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Research Article
26 November 2019

Functional Identification and Characterization of the Nuclear Egress Complex of a Gammaherpesvirus

ABSTRACT

The herpesvirus nuclear egress complex (NEC) is composed of two viral proteins. They play key roles in mediating the translocation of capsids from the nucleus to the cytoplasm by facilitating the budding of capsids into the perinuclear space (PNS). The NEC of alphaherpesvirus can induce the formation of virion-like vesicles from the nuclear membrane in the absence of other viral proteins. However, whether the NEC of gammaherpesvirus harbors the ability to do so in mammalian cells remains to be determined. In this study, we first constructed open reading frame 67 (ORF67)-null and ORF69-null mutants of murine gammaherpesvirus 68 (MHV-68) and demonstrated that both ORF67 and ORF69 play critical roles in nuclear egress and hence viral lytic replication. Biochemical and bioimaging analyses showed that ORF67 and ORF69 interacted with each other and were sufficient to induce the formation of virion-like vesicles from the nuclear membrane in mammalian cells. Thus, we designated ORF67 and ORF69 components of MHV-68 NEC. Furthermore, we identified amino acids critical for mediating the interaction between ORF67 and ORF69 through homology modeling and verified their function in nuclear egress, providing insights into the molecular basis of NEC formation in gammaherpesviruses.
IMPORTANCE Increasing amounts of knowledge indicate that the nuclear egress complex (NEC) is critical for the nuclear egress of herpesvirus capsids, which can be viewed as a vesicle-mediated transport pathway through the nuclear membrane. In this study, we identified open reading frame 67 (ORF67) and ORF69 as components of the NEC in murine gammaherpesvirus 68 (MHV-68) and demonstrated that they efficiently induce virion-like vesicles from the nuclear membrane in mammalian cells. This is the first time that the NEC of a gammaherpesvirus has been found to demonstrate such an essential characteristic. In addition, we identified amino acids critical for mediating the interaction between ORF67 and ORF69 as well as nuclear egress. Notably, these amino acids are conserved in Kaposi’s sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV), providing a structural basis to design antigammaherpesvirus drugs.

INTRODUCTION

Herpesviruses are large enveloped viruses which exist widely in nature. A mature herpesvirus particle consists of a double-stranded DNA genome, a capsid enclosing the genome, tegument, and an envelope, from the inside out. During viral lytic replication, nucleocapsids are first assembled in the nucleus and then transported to the cytoplasm for the subsequent morphogenesis process. The nucleocapsid is about 120 nm in diameter, too large to be transported to the cytoplasm through the nuclear pore. Instead, it completes nuclear export through a unique process called nuclear egress. In this process, the nucleocapsid first buds at the inner nuclear membrane to form a primary virion in the perinuclear space (PNS), and then the envelope of the primary virion fuses with the outer nuclear membrane, releasing the nucleocapsid into the cytoplasm (13).
Nuclear egress can be viewed as a vesicle-mediated transport of cargo through the nuclear membrane, similar to the nuclear export pathway of large ribonucleoprotein complexes that was identified in Drosophila (1, 4). Two viral proteins, UL34 and UL31 in alphaherpesviruses (herpes simplex virus [HSV] and pseudorabies virus [PrV]) or their homologues in betaherpesviruses (UL50 and UL53 in human cytomegalovirus [HCMV]; M50 and M53 in murine cytomegalovirus [MCMV]), play key roles in mediating this process (58) and are designated the nuclear egress complex (NEC). Mechanistically, coexpression of the NEC from PrV is sufficient to induce the formation of virion-like vesicles from the inner nuclear membrane in mammalian cells (9). Recently, it was shown that HSV-1 NEC or artificial membrane tethering of PrV UL31 alone mediates budding and scission of vesicles from synthetic membranes in vitro (10, 11). In contrast, the mechanisms underlying the nuclear egress of gammaherpesviruses were much less characterized. In Epstein-Barr virus (EBV), knocking out BFRF1 or BFLF2 (homologues of UL34 and UL31, respectively, in alphaherpesviruses) from the viral genome resulted in the reduction of viral titers, which was shown to be caused by the nuclear sequestration of capsids (12, 13). In HeLa cells, exogenous BFRF1 recruited cellular endosomal sorting complex required for transport (ESCRT) machinery to induce nuclear envelope-derived cytoplasmic vesicles with a diameter of 1.64 ± 0.42 μm, which are much bigger than virions (14, 15). In Kaposi’s sarcoma-associated herpesvirus (KSHV), coexpression of open reading frame 67 (ORF67) and ORF69 (homologues of UL34 and UL31, respectively, in alphaherpesviruses) induced nuclear membrane deformation and vesicle formation in insect cells but not in mammalian cells (16, 17). Therefore, it is unclear whether NECs of gammaherpesviruses that can induce virion-like vesicles from the nuclear membrane in mammalian cells exist. Furthermore, the definitive role of the NEC in the lytic replication of most gammaherpesviruses remains to be functionally demonstrated.
Murine gammaherpesvirus 68 (MHV-68) is a natural parasite of murid rodents. It infects and replicates efficiently in many laboratory cell lines, providing an excellent tractable model to study the lytic replication of gammaherpesviruses (18). We and others have previously observed dramatic deformation of nuclear membranes during MHV-68 replication (19, 20), but the viral protein(s) responsible for this phenomenon has not been determined. The sequence homologues of the NEC in MHV-68 are ORF67 and ORF69 (21). Interaction between these two proteins was reported in a genome-wide yeast two-hybrid screening study which mapped the protein interaction network of MHV-68 (22). We therefore aimed to investigate whether ORF67 and ORF69 work together as MHV-68 NEC and whether coexpression of them is sufficient to deform the nuclear membrane and produce virion-like vesicles in mammalian cells.
In this study, we first identified ORF67 and ORF69 as the NEC of MHV-68. Lack of ORF67 or ORF69 expression during MHV-68 lytic replication resulted in the accumulation of capsids in the nucleus. ORF67 and ORF69 interacted with each other in the absence of other viral proteins, colocalized at the inner nuclear membrane, and induced the formation of virion-like vesicles in mammalian cells. Furthermore, we predicted the structure of MHV-68 NEC through homology modeling and identified amino acids which are critical for mediating the interaction between ORF67 and ORF69, providing insights into the molecular basis of NEC formation in gammaherpesviruses.

