The α-TIF (VP16) Homologue (ETIF) of Equine Herpesvirus 1 Is Essential for Secondary Envelopment and Virus Egress

me53 is an immediate-early/late gene found in all lepidopteran baculoviruses sequenced to date. Deletion of me53 results in a greater-than-1,000-fold reduction in budded-virus production in tissue culture (J. de Jong, B. M. Arif, D. A. Theilmann, and P. J. Krell, J. Virol. 83:7440-7448, 2009). We investigated the localization of ME53 using an ME53 construct fused to green fluorescent protein (GFP). ME53:GFP adopted a primarily cytoplasmic distribution at early times postinfection and a primarily nuclear distribution at late times postinfection. Additionally, at late times ME53:GFP formed distinct foci at the cell periphery. These foci colocalized with the major envelope fusion protein GP64 and frequently with VP39 capsid protein, suggesting that these cell membrane regions may represent viral budding sites. Deletion of vp39 did not influence the distribution of ME53:GFP; however, deletion of gp64 abolished ME53:GFP foci at the cell periphery, implying an association between ME53 and GP64. Despite the association of ME53 and GP64, ME53 fractionated with the nucleocapsid only after budded-virus fractionation. Together these findings suggest that ME53 may be providing a scaffold that bridges the viral envelope and nucleocapsid.Autographa californica multiple nucleopolyhedrovirus (AcMNPV), a member of the Baculoviridae, is the type species for the lepidopteran baculovirus genus Alphabaculovirus. A hallmark of lepidopteran baculoviruses is the production of two morphologically distinct virion phenotypes, occlusion-derived virus (ODV) and budded virus (BV). ODVs are responsible for horizontal insect-to-insect transmission of virus, whereas BVs establish a systemic infection within a susceptible host. Budded virus can enter a wide variety of cell types, including mammalian cells (22,25). Entry of BVs into insect cells involves clathrin-mediated endocytosis, and release of budded-virus nucleocapsids from endosomes is pH-dependent (3, 39). However, evidence for entry into mammalian cells supports both clathrin-dependent and clathrin-independent pathways (22,25). Once free of the endosome, nucleocapsids associate with Factin, which facilitates transport to the nucleus (4, 5, 23). After nuclear entry, viral early transcription, DNA replication, and late transcription occur in a cascade fashion. At late times, de novo nucleocapsids are transported from the nucleus to the plasma membrane, where they acquire a lipid envelope and associated viral proteins and bud from the cell, forming BVs. During the very late phase of infection, progeny nucleocapsids are retained in the nucleus, become enveloped, and are embedded into occlusion bodies (OBs).me53 is a conserved gene found in all lepidopteran alphaand betabaculoviruses sequenced to date. me53 is transcribed from a dual early/late promoter and was originally identified as a major early transcript (19,20). In contrast to a report that deletion of me53 resulted in complete abrogation of BV production and DNA replication (45), we have shown that deletion of me53 did not abrogate B...

Section: Discussionmentioning

confidence: 99%

Immediate-Early Protein ME53 Forms Foci and Colocalizes with GP64 and the Major Capsid Protein VP39 at the Cell Membranes of Autographa californica Multiple Nucleopolyhedrovirus-Infected Cells

Jong

Theilmann

Arif

et al. 2011

“…Understanding the mechanism of ORF52's function(s) requires a better understanding of the protein's interactions with other viral proteins and cellular machinery involved in the formation and release of virions from the infected cell. Studies have identified alphaherpesvirus tegument proteins that are specifically required for virion morphogenesis in the cytoplasm, such as pseudorabies virus UL37 (25) and UL48 in both pseudorabies virus and HSV-1 (16,49). Undoubtedly, other tegument proteins are also involved in these processes.…”

Section: Discussionmentioning

confidence: 99%

Murine Gammaherpesvirus 68 ORF52 Encodes a Tegument Protein Required for Virion Morphogenesis in the Cytoplasm

