Random Transposon-Mediated Mutagenesis of the Essential Large Tegument Protein pUL36 of Pseudorabies Virus

Homologs of the pseudorabies virus (PrV) essential large tegument protein pUL36 are conserved throughout the Herpesviridae. pUL36 functions during transport of the nucleocapsid to and docking at the nuclear pore as well as during virion formation after nuclear egress in the cytoplasm. Deletion analyses revealed several nonessential regions within the 3,084-amino-acid PrV pUL36 (S. Böttcher, B. G. Klupp, H. Granzow, W. Fuchs, K. Michael, and T. C. Mettenleiter, J. Virol. 80:9910-9915, 2006; S. Böttcher, H. Granzow, C. Maresch, B. Möhl, B. G. Klupp, and T. C. Mettenleiter, J. Virol. 81:13403-13411, 2007), while the C-terminal 62 amino acids are essential for virus replication (K. Coller, J. Lee, A. Ueda, and G. Smith, J. Virol. 81:11790-11797, 2007). To identify additional functional domains, we performed random mutagenesis of PrV pUL36 by transposon-mediated insertion of a 15-bp linker. By this approach, 26 pUL36 insertion mutants were selected and tested in transient transfection assays for their ability to complement one-step growth and/or viral spread of a PrV UL36 null mutant. Ten insertion mutants in the N-terminal half and 10 in the C terminus complemented both, whereas six insertion mutants clustering in the center of the protein did not complement in either assay. Interestingly, several insertions within conserved parts yielded positive complementation, including those located within the essential C-terminal 62 amino acids. For 15 mutants that mediated productive replication, stable virus recombinants were isolated and further characterized by plaque assay, in vitro growth analysis, and electron microscopy. Except for three mutant viruses, most insertion mutants replicated like wild-type PrV. Two insertion mutants, at amino acids (aa) 597 and 689, were impaired in one-step growth and viral spread and exhibited a defect in virion maturation in the cytoplasm. In contrast, one functional insertion (aa 1800) in a region which otherwise yielded only nonfunctional insertion mutants was impaired in viral spread but not in one-step growth without a distinctive ultrastructural phenotype. In summary, these studies extend and refine previous analyses of PrV pUL36 and demonstrate the different sensitivities of different regions of the protein to functional loss by insertion.The herpesvirus particle is composed of four structural elements. The DNA genome-containing core is enclosed in an icosahedral capsid, which, in turn, is embedded in a proteinaceous layer termed the tegument and enveloped by a cell-derived membrane containing viral glycoproteins (35). The tegument of the Alphaherpesvirinae contains more than 15 different viral and several cellular proteins and can be structurally and functionally separated into at least two layers: a capsid-proximal “inner” part and an envelope-associated “outer” part (reviewed in references 34 and 35). The largest tegument proteins in all herpesviruses analyzed so far are homologs of herpes simplex virus type 1 (HSV-1) pUL36, which are essential for viral replication. pUL36, its interaction partner, pUL37, and the pUS3 kinase are part of the inner tegument and remain associated with nucleocapsids during their transport along microtubules to the nuclear pore (2, 3, 19, 31). In contrast, other tegument proteins like pUL46, pUL47, and pUL49 rapidly diffuse in the cytoplasm after fusion of the virion envelope with the plasma membrane. Proteolytic cleavage of HSV-1 pUL36 after docking of the nucleocapsid to the nuclear pore appears to be required for release of viral DNA into the nucleus (22). Besides these roles early in infection, pUL36 also functions during later stages of replication in virion maturation. After assembly in the nucleus, nucleocapsids are translocated to the cytoplasm by budding at the inner nuclear membrane and fusion with the outer nuclear membrane (34). Although functional nuclear localization motifs have been described for pseudorabies virus (PrV) and HSV-1 pUL36 (1, 37), in PrV-infected cells, pUL36 was never detected in the nucleus but was added to nascent virions early after nuclear egress (18, 27, 31, 37). It has been suggested that pUL36 interacts either directly (9, 32, 42, 