RESULTS

Disruption of ORF67 or ORF69 in MHV-68 BAC inhibits the production of infectious viruses.

To functionally examine the roles of ORF67 and ORF69 in viral lytic replication and especially in the nuclear egress of capsids, we first took a genetic approach. We utilized the MHV-68 bacterial artificial chromosome (BAC) system to generate an ORF67-null (67S) or ORF69-null (69S) BAC (Fig. 1A). Revertants of the 67S BAC (67S-R) and 69S BAC (69S-R) were also generated in order to verify that the phenotypes observed for 67S or 69S BAC were not due to unwanted changes in the viral genome. Wild-type (WT) BAC, 67S BAC, 67S-R BAC, 69S BAC, and 69S-R BAC were individually transfected into 293T cells. At 60 h posttransfection, cells transfected with WT BAC, 67S-R BAC, or 69S-R BAC showed severe cytopathic effect (CPE), but no CPE was observed in cells transfected with 67S BAC or 69S BAC (Fig. 1B). Consistently, lack of ORF67 or ORF69 expression resulted in a dramatic reduction in the number of viral genome copies in the culture supernatant (Fig. 1C).
FIG 1
FIG 1 MHV-68 ORF67 and ORF69 play critical roles in MHV-68 lytic replication. (A) Construction of an ORF67-null (67S) or an ORF69-null (69S) BAC. A schematic diagram of the ORF67 and ORF69 loci and their flanking ORFs on the WT BAC is shown. Sequences composed of triple-frame stop codons and a SmaI site were individually inserted at the indicated positions. (B) 293T cells were individually transfected with WT BAC, 67S BAC, 69S BAC, or their revertants (67S-R and 69S-R, respectively). At 60 h posttransfection, CPEs were detected with an optical microscope. (C) Viral DNA was extracted from the cell culture supernatants from cells shown in panel B, and viral genome copies were examined by real-time PCR. The results are representative of three independent experiments. ***, P < 0.001. (D) 293T cells were individually transfected with different BACs as described for panel B. At 26 h or 40 h posttransfection, cells were analyzed by indirect immunofluorescence assay with a confocal microscope (Olympus FV1200). ORF65 was stained using anti-ORF65 polyclonal antibody and Alexa Fluor 488-conjugated secondary antibody (green). Nuclei were stained with DAPI (blue).
To further examine these two proteins’ roles in viral infection, we next conducted an immunofluorescence assay to follow the propagation of virus in the absence of ORF67 or ORF69 protein by staining for a late gene product, the small capsid protein ORF65. At 26 h posttransfection, the first round of progeny viruses produced by transfected BACs were released and began to infect nearby cells. At this time, late genes were expressed in cells transfected with BACs but not yet in cells infected with progeny viruses produced by transfected cells. Because of the low transfection efficiency for BAC DNA, fluorescence signals were observed only as scattered dots (Fig. 1D, second left column). At 40 h, viral late genes were expressed in newly infected cells, and many large agglomerates of fluorescence signals were detected in samples transfected with WT BAC or revertants; in contrast, in cells transfected with 67S or 69S BAC, most fluorescence signals were still in an isolated fashion, indicating that few virions were produced and spread by 67S or 69S BAC (Fig. 1D, right column). Similar results were obtained when staining for a tegument protein ORF33 (data not shown). These results demonstrated that both ORF67 and ORF69 played critical roles in viral lytic replication.