Bortz¹,

Jia²,

Wu³

et al. 2007

The tegument, a semiordered matrix of proteins overlying the nucleocapsid and underlying the virion envelope, in viruses in the gamma subfamily of Herpesviridae is poorly understood. Murine gammaherpesvirus 68 (MHV-68) is a robust model for studying gammaherpesvirus virion structure, assembly, and composition, as MHV-68 efficiently completes the lytic phase and productively infects cultured cells. We have found that MHV-68 ORF52 encodes an abundant tegument protein conserved among gammaherpesviruses. Detergent sensitivity experiments revealed that the MHV-68 ORF52 protein is more tightly bound to the virion nucleocapsid than the ORF45 tegument protein but could be dissociated from particles that retained the ORF65 small capsomer protein. ORF52, tagged with enhanced green fluorescent protein or FLAG epitope, localized to the cytoplasm. A recombinant MHV-68 bacterial artificial chromosome mutant with a nonsense mutation incorporated into ORF52 exhibited viral DNA replication, expression of late lytic genes, and capsid assembly and packaging at levels near those of the wild type. However, the MHV-68 ORF52-null virus was deficient in the assembly and release of infectious virion particles. Instead, partially tegumented capsids produced by the ORF52-null mutant accumulated in the cytoplasm, containing conserved capsid proteins, the ORF64 and ORF67 tegument proteins, but virtually no ORF45 tegument protein. Thus, ORF52 is essential for the tegumentation and egress of infectious MHV-68 particles in the cytoplasm, suggesting an important conserved function in gammaherpesvirus virion morphogenesis.Virion morphogenesis among the herpesviruses is a multistep process. Nucleocapsid assembly and packaging of the viral DNA occurs in the nucleus, and nascent nucleocapsids are thought to bud into the cytoplasm in an envelopment/de-envelopment process through the nuclear inner and outer envelopes (29). The nucleocapsids are transported, in association with primary tegument proteins, through the cytoplasm to a distinct site of virion assembly. Transmission electron microscopy (TEM) studies have indicated that the major tegumentation and envelopment process occurs here, as the nascent nucleocapsids associate with electrondense tegument/glycoprotein densities conjunct to Golgi apparatus-derived vesicular membranes (29,30,46). Tegumentation results in a "wrapping" of the nascent nucleocapsid and budding into the lumen of the cytoplasmic compartments, forming nearly complete virions within the lumen. These particles then egress in a manner resembling exocytosis, with fusion of the vesicular and plasma membranes and release of virions into the extracellular space. As different tegument proteins are seemingly involved in a number of steps post-nucleocapsid assembly, identifying the role of a particular tegument protein in virion morphogenesis necessarily involves study of when and where during the egress process the protein associates with nascent virions. While the nuclear steps are thought to involve conserved tegument proteins, such ...

“…For example, both glycoprotein E (gE) and gI, which form a noncovalently linked complex in infected cells and virions, are essential for the growth of both viruses, at least in certain cell types, while they are dispensable in all other alphaherpesviruses tested (3,8,25,37,42,50,63,65). In contrast, the major viral transactivator encoded by VZV ORF10 (MDV UL48) is dispensable for the growth of either virus, but is essential for most related viruses (17,19,20,58,61). In this context, it must be noted that the MDV UL49 product, the ORF9 homologue of VZV, was shown to be absolutely required for the growth of the chicken virus.…”

Section: Varicella-zoster Virus (Vzv)mentioning

confidence: 99%

A Self-Excisable Infectious Bacterial Artificial Chromosome Clone of Varicella-Zoster Virus Allows Analysis of the Essential Tegument Protein Encoded by ORF9

Tischer

Kaufer

Sommer

et al. 2007

Self Cite

115

150

In order to facilitate the generation of mutant viruses of varicella-zoster virus (VZV), the agent causing varicella (chicken pox) and herpes zoster (shingles), we generated a full-length infectious bacterial artificial chromosome (BAC) clone of the P-Oka strain. First, mini-F sequences were inserted into a preexisting VZV cosmid, and the SuperCos replicon was removed. Subsequently, mini-F-containing recombinant virus was generated from overlapping cosmid clones, and full-length VZV DNA recovered from the recombinant virus was established in Escherichia coli as an infectious BAC. An inverted duplication of VZV genomic sequences within the mini-F replicon resulted in markerless excision of vector sequences upon virus reconstitution in eukaryotic cells. Using the novel tool, the role in VZV replication of the major tegument protein encoded by ORF9 was investigated. A markerless point mutation introduced in the start codon by two-step en passant Red mutagenesis abrogated ORF9 expression and resulted in a dramatic growth defect that was not observed in a revertant virus. The essential nature of ORF9 for VZV replication was ultimately confirmed by restoration of the growth of the ORF9-deficient mutant virus using trans-complementation via baculovirus-mediated gene transfer. Varicella-zoster virus (VZV), the causative agent of chicken pox (varicella) and shingles (herpes zoster), is a highly cellassociated herpesvirus both in vitro and in vivo (7, 23). Similar to the situation with a closely related alphaherpesvirus, Marek's disease virus (MDV), VZV spreads directly from cell to cell, presumably utilizing the machinery involved in adherens junction modeling and architecture. Infectious virus is released into the environment only after lytic VZV replication in the skin and respiratory mucous membranes of infected individuals (2, 43). Efficient and timely coordinated interaction between the tegument, a proteinaceous structure surrounding the icosahedral nucleocapsid, and envelope membrane (glyco) proteins plays a crucial role in the secondary envelopment and spread of herpesviruses in general and VZV in particular (35). It has been shown for related viruses, such as herpes simplex virus, that the inner layer of tegument is tightly associated with the nucleocapsid, whereas the outer layer of tegument provides the link to envelope proteins (34, 36). The current model of herpesviral tegumentation predicts that the inner layer of tegument is added to the nucleocapsid in the nucleus and deenveloped particles in the cytoplasm, while proteins representing the outer layer are thought to accumulate at cytoplasmic membranes where they make contact with their respective partners, either other tegument proteins or membrane proteins or both (34-36). Through these intricately regulated interactions, final secondary envelopment of particles is facilitated and, ultimately, infectious virus is produced and released.Determination of the role of individual tegument or membrane (glyco)proteins in the replication cycle of various herpesviru...