44) or indirectly via capsid-associated pUL25 (10) with the capsid shell starting the tegumentation process in the cytosol.In PrV, pUL36 is the only tegument protein which has been shown to be truly essential. It consists of 3,084 amino acids (aa), resulting in a molecular mass of more than 300 kDa (27). Deletion of pUL36 in HSV-1 and PrV abolished viral replication. Ultrastructurally, similar phenotypes with nonenveloped nucleocapsids present in the cytoplasm and the lack of extracellular particles indicated a defect in virion maturation in the cytoplasm (13, 16). Several functional domains have been identified in pUL36. The interaction domain of pUL36 with pUL37 (5, 16, 20, 27, 36, 42) could be located in the N-terminal part of PrV and HSV-1 pUL36 (16, 36) (Fig. ​(Fig.1).1). Deletion of the pUL37 binding site in PrV pUL36 (PrV-UL36BSF) resulted in a similar phenotype to deletion of pUL37 with an impairment of secondary envelopment in the cytoplasm (16, 26). Unlike in PrV, pUL37 is essential for replication in HSV-1 (14, 30).Open in a separate windowFIG. 1.Schematic overview of PrV pUL36 and corresponding insertion mutants. (A) Diagram of the PrV genome with the unique long (U_L) and unique short (U_S) regions as well as repeat regions (internal repeat, IR; terminal repeat, TR). The positions of BamHI restriction sites are indicated, and restriction fragments are numbered according to their size. (B) Schematic diagram of the UL36 open reading frame with conserved regions. Pfam analysis (4; http://www.sanger.ac.uk/Software/Pfam/) delineated two highly conserved PfamA domains within pUL36 homologs of herpesviruses of all three herpesvirus subfamilies [box I, Herpes_teg_N PrV (p)UL36, aa 11 to 178] and of alphaherpesviruses [box II, Herpes_UL36 PrV (p)UL36, aa 1000 to 1251] as well as PfamB domains (hatched rectangles) (6) (C) Known essential and nonessential regions in PrV pUL36. Nonessential regions are shown in gray, with the positions of the amino acids deleted in the corresponding constructs (6, 8). Deletions tested by Lee et al. (28) are shown below, marked by arrows. The essential C terminus is shown in black. Besides the N-terminal deletion Δ6-225, none of the truncated proteins was functional. (D) Predicted or identified motifs in pUL36: USP (Cys26), active-site cysteine of the deubiquitinating activity (24); pUL37 interaction domain (16, 27); NLS, nuclear localization signal (37); leucine zipper (27); and late domain motifs PPKY and PSAP (6). (E) Locations of linker insertions in pUL36 are indicated by arrows and the position of the amino acid immediately preceding the insertion. Insertions shown by arrows pointing upwards yielded functional proteins, while arrows pointing downwards indicate nonfunctional mutants. Insertions resulting in proteins which were impaired but not fully deficient in complementation are underlined. For orientation, the BamHI site separating BamHI fragments 1 and 2 is indicated.A second functional domain in the N terminus of pUL36 comprises a ubiquitin-specific cysteine protease (USP) activity which could be identified in all three herpesvirus subfamilies (24, 40, 41). Interestingly, the USP activity is not essential for virus replication in cell culture (7, 21, 25, 43). However, it is relevant for oncogenicity of Marek′s disease virus (MDV) (21) and for virion maturation and neuroinvasion of PrV (7, 8, 29).Several other regions in PrV pUL36 were deleted without abolishing virus replication (6, 8, 28). While deletion of nearly 1/3 of the protein in the C-terminal part (aa 2087 to 2981) had only a slight effect, deletion of a region containing two leucine zipper motifs impaired virus replication and spread more strongly (8). The highly conserved C-terminal 62 amino acids, except for the extreme C-terminal 6 amino acids, are essential for virus replication (6, 28). Due to the size of the protein, a more detailed mutagenesis analysis has, however, not yet been undertaken.Therefore, the aim of our study was to construct random insertion mutants of PrV pUL36 using transposon-mediated insertion mutagenesis resulting in a 5-amino-acid linker insertion. Mutant proteins were analyzed functionally in transient transfection assays for complementation, and stable recombinants were isolated and further characterized.     