ORF67 and ORF69 play key roles in nuclear egress.

To test which specific stage of viral lytic replication was impaired in 67S- or 69S-transfected cells, we analyzed their virion morphogenesis process through thin-section transmission electron microscopy (TEM). In cells transfected with WT BAC, virion morphogenesis took place normally, and viral particles in the cytoplasm and mature virions in the extracellular space were frequently detected (Fig. 2A). However, in cells transfected with 67S or 69S BAC, a large amount of capsids accumulated in the nucleus, and viral particles were hardly observed in the cytoplasm or extracellular space (Fig. 2B to E). These data demonstrated that both ORF67 and ORF69 were critical for the nuclear egress of capsids.
FIG 2
FIG 2 Both ORF67 and ORF69 play critical roles in nuclear egress. (A to E) 293T cells were individually transfected with WT BAC, 67S BAC, or 69S BAC. Approximately 70-nm thin sections were prepared at 74 h posttransfection and examined with a 120-kV transmission electron microscope. Putative A capsids, B capsids, and C capsids in the nucleus (indicated as a, b, or c), viral particles in the cytoplasm (indicated with the arrowhead), and extracellular virions associated with the cell surface (indicated with the arrow) were observed in cells transfected with WT BAC. Insets in panels B and D are magnified in C and E, respectively, showing the accumulation of capsids in 67S BAC- or 69S BAC-transfected cells. Nu, nucleus; NM, nuclear membrane; Cyto, cytoplasm.

ORF67 and ORF69 interact with each other in vivo and directly in vitro.

If ORF67 and ORF69 function as an NEC in MHV-68, they should form complexes with each other. To detect whether ORF67 and ORF69 interact with each other during MHV-68 infection, we inserted a FLAG tag sequence at the N terminus of ORF67 in the MHV-68 genome, constructing a FLAG-ORF67 BAC which produced infectious virions normally (data not shown). In cells infected with this FLAG-ORF67 virus, ORF69 was pulled down by ORF67 (Fig. 3A). Interaction between ORF67 and ORF69 was also detected when they were coexpressed in cells by transfection, indicating that no other viral protein is required for their interaction (Fig. 3B). Furthermore, when ORF67 and ORF69 were expressed by a prokaryotic expression system and purified in vitro, a gel filtration assay demonstrated that they directly interacted with each other and formed heterodimers (Fig. 3C).
FIG 3
FIG 3 ORF67 and ORF69 interact with each other in vivo and in vitro. (A) BHK-21 cells were infected with WT or Flag-ORF67 viruses. At 48 h postinfection, cells were harvested and lysates were immunoprecipitated with anti-FLAG M2 agarose. Samples were analyzed by Western blotting (WB) with rabbit anti-ORF67 antibody and rabbit anti-ORF69 antibody. (B) 293T cells were cotransfected with an HA-tagged ORF67 plasmid plus a FLAG-tagged ORF69 plasmid or a control vector. At 48 h posttransfection, cells lysates were immunoprecipitated with anti-FLAG M2 agarose, and samples were analyzed by WB with anti-FLAG antibody or anti-HA antibody. (C) Gel filtration assay of ORF67Δ40 and ORF69Δ45 complex. ORF67Δ40 and ORF69Δ45 proteins purified by Ni-NTA column were loaded on the Column Superdex 200 10/300 GL (GE Healthcare). The horizontal axis indicates the elution volume of proteins, and the vertical axis indicates the absorbance of the sample at the 280 nm wavelength, reflecting the amount of proteins. The elution volumes of 35 kDa and 67 kDa are marked in the diagram. Protein components at different elution volumes were subjected to SDS-PAGE and stained with Coomassie brilliant blue.

