Functions of the C-Terminal Domain of Varicella-Zoster Virus Glycoprotein E in Viral Replication In Vitro and Skin and T-Cell Tropism In Vivo

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The protein product of varicella-zoster virus (VZV) ORF47 is a serine/threonine protein kinase and tegument component. Evaluation of two recombinants of the Oka strain, rOka47⌬C, with a C-terminal truncation of ORF47, and rOka47D-N, with a point mutation in the conserved kinase motif, showed that ORF47 kinase function was necessary for optimal VZV replication in human skin xenografts in SCID mice but not in cultured cells. We now demonstrate that rOka47⌬C and rOka47D-N mutants do not infect human T-cell xenografts. Differences in the growth of kinase-defective ORF47 mutants allowed an examination of requirements for VZV pathogenesis in skin and T cells in vivo. Although virion assembly was reduced and no virion transport to cell surfaces was observed, epidermal cell fusion persisted, and VZV polykaryocytes were generated by rOka47⌬C and rOka47D-N in skin. Virion assembly was also impaired in vitro, but VZV-induced cell fusion continued to cause syncytia in cultured cells infected with rOka47⌬C or rOka47D-N. Intracellular trafficking of envelope glycoprotein E and the ORF47 and IE62 proteins, components of the tegument, was aberrant without ORF47 kinase activity. In summary, normal VZV virion assembly appears to require ORF47 kinase function. Cell fusion was induced by ORF47 mutants in skin, and cell-cell spread occurred even though virion formation was deficient. VZV-infected T cells do not undergo cell fusion, and impaired virion assembly by ORF47 mutants was associated with a complete elimination of T-cell infectivity. These observations suggest a differential requirement for cell fusion and virion formation in the pathogenesis of VZV infection in skin and T cells.Varicella-zoster virus (VZV) is a ubiquitous human herpes virus and the causative agent of varicella (chicken pox) and zoster (shingles) (1,8). Varicella is characterized by viremia and skin lesions. VZV infects T cells, which may be a mechanism for its transport from respiratory epithelial sites of inoculation to dermal and epidermal cells (1,19,20,38). Thus, T cells as well as skin are critical targets for VZV pathogenesis. VZV establishes latency in sensory ganglia and causes zoster upon reactivation. Two major advances have provided new opportunities for understanding the molecular mechanisms of VZV pathogenesis. First, VZV cosmids permit the construction of VZV recombinant viruses with targeted genetic mutations (4,15,22). Second, the SCIDhu mouse model, in which skin and T-cell xenografts are infected in vivo, makes it possible to define the effects of genetic mutations in VZV on virulence for differentiated human cells within their unique tissue microenvironments (2, 3, 13, 24-28, 31, 34, 35, 37).VZV virion production begins with the assembly of capsids, which appear to undergo initial envelopment at the nuclear membrane and de-envelopment in the cytoplasm. Final envelopment in the trans-Golgi network (TGN) (7) is followed by virion transport to cell surfaces. Syncytium formation, a hallmark of VZV infection in cultured cells, reflects cell fu...

Section: Discussionmentioning

confidence: 99%

Differential Requirement for Cell Fusion and Virion Formation in the Pathogenesis of Varicella-Zoster Virus Infection in Skin and T Cells

Besser¹,

Ikoma

Fabel

et al. 2004

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“…In other herpesviruses, inhibition of endocytosis of glycoproteins has been reported to have different effects on replication of the specific members of this family of viruses. For example, complete inhibition of VZV replication has been observed for a virus encoding gE that cannot undergo endocytosis (37). Moderate reduction in virus replication for gB of HSV-1 and development of a small-plaque phenotype in cultured cells for gE of PRV have been associated with the respective envelope proteins with defects in endocytosis (3).…”

Section: Vol 84 2010 Hcmv Glycoprotein Endocytosis 7047mentioning

confidence: 99%

“…For herpesviral glycoproteins that have been studied in detail, the results differ significantly between individual herpesviruses and specific viral glycoproteins. For example, endocytosis of gE of varicellazoster virus (VZV) has been shown to be essential for viral replication, whereas it is irrelevant for replication of pseudorabies virus (PRV) (37,55). For gB of several herpesviruses, endocytosis seems to be dispensable for virion formation since its inhibition has little effect on replication kinetics (3,40).…”

mentioning

confidence: 99%

Optimal Replication of Human Cytomegalovirus Correlates with Endocytosis of Glycoprotein gpUL132

