Reconstitution of the Kaposi's Sarcoma-Associated Herpesvirus Nuclear Egress Complex and Formation of Nuclear Membrane Vesicles by Coexpression of ORF67 and ORF69 Gene Products

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All herpesviruses encode a complex of two proteins, referred to as the nuclear egress complex (NEC), which together facilitate the exit of assembled capsids from the nucleus. Previously, we showed that the Kaposi's sarcoma-associated herpesvirus (KSHV) NEC specified by the ORF67 and ORF69 genes when expressed in insect cells using baculoviruses for protein expression forms a complex at the nuclear membrane and remodels these membranes to generate nuclear membrane-derived vesicles. In this study, we have analyzed the functional domains of the KSHV NEC proteins and their interactions. Site-directed mutagenesis of gammaherpesvirus conserved residues revealed functional domains of these two proteins, which in many cases abolish the formation of the NEC and remodeling of nuclear membranes. Small in-frame deletions within ORF67 in all cases result in loss of the ability of the mutant protein to induce cellular membrane proliferation as well as to interact with ORF69. Truncation of the C terminus of ORF67 that resides in the perinuclear space does not impair the functions of ORF67; however, deletion of the transmembrane domain of ORF67 produces a protein that cannot induce membrane proliferation but can still interact with ORF69 in the nucleus and can be tethered to the nuclear membrane by virtue of its interaction with the wild-type-membrane-anchored ORF67. In-frame deletions in ORF69 have varied effects on NEC formation, but all abolish remodeling of nuclear membranes into circular structures. One mutant interacts with ORF67 as well as the wild-type protein but cannot function in membrane curvature and fission events that generate circular vesicles. These studies genetically confirm that ORF67 is required for cellular membrane proliferation and that ORF69 is the factor required to remodel these duplicated membranes into circular-virion-size vesicles. Furthermore, we also investigated the NEC encoded by Epstein-Barr virus (EBV). The EBV complex comprised of BFRF1 and BFLF2 was visualized at the nuclear membrane using autofluorescent protein fusions. BFRF1 is a potent inducer of membrane proliferation; however, BFLF2 cannot remodel these membranes into circular structures. What was evident is the superior remodeling activity of ORF69, which could convert the host membrane proliferations induced by BFRF1 into circular structures.

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confidence: 52%

Interactions of the Kaposi's Sarcoma-Associated Herpesvirus Nuclear Egress Complex: ORF69 Is a Potent Factor for Remodeling Cellular Membranes

Luitweiler

Henson

Pryce

et al. 2013

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“…Although lytic events and distortions of nuclear pores have been documented, the nuclear release of these viruses appears to predominantly occur by a budding mechanism (34,41,76). In support of the budding egress model, cooverexpression of nuclear egress proteins pUL31 and pUL34 results in the accumulation of vesicles between the inner and outer nuclear membranes, which may reflect an activity associated with budding during infection (21,32). …”

Section: Discussionmentioning

confidence: 88%

Nuclear Egress of Pseudorabies Virus Capsids Is Enhanced by a Subspecies of the Large Tegument Protein That Is Lost upon Cytoplasmic Maturation

Leelawong

Lee

Smith

2012

Herpesviruses morphogenesis occurs stepwise both temporally and spatially, beginning in the nucleus and concluding with the emergence of an extracellular virion. The mechanisms by which these viruses interact with and penetrate the nuclear envelope and subsequent compartments of the secretory pathway remain poorly defined. In this report, a conserved viral protein (VP1/2; pUL36) that directs cytoplasmic stages of egress is identified to have multiple isoforms. Of these, a novel truncated VP1/2 species translocates to the nucleus and assists the transfer of DNA-containing capsids to the cytoplasm. The capsids are handed off to full-length VP1/2, which replaces the nuclear isoform on the capsids and is required for the final cytoplasmic stages of viral particle maturation. These results document that distinct VP1/2 protein species serve as effectors of nuclear and cytoplasmic egress.

“…In the absence of either UL31 or UL34, viral replication is impaired and most capsids are retained in the nucleus (6, 7). The NEC is also sufficient to drive the formation of perinuclear vesicles in transfected cells (8,9), which demonstrated that UL31 and UL34 are the only viral proteins necessary for vesiculation. But these experiments left the function of the NEC in membrane budding unclear.…”

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confidence: 98%

The Great (Nuclear) Escape: New Insights into the Role of the Nuclear Egress Complex of Herpesviruses

Bigalke

Heldwein

2015

Herpesviruses are unusual among enveloped viruses because they bud twice yet acquire a single envelope. They are also the only known viruses that bud into the nuclear envelope. We discovered that the herpesvirus nuclear egress complex could bud membranes without the help of other proteins by forming a coat-like hexagonal scaffold inside the budding membrane. This finding raises the possibility that a phenotypically similar nuclear export of large RNAs is cargo driven. Most enveloped viruses acquire their envelopes by capsid budding into a cellular membrane. Some viruses, such as HIV or influenza virus, bud into the plasma membrane while other viruses, such as flaviviruses, bud into intracellular membranes such as the endoplasmic reticulum, the Golgi compartment, or others, depending on the virus. Herpesviruses represent an unusual case. Despite having a single envelope, these double-stranded DNA viruses undergo two rounds of budding; they bud first into the inner nuclear membrane (INM) and later into cytoplasmic membranes derived from the trans-Golgi network or early endosomes (1). This also makes them the only known viruses to use the nuclear membrane for budding. Yet, the envelope acquired during the first budding event at the INM does not end up in the mature viral particle. Only the second and final round of budding in the cytosol generates the single-layer envelope of the mature virus. Instead, the unusual nuclear budding allows the viral capsid to escape from the nucleus.Herpesvirus genomes are replicated and encapsidated in the nucleus. The nucleus is surrounded by the INM and outer nuclear membrane (ONM), and most traffic in and out of the nucleus occurs through the nuclear pores. The diameter of herpesvirus capsids (125 nm in HSV-1) is considerably larger than that of the nuclear pore channels (52 nm), and thus, the capsids cannot fit through them. To escape from the nucleus, nucleocapsids bud into the INM, forming perinuclear immature viral particlesintermediates that are different from the mature, infectious viruses-that then fuse with the ONM, releasing the naked capsids into the cytosol (Fig. 1). As a result of this process, termed nuclear egress, nucleocapsids are translocated from the nucleus into the cytoplasm, where they mature into final, infectious virions.Efficient exit of nascent capsids from the nucleus requires the virus-encoded nuclear egress complex (NEC) (reviewed in reference 1). The NEC consists of conserved viral proteins UL31 and UL34. UL34 is anchored to the INM by a C-terminal transmembrane (TM) helix with three residues extending into the perinuclear space (3). UL31 is a nuclear phosphoprotein that localizes to the INM through interaction with UL34 (4, 5). Formation of the NEC is a prerequisite for efficient nuclear egress. In the absence of either UL31 or UL34, viral replication is impaired and most capsids are retained in the nucleus (6, 7). The NEC is also sufficient to drive the formation of perinuclear vesicles in transfected cells (8,9), which demonstrated that UL31 and UL3...