The glycoprotein E (gE) of varicella zoster virus (VZV), encoded by ORF68, is the most abundant viral glycoprotein. In the current experiments, we demonstrated that ORF68 deletion was incompatible with recovery of infectious virus from VZV cosmids. Replacing ORF68 at a nonnative AvrII site in the genome restored infectivity. Further, we found that VZV gE could be expressed under the control of the Tet-On promoter in stably transfected melanoma cell lines (Met-gE cells) without evidence of toxicity. In these Met-gE cells, gE colocalized with gamma-adaptin, a trans Golgi network marker, in perinuclear sites, but did not reach plasma membranes. In order to investigate how infection altered gE localization, we made a recombinant virus, vOka-MSPgE, with ORF68 from the VZV MSP strain. VZV MSP encodes a mutant gE protein (D150N) that lacks the mAb epitope, 3B3 (Santos et al., Virology 275, 306-317, 2000), whereas Met-gE protein binds mAb 3B3. Within 48 h after Met-gE cells were infected with vOka-MSPgE, the steady-state distribution of Met-gE protein extended beyond the perinuclear areas to other cytoplasmic sites and to plasma membranes. A second recombinant, vOka-MSPgE without gI (vOka-MSPgEdeltagI), was constructed to investigate Met-gE protein distribution in the absence of gI. The redistribution of Met-gE protein which was observed by 48 h after vOka-MSPgE infection did not occur until 5 days (140 h) within vOka-MSPgEdeltagI infected cells. After vOka-MSPgE infection of Met-gE cells, most Met-gE protein was in the final 94K mature form by 72 h. However, progression to predominance of mature gE was delayed in Met-gE cells infected with vOka-MSPgEdeltagI. These observations confirm our hypothesis that VZV gE is essential, based upon the demonstration of restored infectivity after replacing ORF68 in a nonnative site in the genome, and provide further evidence of the role of gI in facilitating the maturation and intracellular distribution of this critical VZV glycoprotein.
Varicella-zoster virus (VZV) glycoprotein E (gE) is essential for VZV replication.To further analyze the functions of gE in VZV replication, a full deletion and point mutations were made in the 62-amino-acid (aa) C-terminal domain. Targeted mutations were introduced in YAGL (aa 582 to 585), which mediates gE endocytosis, AYRV (aa 568 to 571), which targets gE to the trans-Golgi network (TGN), and SSTT, an "acid cluster" comprising a phosphorylation motif (aa 588 to 601). Substitutions Y582G in YAGL, Y569A in AYRV, and S593A, S595A, T596A, and T598A in SSTT were introduced into the viral genome by using VZV cosmids. These experiments demonstrated a hierarchy in the contributions of these C-terminal motifs to VZV replication and virulence. Deletion of the gE C terminus and mutation of YAGL were lethal for VZV replication in vitro. Mutations of AYRV and SSTT were compatible with recovery of VZV, but the AYRV mutation resulted in rapid virus spread in vitro and the SSTT mutation resulted in higher virus titers than were observed for the parental rOka strain. When the rOka-gE-AYRV and rOka-gE-SSTT mutants were evaluated in skin and T-cell xenografts in SCIDhu mice, interference with TGN targeting was associated with substantial attenuation, especially in skin, whereas the SSTT mutation did not alter VZV infectivity in vivo. These results provide the first information about how targeted mutations of this essential VZV glycoprotein affect viral replication in vitro and VZV virulence in dermal and epidermal cells and T cells within intact tissue microenvironments in vivo.Varicella-zoster virus (VZV) is an alphaherpesvirus with a genome of ϳ125,000 bp, encoding at least 70 unique open reading frames (ORFs) (3,8,10). Primary VZV infection causes varicella, which is characterized by cell-associated viremia and the formation of vesicular skin lesions that contain high concentrations of cell-free virions (3,19,35). It is believed that VZV is transmitted by aerosolized virions and infected cell material shed from skin and respiratory epithelium, although this process has not been tested in animal models. VZV preferentially infects memory T cells that have skin homing markers, as a mechanism for its transfer from respiratory epithelial sites of inoculation to dermal and epidermal cells (19). VZV establishes latency in sensory ganglia and causes herpes zoster upon reactivation. Thus, VZV pathogenesis requires infection of circulating lymphocytes, skin, and neural cells. The highly cell-associated nature of VZV replication in vitro and its very restricted infectivity in nonhuman species have been obstacles to understanding how viral gene products contribute to VZV replication and to the infection of specific target cells that are important for the viral life cycle in the host. Two advances have provided new opportunities to analyze the molecular mechanisms of VZV infectivity in vitro and in vivo. First, the use of VZV cosmids permits the identification of VZV genes, or regions within the coding sequence for particular viral prot...
