Differential requirement for cell fusion and virion formation in the pathogenesis of varicella-zoster virus infection in skin and T cells

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, rOka47DeltaC, 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 rOka47DeltaC 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 rOka47DeltaC 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 rOka47DeltaC 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.     

Unusual phosphorylation sequence in the gpIV (gI) component of the varicella-zoster virus gpI-gpIV glycoprotein complex (VZV gE-gI complex).

Varicella-zoster virus (VZV) glycoprotein gpIV, to be renamed VZV gI, forms a heterodimer with glycoprotein gpI (gE) which functions as an Fc receptor in virus-infected cells. Like VZV gpI (gE), this viral glycoprotein is phosphorylated in cell culture during biosynthesis. In this report, we investigated the nature and specificity of the phosphorylation event involving VZV gpIV (gI). Phosphoamino acid analysis indicated that gpIV (gI) was modified mainly on serine residues. To identify the precise location of the phosphorylation site on the 64-kDa protein, a step-by-step mutagenesis procedures was followed. Initially a tailless mutant was generated, and this truncated product was no longer phosphorylated. Thereafter, point mutations were made within the cytoplasmic tail of gpIV (gI) at potential phosphorylation sites. The phosphorylation site was localized to the following sequence: Ser-Pro-Pro (amino acids 343 to 345). Examination of the point mutants established that serine 343 in the cytoplasmic tail was the major phosphoacceptor. In addition, we found that the prolines located immediately to the C terminus of serine 343 were an integral part of the kinase recognition sequence. This site was located immediately N terminal to a predicted beta-turn secondary structure. By comparison with known substrate consensus sequences for various protein kinases, these data suggested that the phosphorylation of VZV gpIV (gI) was catalyzed by a proline-directed protein kinase. Computer homology analysis of other alphaherpesviruses demonstrated that a similar potential phosphorylation site was highly conserved in the cytoplasmic tails of herpes simplex virus type 1 gI, equine herpesvirus type 1 gI, and pseudorabies virus gp63.     

Varicella-Zoster Virus Fc Receptor Component gI Is Phosphorylated on Its Endodomain by a Cyclin-Dependent Kinase

Varicella-zoster virus (VZV) glycoprotein gI is a type 1 transmembrane glycoprotein which is one component of the heterodimeric gE:gI Fc receptor complex. Like VZV gE, VZV gI was phosphorylated in both VZV-infected cells and gI-transfected cells. Preliminary studies demonstrated that a serine 343-proline 344 sequence located within the gI cytoplasmic tail was the most likely phosphorylation site. To determine which protein kinase catalyzed the gI phosphorylation event, we constructed a fusion protein, consisting of glutathione-S-transferase (GST) and the gI cytoplasmic tail, called GST-gI-wt. When this fusion protein was used as a substrate for gI phosphorylation in vitro, the results demonstrated that GST-gI-wt fusion protein was phosphorylated by a representative cyclin-dependent kinase (CDK) called P-TEFb, a homologue of CDK1 (cdc2). When serine 343 within the serine-proline phosphorylation site was replaced with an alanine residue, the level of phosphorylation of the gI fusion protein was greatly reduced. Subsequent experiments with individually immunoprecipitated mammalian CDKs revealed that the VZV gI fusion protein was phosphorylated best by CDK1, to a lesser degree by CDK2, and not at all by CDK6. Transient-transfection assays carried out in the presence of the specific CDK inhibitor roscovitine strongly supported the prior results by demonstrating a marked decrease in gI phosphorylation while gI protein expression was unaffected. Finally, the possibility that VZV gI contained a CDK phosphorylation site in its endodomain was of further interest because its partner, gE, contains a casein kinase II phosphorylation site in its endodomain; prior studies have established that CDK1 can phosphorylate casein kinase II.     

The varicella-zoster virus (VZV) open reading frame 47 (ORF47) protein kinase is dispensable for viral replication and is not required for phosphorylation of ORF63 protein, the VZV homolog of herpes simplex virus ICP22.

