 null mutants, and mixedinfection studies of rats, we conclude that the restricted-spread phenotype after infection of the rat nervous system reflects the loss of Us9 and is not an indirect effect of the Us9 mutations on expression of glycoproteins gE and gI. Therefore, at least three viral envelope proteins, Us9, gE, and gI, function together to promote efficient anterograde transneuronal infection by PRV in the rat central nervous system.Pseudorabies virus (PRV) is a neurotropic alphaherpesvirus of the Herpesviridae family that is able to infect neurons of both the peripheral and central nervous systems of a wide variety of mammals and birds (4, 39). When PRV replicates and spreads in the nervous system, it acts as a self-amplifying tracer of synaptically connected neurons (10, 16). Integral to this process is the demonstrated ability of the virus to exploit the polarized cellular architecture of neurons and move throughout the brain of a host organism. Thus, assays of the spread of virus through the brain naturally incorporate a determination of the direction of viral transport through a circuit. Direction in the nervous system is defined by the terms "anterograde" and "retrograde," which can be used to describe directional movement of virus inside a cell as well as the spread of virus between synaptically connected neurons. We use the terms anterograde spread and retrograde spread in this report to describe movement of virus between synaptically connected neurons. Spread from the primary infected neuron to the second-order uninfected neuron in...

Viral regulation of the long distance axonal transport of herpes simplex virus nucleocapsid

“…As found by others, Us9 is not necessary for the polarized retrograde transport of virus (Polcicova et al, 2005;Brideau et al, 2000). We found that the anterograde transport of viral DNA is reduced about 93% when Us9 is deleted.…”

“…Our HSV results are primarily limited to assays of mature neurons and focus on the characteristics of transport within the axon compartment, rather than the whole cell. Clearly, understanding the precise molecular regulation of long distance axonal transport of HSV nucleocapsid is likely to be complicated, and will be facilitated by comparisons with other herpesvirus strains and model systems (Chowdhury et al, 2006;Brideau et al, 2000;Cohen et al, 2001).…”

“…It is important to note that the Us9 protein in pseudorabies virus (PRV) has also been identified as essential for normal anterograde transneuronal transport (Brideau et al, 2000) and has been characterized as a unique type II tail-anchored membrane protein (Brideau et al, 1998). Based on its primary sequence and similarity to the homologous protein Us9 of PRV (Brideau et al, 1998), one would predict that the HSV-1 Us9 is also a type II, integral membrane protein, and it might be expected to localized in the viral membrane.…”

Two Modes of Pseudorabies Virus Neuroinvasion and Lethality in Mice

“…U S 9, gI, and gE each contribute to virulence, but the relationship of their role in neuroinvasion to virulence remains to be explained (4,15).…”

“…Spread of infection of wild-type virus in the anterograde direction (from presynaptic to postsynaptic neurons) requires the PRV U S 9, gE, and gI proteins (4,12). These genes are deleted in the PRV-Bartha strain (3,14,16,18,19,22).…”