伪狂犬病病毒ul24基因表达蛋白的胞内定位研究

根据GenBank已发表的PrVul24基因序列(NC006151),设计并合成一对引物,PCR扩增出ul24基因编码区,克隆于pEGFP-N1载体,得到重组质粒pUL24-GFP。酶切鉴定,测序及WesternBlot验证重组质粒。ul24基因序列测定结果已提交GenBank,登录号DQ226544。Westernblot分析结果表明UL24-GFP融合蛋白为45KD。将pUL24-GFP转染真核细胞,激光共聚焦显微镜观察融合蛋白的细胞内定位,结果表明UL24-GFP融合蛋白定位于细胞核。     

伪狂犬病病毒ul24基因表达蛋白的胞内定位研究

根据GenBank已发表的PrV ul24基因序列(NC006151),设计并合成一对引物,PCR扩增出ul24基因编码区,克隆于pEGFP-N1载体,得到重组质粒pUL24-GFP.酶切鉴定,测序及Western Blot验证重组质粒.ul24基因序列测定结果已提交GenBank,登录号DQ226544.Western blot分析结果表明UL24-GFP融合蛋白为45KD.将pUL24-GFP转染真核细胞,激光共聚焦显微镜观察融合蛋白的细胞内定位,结果表明UL24-GFP融合蛋白定位于细胞核.     

Structure and Capsid Association of the Herpesvirus Large Tegument Protein UL36

The tegument of all herpesviruses contains a high-molecular-weight protein homologous to herpes simplex virus (HSV) UL36. This large (3,164 amino acids), essential, and multifunctional polypeptide is located on the capsid surface and present at 100 to 150 copies per virion. We have been testing the idea that UL36 is important for the structural organization of the tegument. UL36 is proposed to bind directly to the capsid with other tegument proteins bound indirectly by way of UL36. Here we report the results of studies carried out with HSV type 1-derived structures containing the capsid but lacking a membrane and depleted of all tegument proteins except UL36 and a second high-molecular-weight protein, UL37. Electron microscopic analysis demonstrated that, compared to capsids lacking a tegument, these capsids (called T36 capsids) had tufts of protein located at the vertices. Projecting from the tufts were thin, variably curved strands with lengths (15 to 70 nm) in some cases sufficient to extend across the entire thickness of the tegument (∼50 nm). Strands were sensitive to removal from the capsid by brief sonication, which also removed UL36 and UL37. The findings are interpreted to indicate that UL36 and UL37 are the components of the tufts and of the thin strands that extend from them. The strand lengths support the view that they could serve as organizing features for the tegument, as they have the potential to reach all parts of the tegument. The variably curved structure of the strands suggests they may be flexible, a property that could contribute to the deformable nature of the tegument.All herpesviruses have a tegument, a layer of protein located between the virus capsid and membrane. The tegument accounts for a substantial proportion of the overall virus structure. Its thickness (30 to 50 nm), for example, may be comparable to the capsid radius, and tegument proteins can account for 40% or more of the total virion protein. Herpesvirus tegument proteins are thought to function promptly after initiation of infection, before expression of virus genes can take place (11, 13, 14, 21, 33, 37).Electron microscopic analysis of virions has demonstrated that the tegument is not highly structured (9, 22). It does not have icosahedral symmetry like the capsid, and it may be uniformly or asymmetrically arranged around the capsid (26). Tegument structure is described as fibrous or granular, and its morphology is found to change as the virus matures. Studies with herpes simplex virus type 1 (HSV-1), for example, indicate that the tegument structure is altered in cell-associated compared to extracellular virus (26).The tegument has been most thoroughly studied in HSV-1, where biochemical analyses indicate that it is composed of approximately 20 distinct, virus-encoded protein species. The predominant components are the products of the genes UL47, UL48, and UL49, with each protein present in 800 or more copies per virion (12, 40). Other tegument proteins can occur in 100 or fewer copies, and trace amounts of cell-encoded proteins are also present (17). Tegument proteins are classified as inner or outer components based on their association with the capsid after it enters the host cell cytoplasm. The inner tegument proteins (UL36, UL37, and US3) are those that remain bound to the capsid after entry, while the others (the outer tegument proteins) become detached (7, 18).The HSV-1 UL36 protein has the potential to play a central role in organizing the overall structure of the tegument. With a length of 3,164 amino acids, UL36 could span the thickness of the tegument multiple times. One hundred to 150 UL36 molecules are present in the tegument (12), and they are bound to the capsid by way of an essential C-terminal domain (2, 16). UL36 is able to bind the major tegument components by way of documented direct (UL37 and UL48) and indirect (UL46, UL47, and UL49) contacts (6, 15, 24, 38).Here we describe the results of studies designed to test the idea that UL36 serves to organize the tegument structure. Beginning with infectious virus, a novel method has been used to isolate capsids that contain UL36 and UL37 but lack the virus membrane and are depleted of all other tegument proteins. These capsids (T36 capsids) were examined by electron microscopy to clarify the structure of UL36 and UL37 molecules and their location on the capsid surface.     