To investigate the role of varicella-zoster virus (VZV) open reading frame 47 (ORF47) protein kinase during infection, a VZV mutant was generated in which two contiguous stop codons were introduced into ORF47, thus eliminating expression of the ORF47 kinase. ORF47 kinase was not essential for the growth of VZV in cultured cells, and the growth rate of the VZV mutant lacking ORF47 protein was indistinguishable from that of parental VZV. Nuclear extracts from cells infected with parental VZV contained several phosphorylated proteins which were not detected in extracts from cells infected with the ORF47 mutant. The herpes simplex virus type 1 (HSV-1) UL13 protein (the homolog of VZV ORF47 protein) is responsible for the posttranslational processing associated with phosphorylation of HSV-1 ICP22 (the homolog of VZV ORF63 protein). Immunoprecipitation of 32P-labeled proteins from cells infected with parental virus and those infected with ORF47 mutant virus yielded similar amounts of the VZV phosphoproteins encoded by ORF4, ORF62, ORF63, and ORF68 (VZV gE), and the electrophoretic migration of these proteins was not affected by the lack of ORF47 kinase. Therefore, while the VZV ORF47 protein is capable of phosphorylating several cellular or viral proteins, it is not required for phosphorylation of the ORF63 protein in virus-infected cells.     

Identification of the phosphorylation sequence in the cytoplasmic tail of the varicella-zoster virus Fc receptor glycoprotein gpI.

Varicella-zoster virus (VZV) glycoprotein gpI, the homolog of herpes simplex virus gE, functions as a receptor for the Fc portion of immunoglobulin G. Like other cell surface receptors, this viral receptor is highly phosphorylated in cell culture. To identify the precise location of the cellular kinase-mediated phosphorylation, we generated a tailless deletion mutant and several point mutants which had altered serine and threonine residues within the cytoplasmic domain of gpI. The mutated and wild-type genes of gpI were transfected and expressed within a vaccinia virus-T7 polymerase transfection system in order to determine what effect these mutations had on the phosphorylation state of the protein in vivo and in vitro. Truncation of the cytoplasmic domain of gpI diminished the phosphorylation of gpI in vivo. Examination of the point mutants established that the major phosphorylation sequence of gpI was located between amino acids 593 and 598, a site which included four phosphorylatable serine and threonine residues. Phosphorylation analyses of the mutant and wild-type glycoproteins confirmed that gpI was a substrate for casein kinase II, with threonines 596 and 598 being critical residues. Although the mutant glycoproteins were phosphorylated by casein kinase I, protease V8 partial digestion profiles suggested that casein kinase II exerted the major effect. Thus, these mutagenesis studies demonstrated that the gpI cytoplasmic sequence Ser-Glu-Ser-Thr-Asp-Thr was phosphorylated in mammalian cells in the absence of any other herpesvirus products. Since the region defined by transfection was consistent with results obtained with in vitro phosphorylation by casein kinase II, we propose that VZV gpI is a physiologic substrate for casein kinase II. Immunofluorescence and pulse-chase experiments demonstrated that the mutant glycoproteins were processed and transported to the outer cell membrane.     

Varicella-zoster virus ORF47 protein serine kinase: characterization of a cloned, biologically active phosphotransferase and two viral substrates, ORF62 and ORF63

Varicella-zoster virus (VZV) codes for a protein serine kinase called ORF47; the herpes simplex virus (HSV) homolog is UL13. No recombinant alphaherpesvirus serine kinase has been biologically active in vitro. We discovered that preservation of the intrinsic kinase activity of recombinant VZV ORF47 required unusually stringent in vitro conditions, including physiological concentrations of polyamines. In this assay, ORF47 phosphorylated two VZV regulatory proteins: the ORF62 protein (homolog of HSV ICP4) and the ORF63 protein (homolog of HSV ICP22). Of interest, ORF47 kinase also coprecipitated ORF63 protein from the kinase assay supernatant.     

Endocytosis and recycling of varicella-zoster virus Fc receptor glycoprotein gE: internalization mediated by a YXXL motif in the cytoplasmic tail.

