Dissemination of herpes simplex virus (HSV) during recurrent disease in the host is dependent upon efficient viral replication and on the ability of the virus to spread from cell to cell in the face of the host innate and adaptive immune defenses. Cytoplasmic envelopment of HSV-1 virions is followed by vesicular transport of virions to the cell surface and secretion by fusion of the vesicle membrane with the plasma membrane (8
). Transport of virions to cell membranes in contact with the extracellular medium results in release of free virions. Transport to surfaces apposed to other cells results in cell-to-cell spread of virus infection. The mechanism by which virions are sorted to junctional or basolateral surfaces in epithelial and fibroblast cells is poorly characterized. About half of the virus-encoded proteins play critical roles in virus replication, but relatively few have been found to have specific functions in cell-to-cell spread of virus. The essential components of the virion entry apparatus, gB, gD, and gH/gL, are required for cell-to-cell spread (7
). It is likely that this is because cell-to-cell spread requires interaction of the virus entry proteins with cellular receptors and subsequent fusion of the virion envelope with the plasma membrane of the naïve host cell. A few additional viral proteins have been shown to be required for efficient cell-to-cell spread at least in some cell types. HSV-1 gE and gI form a heterodimeric complex that is required for efficient cell-to-cell spread in the nervous system in vivo
). The gE/gI complex is also required for spread in cultured neuronal cells and in epithelial and fibroblast cells that form well-defined cell junctions (1
). The gE spread phenotype in epithelial cells requires sequences in the cytoplasmic tail of gE and also requires sorting of gE to basolateral cell surfaces and adherens junctions, where it colocalizes with β-catenin (12
). Deletion of US9 in pseudorabies virus (PRV) is associated with failure of viral spread in neuronal cultures and in vivo
). In HSV-1, the effect of US9 deletion on neuronal spread is less clear, and the degree of inhibition of neuronal spread may depend on the experimental system (36
). The function of US9 appears to be tied in neurons to sorting of virus components from the neuronal cell body into axons (5
The HSV-1 UL34 gene, along with its homologs in other herpesviruses, is required for efficient viral replication in all cultured cells tested, presumably because it is required for efficient egress of capsids from the infected cell nucleus (15
). The UL34 protein (pUL34) is targeted specifically to the inner nuclear membrane (INM) by a mechanism that requires its interaction with HSV pUL31 (48
), and this dependence is a conserved feature of herpesvirus envelopment (18
). In addition to their localization at the nuclear envelope in infected cells, pUL31 and pUL34 of HSV and PRV have been shown to be structural components of the perinuclear virion (18
). The proteins are lost from the egressing virion upon deenvelopment at the outer nuclear membrane (ONM), and pUL34 and pUL31 and their homologs are not detected in mature virions (15
). Localization of these two proteins at the INM results in the recruitment of other proteins, including protein kinase C delta and pUS3, to the nuclear membrane to form a nuclear envelopment complex (NEC) (41
). Deletion of the HSV UL34 gene causes failure to disrupt the nuclear lamina and essentially complete failure of nuclear egress, with accumulation of nucleocapsids in the infected cell nucleus (3
). The concentration of pUL34 and pUL31 at the nuclear membrane during infection suggests that the nuclear envelope (NE) is likely to be their most important functional site.
Complete deletion of any gene whose product is required at multiple steps in infection will result in arrest of infection at the first of those steps, making identification and analysis of later events impossible. Point mutations in that gene will sometimes result in proteins with full or partial function at early steps and failure of function at later steps, thereby allowing characterization of those later steps. This strategy has been useful in analysis of UL34 gene function, since careful analysis of point mutations has allowed identification of UL34 gene functions in nuclear egress that follow nuclear lamina disruption, including mediation and regulation of membrane curvature around capsids (50
). Analysis of point mutations has the additional advantage that extragenic suppressors of the mutant phenotypes can be selected and mapped, allowing identification of functionally important interactions. This genetic approach has had limited use in analysis of herpesvirus morphogenesis, principally because of the difficulty of mapping the extragenic suppressor mutations by marker transfer. Application of the method so far has yielded useful results only when the position of the suppressor could be predicted based on already known or suspected interactions (9
). Two recent technical advances allow for more extensive and powerful use of extragenic suppressor analysis. The first of these is the use of high-throughput sequencing methods for rapid and convenient sequencing of whole herpesvirus genomes for identification of single nucleotide polymorphisms (SNPs) between the parental and suppressor mutant viruses (61
). The second is the use of molecular clones of herpesvirus genomes in the form of bacterial artificial chromosomes (BACs) as the parent genomes for marker transfer identification of the relevant SNP.
Here, we show that a mutation in UL34 that results in substitution of alanine for a highly conserved tyrosine at pUL34 position 68 results in a surprising phenotype. This mutation is associated with a major virus replication defect and inhibition of nuclear egress but also results in a profound defect in virus cell-to-cell spread and in trafficking of gE. We isolated extragenic suppressors of the cell-to-cell spread phenotype and showed by whole-genome sequencing and PCR-based screening that phenotypic suppression is correlated with a nonsense mutation in the US9 gene. This mutation alone is not sufficient to suppress the UL34 Y68A phenotype.