Intracellular Trafficking of the UL11 Tegument Protein of Herpes Simplex Virus Type 1

Growing evidence indicates that herpes simplex virus type 1 (HSV-1) acquires its final envelope in the trans-Golgi network (TGN). During the envelopment process, the viral nucleocapsid as well as the envelope and tegument proteins must arrive at this site in order to be incorporated into assembling virions. To gain a better understanding of how these proteins associate with cellular membranes and target to the correct compartment, we have been studying the intracellular trafficking properties of the small tegument protein encoded by the U(L)11 gene of HSV-1. This 96-amino-acid, myristylated protein accumulates on the cytoplasmic face of internal membranes, where it is thought to play a role in nucleocapsid envelopment and egress. When expressed in the absence of other HSV-1 proteins, the UL11 protein localizes to the Golgi apparatus, and previous deletion analyses have revealed that the membrane-trafficking information is contained within the first 49 amino acids. The goal of this study was to map the functional domains required for proper Golgi membrane localization. In addition to N-terminal myristylation, which allows for weak membrane binding, UL11 appears to be palmitylated on one or more of three consecutive N-terminal cysteines. Using membrane-pelleting experiments and confocal microscopy, we show that palmitylation of UL11 is required for both Golgi targeting specificity and strong membrane binding. Furthermore, we found that a conserved acidic cluster within the first half of UL11 is required for the recycling of this tegument protein from the plasma membrane to the Golgi apparatus. Taken together, our results demonstrate that UL11 has highly dynamic membrane-trafficking properties, which suggests that it may play multiple roles on the plasma membrane as well as on the nuclear and TGN membranes.     

The Novel Structural Protein of Human Cytomegalovirus, pUL25, Is Localized in the Viral Tegument

Human cytomegalovirus UL25 codes for a structural phosphoprotein of 85 kDa (C. J. Baldick and T. Shenk, J. Virol. 70:6097-6105, 1996; M. C. Battista et al., J. Virol. 73:3800-3809, 1999). In this study we analyzed the intracellular and intraviral localization of pUL25 by confocal and immunoelectron microscopy and found that pUL25 is a component of the viral tegument and the dense body matrix, acquired during the late cytoplasmic phase of virus maturation.     

A Network of Protein Interactions around the Herpes Simplex Virus Tegument Protein VP22

Assembly of the herpesvirus tegument is poorly understood but is believed to involve interactions between outer tegument proteins and the cytoplasmic domains of envelope glycoproteins. Here, we present the detailed characterization of a multicomponent glycoprotein-tegument complex found in herpes simplex virus 1 (HSV-1)-infected cells. We demonstrate that the tegument protein VP22 bridges a complex between glycoprotein E (gE) and glycoprotein M (gM). Glycoprotein I (gI), the known binding partner of gE, is also recruited into this gE-VP22-gM complex but is not required for its formation. Exclusion of the glycoproteins gB and gD and VP22''s major binding partner VP16 demonstrates that recruitment of virion components into this complex is highly selective. The immediate-early protein ICP0, which requires VP22 for packaging into the virion, is also assembled into this gE-VP22-gM-gI complex in a VP22-dependent fashion. Although subcomplexes containing VP22 and ICP0 can be formed when either gE or gM are absent, optimal complex formation requires both glycoproteins. Furthermore, and in line with complex formation, neither of these glycoproteins is individually required for VP22 or ICP0 packaging into the virion, but deletion of gE and gM greatly reduces assembly of both VP22 and ICP0. Double deletion of gE and gM also results in small plaque size, reduced virus yield, and defective secondary envelopment, similar to the phenotype previously shown for pseudorabies virus. Hence, we suggest that optimal gE-VP22-gM-gI-ICP0 complex formation correlates with efficient virus morphogenesis and spread. These data give novel insights into the poorly understood process of tegument acquisition.     