Varicella-zoster virus (VZV) encodes a cell surface Fc receptor, glycoprotein gE. VZV gE has previously been shown to display several features common to nonviral cell surface receptors. Most recently, VZV gE was reported to be tyrosine phosphorylated on a dimeric form (J. K. Olson, G. A. Bishop, and C. Grose, J. Virol. 71:110-119, 1997). Thereafter, attention focused on the ability of VZV gE to undergo receptor-mediated endocytosis. The current transient transfection studies demonstrated by confocal microscopy and internalization assays that VZV gE was endocytosed when expressed in HeLa cells. Endocytosis of gE was shown to be dependent on clathrin-coated vesicle formation within the cells. Subsequent colocalization studies showed that endocytosis of VZV gE closely mimicked endocytosis of the transferrin receptor. The gE cytoplasmic tail and more specifically tyrosine residue 582 were determined by mutagenesis studies to be important for efficient internalization of the protein; this tyrosine residue is part of a conserved YXXL motif. The amount of gE internalized at any given time reached a steady state of 32%. In addition, like the transferrin receptor, internalized gE recycled to the cell surface. The finding of gE endocytosis provided insight into earlier documentation of gE serine/threonine and tyrosine phosphorylation, since these phosphorylation events may serve as sorting signals for internalized receptors. Taken together with the previous discovery that both human and simian immunodeficiency virus envelope proteins can undergo endocytosis, the gE findings suggest that endocytosis of envelope components may be a posttranslational regulatory mechanism among divergent families of enveloped viruses.     

Essential functions of the unique N-terminal region of the varicella-zoster virus glycoprotein E ectodomain in viral replication and in the pathogenesis of skin infection

Varicella-zoster virus (VZV) glycoprotein E (gE) is a multifunctional protein important for cell-cell spread, envelopment, and possibly entry. In contrast to other alphaherpesviruses, gE is essential for VZV replication. Interestingly, the N-terminal region of gE, comprised of amino acids 1 to 188, was shown not to be conserved in the other alphaherpesviruses by bioinformatics analysis. Mutational analysis was performed to investigate the functions associated with this unique gE N-terminal region. Linker insertions, serine-to-alanine mutations, and deletions were introduced in the gE N-terminal region in the VZV genome, and the effects of these mutations on virus replication and cell-cell spread, gE trafficking and localization, virion formation, and replication in vivo in the skin were analyzed. In summary, mutagenesis of the gE N-terminal region identified a new functional region in the VZV gE ectodomain essential for cell-cell spread and the pathogenesis of VZV skin tropism and demonstrated that different subdomains of the unique N-terminal region had specific roles in viral replication, cell-cell spread, and secondary envelopment.     

Phosphorylation of varicella-zoster virus open reading frame (ORF) 62 regulatory product by viral ORF 47-associated protein kinase.

Varicella-zoster virus (VZV) encodes within its unique long region a gene product with protein kinase motifs. In a previous study, we demonstrated that immunoprecipitated VZV open reading frame (ORF) 47 protein was associated with a functional protein kinase activity, on the basis of its ability to both autophosphorylate and phosphorylate artificial substrates. To further define potential substrates of ORF 47-associated protein kinase, we analyzed individual viral phosphoproteins to determine whether any were modified by the viral protein kinase. These candidates included gene products of VZV ORFs 4, 61, 62, and 63, which are homologs of herpes simplex virus type 1 (HSV-1) immediate-early proteins. Each of the above VZV proteins was coimmunoprecipitated with ORF 47 kinase, and the immune complex was incubated in a protein kinase assay. Under these conditions, only the VZV immediate-early ORF 62 protein was phosphorylated by ORF 47-associated protein kinase. The specificity of this phosphorylation event was analyzed by a competition assay in which a recombinant ORF 47 protein lacking enzymatic activity was able to reduce the amount of phosphorylation of ORF 62 protein by VZV ORF 47-associated kinase. To provide an additional evaluation of specificity, the experiment was repeated with [32P]GTP instead of [32P]ATP, because the VZV ORF 47 kinase has the distinctive property of using GTP as a phosphate donor. Again the ORF 62 substrate was phosphorylated. In summary, the VZV ORF 47-associated protein kinase (the HSV-1 UL13 homolog) catalyzed the in vitro phosphorylation of the VZV ORF 62 protein, the homolog of the HSV-1 ICP4 regulatory protein.     

