Todd W. Wisner

Alphaherpesviruses spread rapidly through dermal tissues and within synaptically connected neuronal circuitry. Spread of virus particles in epithelial tissues involves movement across cell junctions. Herpes simplex virus (HSV), varicella-zoster virus (VZV), and pseudorabies virus (PRV) all utilize a complex of two glycoproteins, gE and gI, to move from cell to cell. HSV gE/gI appears to function primarily, if not exclusively, in polarized cells such as epithelial cells and neurons and not in nonpolarized cells or cells that form less extensive cell junctions. Here, we show that HSV particles are specifically sorted to cell junctions and few virions reach the apical surfaces of polarized epithelial cells. gE/gI participates in this sorting. Mutant HSV virions lacking gE or just the cytoplasmic domain of gE were rarely found at cell junctions; instead, they were found on apical surfaces and in cell culture fluids and accumulated in the cytoplasm. A component of the AP-1 clathrin adapter complexes, mu1B, that is involved in sorting of proteins to basolateral surfaces was involved in targeting of PRV particles to lateral surfaces. These results are related to recent observations that (i) HSV gE/gI localizes specifically to the trans-Golgi network (TGN) during early phases of infection but moves out to cell junctions at intermediate to late times (T. McMillan and D. C. Johnson, J. Virol., in press) and (ii) PRV gE/gI participates in envelopment of nucleocapsids into cytoplasmic membrane vesicles (A. R. Brack, B. G. Klupp, H. Granzow, R. Tirabassi, L. W. Enquist, and T. C. Mettenleiter, J. Virol. 74:4004-4016, 2000). Therefore, interactions between the cytoplasmic domains of gE/gI and the AP-1 cellular sorting machinery cause glycoprotein accumulation and envelopment into specific TGN compartments that are sorted to lateral cell surfaces. Delivery of virus particles to cell junctions would be expected to enhance virus spread and enable viruses to avoid host immune defenses.

Herpesviruses must traverse the nuclear envelope to gain access to the cytoplasm and, ultimately, to exit cells. It is believed that herpesvirus nucleocapsids enter the perinuclear space by budding through the inner nuclear membrane (NM). To reach the cytoplasm these enveloped particles must fuse with the outer NM and the unenveloped capsids then acquire a second envelope in the trans-Golgi network. Little is known about the process by which herpesviruses virions fuse with the outer NM. Here we show that a herpes simplex virus (HSV) mutant lacking both the two putative fusion glycoproteins gB and gH failed to cross the nuclear envelope. Enveloped virions accumulated in the perinuclear space or in membrane vesicles that bulged into the nucleoplasm (herniations). By contrast, mutants lacking just gB or gH showed only minor or no defects in nuclear egress. We concluded that either HSV gB or gH can promote fusion between the virion envelope and the outer NM. It is noteworthy that fusion associated with HSV entry requires the cooperative action of both gB and gH, suggesting that the two types of fusion (egress versus entry) are dissimilar processes.

Human cytomegalovirus (HCMV) establishes persistent lifelong infections and replicates slowly. To withstand robust immunity, HCMV utilizes numerous immune evasion strategies. The HCMV gene cassette encoding US2 to US11 encodes four homologous glycoproteins, US2, US3, US6, and US11, that inhibit the major histocompatibility complex class I (MHC-I) antigen presentation pathway, probably inhibiting recognition by CD8(+) T lymphocytes. US2 also inhibits the MHC-II antigen presentation pathway, causing degradation of human leukocyte antigen (HLA)-DR-alpha and -DM-alpha and preventing recognition by CD4(+) T cells. We investigated the effects of seven of the US2 to US11 glycoproteins on the MHC-II pathway. Each of the glycoproteins was expressed by using replication-defective adenovirus vectors. In addition to US2, US3 inhibited recognition of antigen by CD4(+) T cells by a novel mechanism. US3 bound to class II alpha/beta complexes in the endoplasmic reticulum (ER), reducing their association with Ii. Class II molecules moved normally from the ER to the Golgi apparatus in US3-expressing cells but were not sorted efficiently to the class II loading compartment. As a consequence, formation of peptide-loaded class II complexes was reduced. We concluded that US3 and US2 can collaborate to inhibit class II-mediated presentation of endogenous HCMV antigens to CD4(+) T cells, allowing virus-infected cells to resist recognition by CD4(+) T cells.

The final assembly of herpes simplex virus (HSV) involves binding of tegument-coated capsids to viral glycoprotein-enriched regions of the trans-Golgi network (TGN) as enveloped virions bud into TGN membranes. We previously demonstrated that HSV glycoproteins gE/gI and gD, acting in a redundant fashion, are essential for this secondary envelopment. To define regions of the cytoplasmic (CT) domain of gE required for secondary envelopment, HSVs lacking gD and expressing truncated gE molecules were constructed. A central region (amino acids 470 to 495) of the gE CT domain was important for secondary envelopment, although more C-terminal residues also contributed. Tandem affinity purification (TAP) proteins including fragments of the gE CT domain were used to identify tegument proteins VP22 and UL11 as binding partners, and gE CT residues 470 to 495 were important in this binding. VP22 and UL11 were precipitated from HSV-infected cells in conjunction with full-length gE and gE molecules with more-C-terminal residues of the CT domain. gD also bound VP22 and UL11. Expression of VP22 and gD or gE/gI in cells by use of adenovirus (Ad) vectors provided evidence that other viral proteins were not necessary for tegument/glycoprotein interactions. Substantial quantities of VP22 and UL11 bound nonspecifically onto or were precipitated with gE and gD molecules lacking all CT sequences, something that is very unlikely in vivo. VP16 was precipitated equally whether gE/gI or gD was present in extracts or not. These observations illustrated important properties of tegument proteins. VP22, UL11, and VP16 are highly prone to binding nonspecifically to other proteins, and this did not represent insolubility during our assays. Rather, it likely reflects an inherent "stickiness" related to the formation of tegument. Nevertheless, assays involving TAP proteins and viral proteins expressed by HSV and Ad vectors supported the conclusion that VP22 and UL11 interact specifically with the CT domains of gD and gE.

Herpesvirus capsids collect along the inner surface of the nuclear envelope and bud into the perinuclear space. Enveloped virions then fuse with the outer nuclear membrane (NM). We previously showed that herpes simplex virus (HSV) glycoproteins gB and gH act in a redundant fashion to promote fusion between the virion envelope and the outer NM. HSV mutants lacking both gB and gH accumulate enveloped virions in herniations, vesicles that bulge into the nucleoplasm. Earlier studies had shown that HSV mutants lacking the viral serine/threonine kinase US3 also accumulate herniations. Here, we demonstrate that HSV gB is phosphorylated in a US3-dependent manner in HSV-infected cells, especially in a crude nuclear fraction. Moreover, US3 directly phosphorylated the gB cytoplasmic (CT) domain in in vitro assays. Deletion of gB in the context of a US3-null virus did not add substantially to defects in nuclear egress. The majority of the US3-dependent phosphorylation of gB involved the CT domain and amino acid T887, a residue present in a motif similar to that recognized by US3 in other proteins. HSV recombinants lacking gH and expressing either gB substitution mutation T887A or a gB truncated at residue 886 displayed substantial defects in nuclear egress. We concluded that phosphorylation of the gB CT domain is important for gB-mediated fusion with the outer NM. This suggested a model in which the US3 kinase is incorporated into the tegument layer (between the capsid and envelope) in HSV virions present in the perinuclear space. By this packaging, US3 might be brought close to the gB CT tail, leading to phosphorylation and triggering fusion between the virion envelope and the outer NM.