ORF67 and ORF69 colocalize during transfection and MHV-68 infection.

Sequence analysis suggested that the C terminus of ORF67 contains a transmembrane domain and the N terminus of ORF69 contains two nuclear localization signals. Thus, ORF67 is expected to be membrane associated, and ORF69 is expected to be located in the nucleus. To experimentally examine the subcellular localization of ORF67 and ORF69, 293A cells were transfected with plasmids expressing each or both proteins and subjected to immunofluorescence assays. Indeed, ORF67 by itself was located at the nuclear rim as well as in the cytoplasmic area adjacent to the nucleus, whereas ORF69 alone was distributed diffusely in the nucleoplasm (Fig. 4A, first and second rows). When they were coexpressed, ORF67 and ORF69 displayed good colocalization. In some cells, they were distributed smoothly at the nuclear rim, and in others, they seemed to form speckles at the nuclear rim or in the nucleoplasm (Fig. 4A, third and fourth rows), which demonstrated mutual relocalization of ORF67 and ORF69.
FIG 4
FIG 4 Localization of ORF67 and ORF69 during transfection and infection. (A) 293A cells were transfected with the HA-tagged ORF67 or FLAG-tagged ORF69 plasmid or both. At 24 h posttransfection, ORF67 was stained using mouse anti-HA antibody followed by Alexa Fluor 555-conjugated secondary antibody (red), and ORF69 was stained using rabbit anti-FLAG antibody followed by Alexa Fluor 488-conjugated secondary antibody (green). Nuclei were stained with DAPI (blue). (B) WT MHV-68-infected BHK-21 cells were stained with rabbit anti-ORF67 (top) or anti-ORF69 antibody (bottom), followed by Alexa Fluor 488-conjugated secondary antibody (green). (C) BHK-21 cells were infected with FLAG-ORF67 viruses. At 24 h postinfection, ORF67 was stained using mouse anti-FLAG antibody followed by Alexa Fluor 555-conjugated secondary antibody (red), and ORF69 was stained using rabbit anti-ORF69 antibody followed by Alexa Fluor 488-conjugated secondary antibody (green).
To examine the localization of ORF67 and ORF69 during MHV-68 infection, cells were infected and stained using either a rabbit anti-ORF67 antibody or a rabbit anti-ORF69 antibody. The result showed that they were both located at the nuclear rim (Fig. 4B). To further test whether they colocalize during viral infection, we infected cells with the FLAG-ORF67 virus, and ORF67 and ORF69 were stained with a mouse anti-FLAG monoclonal antibody and the rabbit anti-ORF69 polyclonal antibody, respectively. As shown in Fig. 4C, endogenously expressed ORF67 and ORF69 colocalized at the nuclear rim.

ORF67 and ORF69 induce virion-like vesicles from the nuclear membrane in mammalian cells.