Varicella-zoster virus glycoprotein E is a critical determinant of virulence in the SCID mouse-human model of neuropathogenesis

Varicella-zoster virus (VZV) is a neurotropic alphaherpesvirus. VZV infection of human dorsal root ganglion (DRG) xenografts in immunodeficient mice models the infection of sensory ganglia. We examined DRG infection with recombinant VZV (recombinant Oka [rOka]) and the following gE mutants: gEΔ27-90, gEΔCys, gE-AYRV, and gE-SSTT. gEΔ27-90, which lacks the gE domain that interacts with a putative receptor insulin-degrading enzyme (IDE), replicated as extensively as rOka, producing infectious virions and significant cytopathic effects within 14 days of inoculation. Since neural cells express IDE, the gE/IDE interaction was dispensable for VZV neurotropism. In contrast, gEΔCys, which lacks gE/gI heterodimer formation, was significantly impaired at early times postinfection; viral genome copy numbers increased slowly, and infectious virus production was not detected until day 28. Delayed replication was associated with impaired cell-cell spread in ganglia, similar to the phenotype of a gI deletion mutant (rOkaΔgI). However, at later time points, infection of satellite cells and other supportive nonneuronal cells resulted in extensive DRG tissue damage and cell loss such that cytopathic changes observed at day 70 were more severe than those for rOka-infected DRG. The replication of gE-AYRV, which is impaired for trans-Golgi network (TGN) localization, and the replication of gE-SSTT, which contains mutations in an acidic cluster, were equivalent to that of rOka, causing significant cytopathic effects and infectious virus production by day 14; genome copy numbers were equivalent to those of rOka. These experiments suggest that the gE interaction with cellular IDE, gE targeting to TGN sites of virion envelopment, and phosphorylation at SSTT are dispensable for VZV DRG infection, whereas the gE/gI interaction is critical for VZV neurovirulence.     

Varicella-zoster virus Fc receptor gE glycoprotein: serine/threonine and tyrosine phosphorylation of monomeric and dimeric forms.

Varicella-zoster virus (VZV) glycoprotein gE is the predominant viral cell surface molecule; it behaves as an Fc receptor for immunoglobulin G, but its central function may be more closely related to viral egress and cell-to-cell spread. To further analyze the receptor properties of VZV gE, the gE gene (also called open reading frame 68) was expressed by a baculovirus vector in insect cells. The recombinant baculovirus gE product had a molecular mass of 64 kDa, smaller than the previously documented 98 kDa of mature gE expressed in mammalian cells. The major reason for the lowered molecular mass was diminished glycosylation. In addition to the 64-kDa form, a larger (130-kDa) form was observed in insect cells and represented dimerized 64-kDa molecules. Both the monomeric and dimeric gE forms were highly phosphorylated in insect cells. Protein kinase assays conducted in vitro with [gamma-32P]ATP and [gamma-32P]GTP indicated that endogenous casein kinase II was phosphorylating monomeric gE, while the dimeric gE form was phosphorylated by another kinase which did not utilize [gamma-32P]GTP. When immobilized recombinant gE molecules were probed with a monoclonal antibody which specifically recognizes a phosphotyrosine linkage, the gE dimer was found to be tyrosine phosphorylated whereas the monomer was not similarly modified. When recombinant gE produced in HeLa cells was probed with the same antiphosphotyrosine antibody, a dimeric gE form at 130 kDa was detected on the cell surface. These results suggested that VZV gE closely resembled other cell surface receptors, being modified on its various forms by both serine/threonine and tyrosine protein kinases. In this case, tyrosine phosphorylation occurred on a previously unrecognized and underglycosylated VZV gE dimeric product.     

T-cell tropism and the role of ORF66 protein in pathogenesis of varicella-zoster virus infection

The pathogenesis of varicella-zoster virus (VZV) involves a cell-associated viremia during which infectious virus is carried from sites of respiratory mucosal inoculation to the skin. We now demonstrate that VZV infection of T cells is associated with robust virion production and modulation of the apoptosis and interferon pathways within these cells. The VZV serine/threonine protein kinase encoded by ORF66 is essential for the efficient replication of VZV in T cells. Preventing ORF66 protein expression by stop codon insertion (pOka66S) impaired the growth of the parent Oka (pOka) strain in T cells in SCID-hu T-cell xenografts in vivo and reduced formation of VZV virions. The lack of ORF66 protein also increased the susceptibility of infected T cells to apoptosis and reduced the capacity of the virus to interfere with induction of the interferon (IFN) signaling pathway following exposure to IFN-gamma. However, preventing ORF66 protein expression only slightly reduced growth in melanoma cells in culture and did not diminish virion formation in these cells. The pOka66S virus showed only a slight defect in growth in SCID-hu skin implants compared with intact pOka. These observations suggest that the ORF66 kinase plays a unique role during infection of T cells and supports VZV T-cell tropism by contributing to immune evasion and enhancing survival of infected T cells.     