Caspase-1 Promotes Epstein-Barr Virus Replication by Targeting the Large Tegument Protein Deneddylase to the Nucleus of Productively Infected Cells

The large tegument proteins of herpesviruses contain N-terminal cysteine proteases with potent ubiquitin and NEDD8-specific deconjugase activities, but the function of the enzymes during virus replication remains largely unknown. Using as model BPLF1, the homologue encoded by Epstein-Barr virus (EBV), we found that induction of the productive virus cycle does not affect the total level of ubiquitin-conjugation but is accompanied by a BPLF1-dependent decrease of NEDD8-adducts and accumulation of free NEDD8. Expression of BPLF1 promotes cullin degradation and the stabilization of cullin-RING ligases (CRLs) substrates in the nucleus, while cytoplasmic CRLs and their substrates are not affected. The inactivation of nuclear CRLs is reversed by the N-terminus of CAND1, which inhibits the binding of BPLF1 to cullins and prevents efficient viral DNA replication. Targeting of the deneddylase activity to the nucleus is dependent on processing of the catalytic N-terminus by caspase-1. Inhibition of caspase-1 severely impairs viral DNA synthesis and the release of infectious virus, pointing a previously unrecognized role of the cellular response to danger signals triggered by EBV reactivation in promoting virus replication.     

Epstein-Barr Virus Large Tegument Protein BPLF1 Contributes to Innate Immune Evasion through Interference with Toll-Like Receptor Signaling

Viral infection triggers an early host response through activation of pattern recognition receptors, including Toll-like receptors (TLR). TLR signaling cascades induce production of type I interferons and proinflammatory cytokines involved in establishing an anti-viral state as well as in orchestrating ensuing adaptive immunity. To allow infection, replication, and persistence, (herpes)viruses employ ingenious strategies to evade host immunity. The human gamma-herpesvirus Epstein-Barr virus (EBV) is a large, enveloped DNA virus persistently carried by more than 90% of adults worldwide. It is the causative agent of infectious mononucleosis and is associated with several malignant tumors. EBV activates TLRs, including TLR2, TLR3, and TLR9. Interestingly, both the expression of and signaling by TLRs is attenuated during productive EBV infection. Ubiquitination plays an important role in regulating TLR signaling and is controlled by ubiquitin ligases and deubiquitinases (DUBs). The EBV genome encodes three proteins reported to exert in vitro deubiquitinase activity. Using active site-directed probes, we show that one of these putative DUBs, the conserved herpesvirus large tegument protein BPLF1, acts as a functional DUB in EBV-producing B cells. The BPLF1 enzyme is expressed during the late phase of lytic EBV infection and is incorporated into viral particles. The N-terminal part of the large BPLF1 protein contains the catalytic site for DUB activity and suppresses TLR-mediated activation of NF-κB at, or downstream of, the TRAF6 signaling intermediate. A catalytically inactive mutant of this EBV protein did not reduce NF-κB activation, indicating that DUB activity is essential for attenuating TLR signal transduction. Our combined results show that EBV employs deubiquitination of signaling intermediates in the TLR cascade as a mechanism to counteract innate anti-viral immunity of infected hosts.     

Identification of Phosphorylation Sites within the Herpes Simplex Virus Tegument Protein VP22

The herpes simplex virus protein VP22 is a major phosphoprotein of infected cells. In this study, we identify two serine phosphorylation sites within VP22 and show that the N-terminal site is a substrate for casein kinase II, while the extreme C-terminal site is a substrate for another, as yet unidentified, cellular kinase. Furthermore, we show that a mutant of VP22 which has both sites altered is unable to incorporate phosphate in vivo, confirming that there are no other phosphorylation sites within VP22.     

Vaccinia Virus Protein F12 Associates with Intracellular Enveloped Virions through an Interaction with A36

Vaccinia virus is the prototypical member of the family Poxviridae. Three morphologically distinct forms are produced during infection: intracellular mature virions (IMV), intracellular enveloped virions (IEV), and extracellular enveloped virions (EEV). Two viral proteins, F12 and A36, are found exclusively on IEV but not on IMV and EEV. Analysis of membranes from infected cells showed that F12 was only associated with membranes and is not an integral membrane protein. A yeast two-hybrid assay revealed an interaction between amino acids 351 to 458 of F12 and amino acids 91 to 111 of A36. We generated a recombinant vaccinia virus that expresses an F12, which lacks residues 351 to 458. Characterization of this recombinant revealed a small-plaque phenotype and a subsequent defect in virus release similar to a recombinant virus that had F12L deleted. In addition, F12 lacking residues 351 to 458 was unable to associate with membranes in infected cells. These results suggest that F12 associates with IEV through an interaction with A36 and that this interaction is critical for the function of F12 during viral egress.     