Formation of primary virions within the perinuclear space requires the NEC to induce vesicles from the inner nuclear membrane. Thus, we speculated that the speckled distribution of ORF67 and ORF69 shown in Fig. 4A may be caused by vesicles formed by themselves. We therefore used three-dimensional structured illumination microscopy (3D-SIM), a method that provides high-resolution three-dimensional images by breaking the diffraction limit of light, to observe speckled structures. In the experiment, the inner nuclear membrane was labeled with an anti-lamin A/C antibody, and because ORF69 was fused with a FLAG tag, the speckled structure was examined with an anti-FLAG-tag antibody. In control cells, lamin A/C was distributed diffusely in the nucleus when stack 3D-SIM (1.875 μm) was performed and the 3D data sets were projected into a two-dimensional (2D) image (Fig. 5A, first row). However, it was mainly located at the nuclear rim in the images of single slices (125 nm) (Fig. 5A, second row) or the reconstructed cross-sectional image of Z slices (see Movie S1 in the supplemental material). In cells coexpressing ORF67 and ORF69, lamin A/C formed small circles which wrapped speckles labeled with ORF69 (Fig. 5A, third and fourth rows). In the single slices or the cross-sectional Z slices, signals of lamin A/C and ORF69 were mainly distributed at the nuclear rim (Fig. 5B and C; see Movie S2). These observations supported our hypothesis that these speckles were vesicles induced by ORF67 and ORF69 at the inner nuclear membrane. In confocal microscopy or stack 3D-SIM imaging, vesicles located on different focal planes were projected onto the same plane, causing the illusion that the vesicles were distributed in the nucleoplasm.
FIG 5
FIG 5 ORF67 and ORF69 form speckles at the inner nuclear membrane. (A) HeLa cells cotransfected with HA-tagged ORF67 and FLAG-tagged ORF69 plasmids or control vectors were fixed at 24 h posttransfection. Cells were stained with mouse anti-lamin A/C antibody and rabbit anti-FLAG antibody simultaneously and imaged by 3D-SIM. For each cell, maximum-intensity projections from fifteen consecutive slices (125 nm each slice) and an image of one Z slice (z = 8) are shown. (B) Enlarged images of the insets in panel A. (C) Cross-sectional image of Z slices from the cell cotransfected with ORF67 and ORF69 in panel A. Bars, 5 μm.
We next directly investigated the ability of ORF67 and ORF69 to deform the nuclear membrane by ultrastructural analysis. 293T cells were transfected with plasmid(s) expressing ORF67 or ORF69 or both and fixed for ultrathin sectioning and TEM at 48 h posttransfection. Expression of ORF67 alone caused the proliferation of the nuclear membrane, altering the bilayer nuclear membrane into a multilayer structure (Fig. 6A to C). Multilayer membranous structures were also observed in the cytoplasm (Fig. 6A and C). Localization of ORF67 to the nuclear membrane and to the membranous structure in the cytoplasm was likely caused by its transmembrane domain, which mediated the subsequent proliferation of the anchored membranes. In contrast, expression of ORF69 alone or the control vector did not cause obvious changes in subcellular structure (data not shown). When ORF67 and ORF69 were simultaneously expressed, numerous vesicles were detected in the PNS or in invaginations from the inner nuclear membrane, but the proliferation of membranes was barely observed. Most vesicles were between 100 nm and 200 nm in diameter, similar to that of the primary enveloped virions (Fig. 6D to F). Notably, the budding process at the inner nuclear membrane was captured (Fig. 6F, white arrow), strongly indicating the origin of these vesicles. In addition, some vesicles were wrapped by membranous structures in the nucleoplasm which appeared to have no connection to the nuclear membrane (Fig. 6D and F). Considering the localization of speckles shown in Fig. 5B and C, these membranous structures were most likely invaginations of the inner nuclear membrane from different planes which showed up as nucleoplasmic in this particular TEM cross section. Collectively, these and the above-described data demonstrated that ORF67 and ORF69 together were capable of inducing virion-like vesicles from the inner nuclear membrane in mammalian cells; thus, they were functionally designated components of the NEC in MHV-68.
FIG 6
FIG 6 Membrane remodeling by ORF67 and ORF69. 293T cells were transfected with a plasmid expressing HA-ORF67 (A) or cotransfected with plasmids expressing HA-ORF67 and HA-ORF69 (D). Approximately 70-nm thin sections were prepared at 48 h posttransfection and examined by a 120-kV transmission electron microscope. (B and C) Magnified images of insets in panel A. (E and F) Magnified images of insets in panel D. Vesicle budding from the inner nuclear membrane is indicated by a white arrow in (F). Nu, nucleus; Cyto, cytoplasm. Bars, 1 μm.

Structure modeling of MHV-68 NEC.