The alphaherpesvirus US3/ORF66 protein kinases direct phosphorylation of the nuclear matrix protein matrin 3

The protein kinase found in the short region of alphaherpesviruses, termed US3 in herpes simplex virus type 1 (HSV-1) and pseudorabies virus (PRV) and ORF66 in varicella-zoster virus (VZV), affects several viral and host cell processes, and its specific targets remain an area of active investigation. Reports suggesting that HSV-1 US3 substrates overlap with those of cellular protein kinase A (PKA) prompted the use of an antibody specific for phosphorylated PKA substrates to identify US3/ORF66 targets. HSV-1, VZV, and PRV induced very different substrate profiles that were US3/ORF66 kinase dependent. The predominant VZV-phosphorylated 125-kDa species was identified as matrin 3, one of the major nuclear matrix proteins. Matrin 3 was also phosphorylated by HSV-1 and PRV in a US3 kinase-dependent manner and by VZV ORF66 kinase at a novel residue (KRRRT150EE). Since VZV-directed T150 phosphorylation was not blocked by PKA inhibitors and was not induced by PKA activation, and since PKA predominantly targeted matrin 3 S188, it was concluded that phosphorylation by VZV was PKA independent. However, purified VZV ORF66 kinase did not phosphorylate matrin 3 in vitro, suggesting that additional cellular factors were required. In VZV-infected cells in the absence of the ORF66 kinase, matrin 3 displayed intranuclear changes, while matrin 3 showed a pronounced cytoplasmic distribution in late-stage cells infected with US3-negative HSV-1 or PRV. This work identifies phosphorylation of the nuclear matrix protein matrin 3 as a new conserved target of this kinase group.     

The Nef protein of human immunodeficiency virus type 1 enhances serine phosphorylation of the viral matrix.

The human immunodeficiency virus type 1 matrix (MA) protein is phosphorylated during virion maturation on its C-terminal tyrosine and on several serine residues. Whereas MA tyrosine phosphorylation facilitates viral nuclear import, the significance of MA serine phosphorylation remains unclear. Here, we report that MA serine but not tyrosine phosphorylation is strongly enhanced by Nef. Mutations that abrogated the membrane association of Nef and its ability to bind a cellular serine/threonine kinase greatly diminished the extent of virion MA serine phosphorylation. Correspondingly, a protein kinase coimmunoprecipitated with Nef could phosphorylate MA on serine in vitro, producing a phosphopeptide pattern reminiscent of that of virion MA. Recombinant p21-activated kinase hPAK65, a recently proposed relative of the Nef-associated kinase, achieved a comparable result. Taken together, these data suggest that MA is a target of the Nef-associated serine kinase.     

The varicella-zoster virus ORF47 kinase interferes with host innate immune response by inhibiting the activation of IRF3

The innate immune response constitutes the first line of host defence that limits viral spread and plays an important role in the activation of adaptive immune response. Viral components are recognized by specific host pathogen recognition receptors triggering the activation of IRF3. IRF3, along with NF-κB, is a key regulator of IFN-β expression. Until now, the role of IRF3 in the activation of the innate immune response during Varicella-Zoster Virus (VZV) infection has been poorly studied. In this work, we demonstrated for the first time that VZV rapidly induces an atypical phosphorylation of IRF3 that is inhibitory since it prevents subsequent IRF3 homodimerization and induction of target genes. Using a mutant virus unable to express the viral kinase ORF47p, we demonstrated that (i) IRF3 slower-migrating form disappears; (ii) IRF3 is phosphorylated on serine 396 again and recovers the ability to form homodimers; (iii) amounts of IRF3 target genes such as IFN-β and ISG15 mRNA are greater than in cells infected with the wild-type virus; and (iv) IRF3 physically interacts with ORF47p. These data led us to hypothesize that the viral kinase ORF47p is involved in the atypical phosphorylation of IRF3 during VZV infection, which prevents its homodimerization and subsequent induction of target genes such as IFN-β and ISG15.     