Dimerization-Induced Allosteric Changes of the Oxyanion-Hole Loop Activate the Pseudorabies Virus Assemblin pUL26N,a Herpesvirus Serine Protease

Herpesviruses encode a characteristic serine protease with a unique fold and an active site that comprises the unusual triad Ser-His-His. The protease is essential for viral replication and as such constitutes a promising drug target. In solution, a dynamic equilibrium exists between an inactive monomeric and an active dimeric form of the enzyme, which is believed to play a key regulatory role in the orchestration of proteolysis and capsid assembly. Currently available crystal structures of herpesvirus proteases correspond either to the dimeric state or to complexes with peptide mimetics that alter the dimerization interface. In contrast, the structure of the native monomeric state has remained elusive. Here, we present the three-dimensional structures of native monomeric, active dimeric, and diisopropyl fluorophosphate-inhibited dimeric protease derived from pseudorabies virus, an alphaherpesvirus of swine. These structures, solved by X-ray crystallography to respective resolutions of 2.05, 2.10 and 2.03 Å, allow a direct comparison of the main conformational states of the protease. In the dimeric form, a functional oxyanion hole is formed by a loop of 10 amino-acid residues encompassing two consecutive arginine residues (Arg136 and Arg137); both are strictly conserved throughout the herpesviruses. In the monomeric form, the top of the loop is shifted by approximately 11 Å, resulting in a complete disruption of the oxyanion hole and loss of activity. The dimerization-induced allosteric changes described here form the physical basis for the concentration-dependent activation of the protease, which is essential for proper virus replication. Small-angle X-ray scattering experiments confirmed a concentration-dependent equilibrium of monomeric and dimeric protease in solution.     

Herpesvirus Tegument Protein pUL37 Interacts with Dystonin/BPAG1 To Promote Capsid Transport on Microtubules during Egress

Herpes simplex virus 1 (HSV-1) is a neurotropic virus that travels long distances through cells using the microtubule network. Its 125-nm-diameter capsid is a large cargo which efficiently recruits molecular motors for movement. Upon entry, capsids reach the centrosome by minus-end-directed transport. From there, they are believed to reach the nucleus by plus-end-directed transport. Plus-end-directed transport is also important during egress, when capsids leave the nucleus to reach the site of envelopment in the cytoplasm. Although capsid interactions with dynein and kinesins have been described in vitro, the actual composition of the cellular machinery recruited by herpesviruses for capsid transport in infected cells remains unknown. Here, we identify the spectraplakin protein, dystonin/BPAG1, an important cytoskeleton cross-linker involved in microtubule-based transport, as a binding partner of the HSV-1 protein pUL37, which has been implicated in capsid transport. Viral replication is delayed in dystonin-depleted cells, and, using video microscopy of living infected cells, we show that dystonin depletion strongly inhibits capsid movement in the cytoplasm during egress. This study provides new insights into the cellular requirements for HSV-1 capsid transport and identifies dystonin as a nonmotor protein part of the transport machinery.     

Role of Envelope Protein gE Endocytosis in the Pseudorabies Virus Life Cycle

Several groups have reported that certain herpesvirus envelope proteins do not remain on the surface of cells that express them but rather are internalized by endocytosis in a recycling process. The biological function of membrane protein endocytosis in the virus life cycle remains a matter of speculation and debate. In this report, we demonstrate that some, but not all, membrane proteins encoded by the alphaherpesvirus pseudorabies virus (PRV) are internalized after reaching the plasma membrane. Glycoproteins gE and gB are internalized from the plasma membrane of cells, while gI and gC are not internalized efficiently. We show for gE that the cytoplasmic domain of the protein is required for endocytosis. While the gI protein is incapable of endocytosis on its own, it can be internalized when complexed with gE. We demonstrate that endocytosis of the gE-gI complex and gB occurs early after infection of tissue culture cells but that this process stops completely after 6 h of infection, a time that correlates with significant shutoff of host protein synthesis. We also show that gE protein internalized at 4 h postinfection is not present in virions formed at a later time. We discuss the differences in PRV gE and gI endocytosis compared to that of the varicella-zoster virus homologs and the possible roles of glycoprotein endocytosis in the virus life cycle.