While we were attempting to solve the crystal structure of the MHV-68 ORF67/ORF69 complex, structures of NECs from alpha- and betaherpesviruses were determined (2328), which showed high similarity to one another. To characterize NECs of gammaherpesviruses in molecular detail, we predicted the structure of MHV-68 NEC through homology modeling. We first built the structures of MHV-68 ORF67 and ORF69 separately, and assessed the global and per-residue model quality using the QMEAN (qualitative model energy analysis) scoring function by SWISS MODEL software (29, 30). Based on the QMEAN scores, we selected the HCMV NEC structure (PDB code 5D5N) as a template to build the MHV-68 ORF67 and ORF69 structures. The predicted structures were then aligned with that of HCMV NEC in PyMOL to construct the structure model of MHV-68 NEC (Fig. 7A). In this model, ORF67 and ORF69 interact with each other at a molar ratio of 1:1, with the α1-α2 helices of ORF69 extending into the cavity formed between the α3 and α5 helices of ORF67. Specifically, aromatic amino acid residues F61 and F65 on the ORF67 α3 helix interact with residues of F52, F65, and L66 on the ORF69 α1-α2 helices, creating a hydrophobic core. The M180 residue on the ORF67 α5 helix also has weak hydrophobic interaction with residues F65 and E68 on the ORF69 α2 helix (Fig. 7B). Among these amino acids, F61 and F65 of ORF67 and F52, F65, L66, and E68 of ORF69 are completely conserved in KSHV and EBV. The counterparts of ORF67 M180 in KSHV and EBV are ORF67 L181 and BFRF1 V180, respectively, which are also hydrophobic amino acids (Fig. 7C).
FIG 7
FIG 7 Structure modeling of MHV-68 NEC. (A) Alignment of the HCMV NEC structure (gray; PDB code 5D5N) with the predicted MHV-68 NEC structure (green, ORF67; orange, ORF69). (B, left) Overview of the predicted structure of MHV-68 NEC. Inset indicates the binding interface between ORF67 and ORF69. (B, right) Closeup view of the binding interface, showing predicted interacting residues. (C) Alignment of amino acid sequences of the interaction regions from ORF67 homologues (MHV-68 ORF67, KSHV ORF67, and EBV BFRF1) and ORF69 homologues (MHV-68 ORF69, KSHV ORF69, and EBV BFLF2). The secondary structure elements of MHV-68 ORF67 and ORF69 are shown at the top, with coils representing α-helices. Black dots indicate amino acids on the interaction surfaces shown in panel B.

Identification of residues critical for mediating the interaction between ORF67 and ORF69.

To experimentally verify the structure model, we made individual alanine substitutions for the predicted key amino acids on the interaction surfaces and examined their effects on ORF67-ORF69 interaction. Immunoprecipitation assay showed that the interaction between ORF67 and ORF69 was abolished by any one of the five mutations (F61A or F65A in ORF67; F52A, F65A, or L66A in ORF69), and weakened by mutation of M180 in ORF67 (Fig. 8A). Consistently, immunofluorescence assay demonstrated that any of these mutations also inhibited the relocalization of ORF69 to the nuclear membrane, indicating failure of ORF67-ORF69 interaction and hence recruitment of ORF69 to the nuclear membrane by ORF67 (Fig. 8B).
FIG 8
FIG 8 Critical sites for mediating the interaction between ORF67 and ORF69. (A) 293T cells transfected with indicated plasmids were lysed at 48 h posttransfection. Cell lysates were immunoprecipitated with anti-FLAG M2 agarose, and samples were analyzed by WB with anti-FLAG and anti-HA antibodies. (B) 293A cells transfected with indicated plasmids were fixed at 24 h posttransfection. ORF67 was stained using mouse anti-HA antibody (red), and ORF69 was stained using rabbit anti-FLAG antibody (green). Nuclei were stained with DAPI (blue).

Disruption of the interaction between ORF67 and ORF69 severely impairs the nuclear egress of MHV-68.