Differentiation of varicella-zoster virus ORF47 protein kinase and IE62 protein binding domains and their contributions to replication in human skin xenografts in the SCID-hu mouse

To investigate the role of the ORF47 protein kinase of varicella-zoster virus (VZV), we constructed VZV recombinants with targeted mutations in conserved motifs of ORF47 and a truncated ORF47 and characterized these mutants for replication, phosphorylation, and protein-protein interactions in vitro and for infectivity in human skin xenografts in the SCID-hu mouse model in vivo. Previous experiments showed that ROka47S, a null mutant that makes no ORF47 protein, did not replicate in skin in vivo (J. F. Moffat, L. Zerboni, M. H. Sommer, T. C. Heineman, J. I. Cohen, H. Kaneshima, and A. M. Arvin, Proc. Natl. Acad. Sci. USA 95:11969-11974, 1998). The construction of VZV recombinants with targeted ORF47 mutations made it possible to assess the effects on VZV infection of human skin xenografts of selectively abolishing ORF47 protein kinase activity. ORF47 mutations that resulted in a C-terminal truncation or disrupted the DYS kinase motif eliminated ORF47 kinase activity and were associated with extensive nuclear retention of ORF47 and IE62 proteins in vitro. Disrupting ORF47 kinase function also resulted in a marked decrease in VZV replication and cutaneous lesion formation in skin xenografts in vivo. However, infectivity in vivo was not blocked completely as long as the capacity of ORF47 protein to bind IE62 protein was preserved, a function that we identified and mapped to the N-terminal domain of ORF47 protein. These experiments indicate that ORF47 kinase activity is of critical importance for VZV infection and cell-cell spread in human skin in vivo but suggest that it is the formation of complexes between ORF47 and IE62 proteins, both VZV tegument components, that constitutes the essential contribution of ORF47 protein to VZV replication in vivo.     

Herpes simplex virus gE/gI must accumulate in the trans-Golgi network at early times and then redistribute to cell junctions to promote cell-cell spread

Herpes simplex virus (HSV) glycoprotein heterodimer gE/gI is necessary for virus spread in epithelial and neuronal tissues. Deletion of the relatively large gE cytoplasmic (CT) domain abrogates the ability of gE/gI to mediate HSV spread. The gE CT domain is required for the sorting of gE/gI to the trans-Golgi network (TGN) in early stages of virus infection, and there are several recognizable TGN sorting motifs grouped near the center of this domain. Late in HSV infection, gE/gI, other viral glycoproteins, and enveloped virions redistribute from the TGN to epithelial cell junctions, and the gE CT domain is also required for this process. Without the gE CT domain, newly enveloped virions are directed to apical surfaces instead of to cell junctions. We hypothesized that the gE CT domain promotes virus envelopment into TGN subdomains from which nascent enveloped virions are sorted to cell junctions, a process that enhances cell-to-cell spread. To characterize elements of the gE CT domain involved in intracellular trafficking and cell-to-cell spread, we constructed a panel of truncation mutants. Specifically, these mutants were used to address whether sorting to the TGN and redistribution to cell junctions are necessary, and sufficient, for gE/gI to promote cell-to-cell spread. gE-519, lacking 32 C-terminal residues, localized normally to the TGN early in infection and then trafficked to cell junctions at late times and mediated virus spread. By contrast, mutants gE-495 (lacking 56 C-terminal residues) and gE-470 (lacking 81 residues) accumulated in the TGN but did not traffic to cell junctions and did not mediate cell-to-cell spread. A fourth mutant, gE-448 (lacking most of the CT domain), did not localize to cell junctions and did not mediate virus spread. Therefore, the capacity of gE/gI to promote cell-cell spread requires early localization to the TGN, but this is not sufficient for virus spread. Additionally, gE CT sequences between residues 495 and 519, which contain no obvious cell sorting motifs, are required to promote gE/gI traffic to cell junctions and cell-to-cell spread.