Kropff¹,

Koedel

Britt

et al. 2010

Envelopment of a herpesvirus particle is a complex process of which much is still to be learned. We previously identified the glycoprotein gpUL132 of human cytomegalovirus (HCMV) as an envelope component of the virion. In its carboxy-terminal portion, gpUL132 contains at least four motifs for sorting of transmembrane proteins to endosomes; among them are one dileucine-based signal and three tyrosine-based signals of the YXXØ and NPXY (where X stands for any amino acid, and Ø stands for any bulky hydrophobic amino acid) types. To investigate the role of each of these trafficking signals in intracellular localization and viral replication, we constructed a panel of expression plasmids and recombinant viruses in which the signals were rendered nonfunctional by mutagenesis. In transfected cells wild-type gpUL132 was mainly associated with the trans-Golgi network. Consecutive mutation of the trafficking signals resulted in increasing fractions of the protein localized at the cell surface, with gpUL132 mutated in all four trafficking motifs predominantly associated with the plasma membrane. Concomitant with increased surface expression, endocytosis of mutant gpUL132 was reduced, with a gpUL132 expressing all four motifs in mutated form being almost completely impaired in endocytosis. The replication of recombinant viruses harboring mutations in single trafficking motifs was comparable to replication of wild-type virus. In contrast, viruses containing mutations in three or four of the trafficking signals showed pronounced deficits in replication with a reduction of approximately 100-fold. Moreover, recombinant viruses expressing gpUL132 with three or four trafficking motifs mutated failed to incorporate the mutant protein into the virus particle. These results demonstrate a role of endocytosis of an HCMV envelope glycoprotein for incorporation into the virion and optimal virus replication.Morphogenesis of herpesviruses is a complex multistep process which requires the assembly of an infectious viral particle containing Ͼ70 proteins. The proteins are organized into distinct regions in the particle, including the capsid containing viral DNA, the tegument, and the envelope of the virion, suggesting an ordered addition of viral proteins during assembly.Most data in the literature favor a model in which the final virion is assembled by multiple budding and fusion events of the nascent particle. According to this model, genome-containing capsids acquire a first envelope by budding at the inner nuclear membrane in a step termed primary envelopment (34).

“…VZV gB is the second most abundant VZV glycoprotein and is also the most conserved among the herpesvirus glycoproteins (41). The gB protein contains three potential endocytosis motifs: two tyrosine-based motifs and one dileucine-type motif.…”

mentioning

confidence: 99%

“…In contrast to PrV gE, VZV gE is an essential glycoprotein that cannot be deleted from the genome (39). Even more importantly, a recent report demonstrated that a single mutation of just the gE YAGL endocytosis motif in a otherwise complete recombinant virus halted replication altogether (41). Because gE endocytosis appears to be an essential component of the VZV life cycle, the postendocytosis trafficking of gE as well as two other essential glycoproteins, gH and gB, in infected cells was investigated in detail.…”

mentioning

confidence: 99%

Incorporation of Three Endocytosed Varicella-Zoster Virus Glycoproteins, gE, gH, and gB, into the Virion Envelope

Marešová

Pasieka

Homan

et al. 2005

The cytoplasmic tails of all three major varicella-zoster virus (VZV) glycoproteins, gE, gH, and gB, harbor functional tyrosine-based endocytosis motifs that mediate internalization. The aim of the present study was to examine whether endocytosis from the plasma membrane is a cellular route by which VZV glycoproteins are delivered to the final envelopment compartment. In this study, we demonstrated that internalization of the glycoproteins occurred in the first 24 h postinfection but was reduced later in infection. Using surface biotinylation of VZV-infected cells followed by a glutathione cleavage assay, we showed that endocytosis was independent of antibody binding to gE, gH, and gB. Subsequently, with this assay, we demonstrated that biotinylated gE, gH, and gB retrieved from the cell surface were incorporated into nascent virus particles isolated after density gradient sedimentation. To confirm and extend this finding, we repeated the above sedimentation step and specifically detected envelopes decorated with Streptavidin-conjugated gold beads on a majority of complete virions through examination by transmission electron microscopy. In addition, a gE-gI complex and a gE-gH complex were found on the virions. Therefore, the above studies established that VZV subsumed a postendocytosis trafficking pathway as one mechanism by which to deliver viral glycoproteins to the site of virion assembly in the cytoplasm. Furthermore, since a recombinant VZV genome lacking only endocytosis-competent gE cannot replicate, these results supported the conclusion that the endocytosisenvelopment pathway is an essential component of the VZV life cycle.Endocytosis has now been demonstrated for numerous herpesvirus type 1 glycoproteins in transfected cell systems. In addition to gE of varicella-zoster virus (VZV), herpes simplex virus type 1 (HSV-1) gE and pseudorabies virus (PrV) gE undergo endocytosis (2,3,47,58,57). Also, PrV gB and human cytomegalovirus (HCMV) gB internalize in transfected cells (51,58,60). Unlike the situation for other human herpesviruses, the predominant VZV glycoprotein present on the envelope of the mature virion is gE (4,23,44). The gE protein traffics from the endoplasmic reticulum (ER) through the Golgi apparatus, where it is extensively processed, to the outer cell membrane. Both the ectodomain and the endodomain of gE have important functions. Based on the fact that a single point mutation in the VZV-MSP gE ectodomain changes the VZV-MSP egress pattern from a typical "viral highway" phenotype to a diffuse pattern similar to that observed in HSV-1-infected cells (53, 54), the ectodomain is likely to be critical for determining virus-cell interactions of mature virions.VZV gE endocytosis is mediated by a YAGL sequence located in the cytoplasmic tail of the protein (47). This sequence fits the consensus tyrosine-based YXX⌽ endocytosis motif (where Y is tyrosine, X is any amino acid, and ⌽ is any bulky hydrophobic amino acid) (6). Recycling of gE back to the plasma membrane postendocytosis has been demonst...