These data suggest that pUL114 associates with ppUL44 and that it functions as part of the viral DNA replication complex to increase the efficiency of both early and late phase viral DNA synthesis.
Varicella-zoster virus (VZV) infection involves the cell-cell spread of virions, but how viral proteins interactwith the host cell membranes that comprise intercellular junctions is not known. Madin-Darby canine kidney (MDCK) cells were constructed to express the glycoproteins gE, gI, or gE/gI constitutively and were used to examine the effects of these VZV glycoproteins in polarized epithelial cells. At low cell density, VZV gE induced partial tight junction (TJ) formation under low-calcium conditions, whether expressed alone or with gI. Although most VZV gE was intracellular, gE was also shown to colocalize with the TJ protein ZO-1 with or without concomitant expression of gI. Freeze fracture electron microscopy revealed normal TJ strand morphology in gE-expressing MDCK cells. Functionally, the expression of gE was associated with a marked acceleration in the establishment of maximum transepithelial electrical resistance (TER) in MDCK-gE cells; MDCK-gI and MDCK-gE/gI cells exhibited a similar pattern of early TER compared to MDCK cells, although peak resistances were lower than those of gE alone. VZV gE expression altered F-actin organization and lipid distribution, but coexpression of gI modulated these effects. Two regions of the gE ectodomain, amino acids (aa) 278 to 355 and aa 467 to 498, although lacking Ca 2؉ binding motifs, exhibit similarities with corresponding regions of the cell adhesion molecules, E-cadherin and desmocollin. These observations suggest that VZV gE and gE/gI may contribute to viral pathogenesis by facilitating epithelial cell-cell contacts.Varicella-zoster virus (VZV) is a human alphaherpesvirus that causes two diseases, varicella (chicken pox) and herpes zoster (shingles); the latter disease is a recurrent infection following prolonged latency in sensory ganglia. Despite genetic similarities, VZV exhibits an extreme host range restriction in vivo and grows poorly in tissue culture, suggesting that its pathogenic mechanisms differ from those of related herpesviruses. The herpesvirus glycoproteins function at several points in the replication cycle, including viral attachment, entry, envelopment, cell-cell spread, and egress. The VZV glycoproteins gB, gC, gE, gH, gI, gL, gK, and the putative gM have some homology with those of herpes simplex virus type 1 (HSV-1), HSV-2, pseudorabies virus (PRV), and the other nonhuman alphaherpesviruses, but VZV lacks gD and gG (12,24). Although the spread of VZV is associated with extensive fusion of cell membranes, information about the processes by which VZV and other herpesviruses move to cell junctions, including tight junctions (TJ) as well as adherens junctions, and invade adjacent, uninfected cells is limited. These processes are important for VZV replication, since syncytium formation is a hallmark of VZV infection in vitro and multinucleated polykaryocytes are common in VZV-infected tissues.The VZV glycoproteins gE and gI form heterodimers and, like the corresponding proteins of HSV and PRV, are likely to be involved in cell-cell spread (3,4...
Herpes simplex virus type 1 glycoprotein K (gK) plays an essential role in viral replication and cell fusion. gK is a very hydrophobic membrane protein that contains a signal sequence and several hydrophobic regions. It has been shown that mutations inducing cell fusion map to two distinct domains of gK, suggesting that these domains are functionally important. To understand the transmembrane topology of gK and the localization of these functional domains, we constructed a set of gK deletion, insertion, and truncation mutants and expressed these by in vitro translation in the presence of microsomal membranes. The transmembrane topology of gK was determined by examination of the post-translational processing and protease sensitivity of the mutant proteins. Our data demonstrate that gK contains three transmembrane domains (amino acids 125-139, 226-239, and 311-325). Another hydrophobic domain (amino acids 241-265), which is relatively less hydrophobic and much longer compared with the transmembrane sequences, is located in the extracellular loop. The analysis showed that the domains containing syncytial mutations are both ectodomains. They may interact with each other to form a complex tertiary structure that is critical for the biological function of gK.
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