To further investigate the functional consequence of these mutations on viral replication and, in particular, virion morphogenesis and egress, we engineered mutations destroying ORF67-ORF69 interaction in the context of the viral genome. Among the five residues which were shown to be critical for mediating ORF67-ORF69 interaction (ORF67-F61, ORF67-F65, ORF69-F52, ORF69-F65, and ORF69-L66) (Fig. 8), we selected three and individually replaced ORF67-F61, ORF69-F52, and ORF69-L66 coding sequences with alanine codes on the viral genome, constructing 67-F61A, 69-F52A, and 69-L66A BACs, respectively (Fig. 9A). To rule out unexpected changes in viral genomes which may have resulted in the nonviability of MHV-68, we also constructed their revertants (67-F61A-R, 69-F52A-R, and 69-L66A-R). When transfected into 293T cells, these three mutant BACs all failed to cause CPE, in contrast to their revertants (Fig. 9B). Each of these mutations led to a significant reduction in viral genome copy number in culture supernatant (Fig. 9C) and also inhibited the spread of ORF65 signals to neighboring cells (Fig. 9D), indicating severe inhibition of viral lytic replication. These phenotypes caused by the defect in ORF67-ORF69 interaction were similar to that displayed by the ORF67S or ORF69S BAC (Fig. 1).
FIG 9
FIG 9 Disruption of the interaction between ORF67 and ORF69 severely impairs the production of infectious virions. (A) Construction of 67-F61A BAC, 69-F52A BAC, and 69-L66A BAC. Nucleotide sequences of each mutant BAC are listed. (B) 293T cells individually transfected with mutant or corresponding revertant BACs were imaged with an optical microscope at 60 h posttransfection. (C) Viral DNA was extracted from the cell culture supernatants from the cells shown in panel B, and viral genome copies were analyzed by real-time PCR. Results are representative of three independent experiments. ***, P < 0.001. (D) 293T cells individually transfected with indicated BACs were analyzed by indirect immunofluorescence assay as described for Fig. 1D.
We next analyzed the ultrastructural phenotype of these mutants by TEM. In WT BAC-transfected cells, viral particles at different stages of morphogenesis were observed in the nucleus, PNS, and cytoplasm, and mature virions were detected on cell surfaces. However, in cells transfected with 67-F61A, 69-F52A, or 69-L66A BAC, most capsids were retained in the nucleus and only a few viral particles were found in the PNS, in the cytoplasm or outside the cell (Fig. 10). Quantitative analysis of TEM images showed that 47.07% of viral particles were distributed in the nucleus in WT samples. In contrast, more than 90% of viral particles were observed in the nucleus in all three mutant samples, indicating a severe defect in translocation of capsids to the cytoplasm (Table 1). These results demonstrated that the interaction between ORF67 and ORF69 played a critical role in the nuclear egress.
FIG 10
FIG 10 Disruption of the interaction between ORF67 and ORF69 severely impairs nuclear egress. 293T cells were individually transfected with WT BAC, 67-F61A BAC, 69-F52A BAC, or 69-L66A BAC. Approximately 70-nm thin sections were prepared at 53 h posttransfection and examined with a 120-kV transmission electron microscope. Putative B capsids and C capsids (indicated by b or c) in the nucleus, primary enveloped virions in the perinuclear space (indicated with triangle), viral particles in the cytoplasm (indicated with arrowhead), and extracellular virions (indicated with the arrow) were observed in cells transfected with WT BAC. In cells transfected with mutant BAC, capsids mostly accumulated in the nucleus. Nu, nucleus; Cyto, cytoplasm. Bars, 500 nm.
TABLE 1
TABLE 1 Distributions of viral particles in cellular compartments
BACNo. of cellsaNo. of particlesb% of viral particles in/on:
NucleuscPerinuclear spacedCytoplasmeCell surfacef
WT652947.073.0346.693.21
67-F61A661092.30g0.167.54g0
69-F52A652593.71g0.385.72g0.19
69-L66A651595.73g04.27g0
a
Number of cells containing viral particles.
b
Total viral and subviral particles enumerated in 6 cells that contained viral particles.
c
Values are calculated as number of capsids in the nucleus/total number of viral particles.
d
Values are calculated as number of primary virions in the perinuclear space/total number of viral particles.
e
Values are calculated as number of mature or immature virions (including capsids) in the cytoplasm/total number of viral particles.
f
Values are calculated as number of mature extracellular virions/total number of viral particles.
g
P < 0.001 versus WT.

DISCUSSION

Nuclear egress of herpesviruses is complex and requires the participation of many viral and host factors. As NECs are vital for this process and mediate a novel membrane-budding machinery, they have received much attention in recent years. The ability to replicate robustly in cell culture systems makes MHV-68 an excellent system to study the lytic replication and, especially, the morphogenesis and egress process of gammaherpesviruses. However, the NEC components of MHV-68 had not yet been identified and characterized. Moreover, it remained to be investigated whether the gammaherpesvirus NEC could induce the formation of virion-like vesicles from the nuclear membrane in mammalian cells. In this study, we functionally identified ORF67 and ORF69 as the MHV-68 NEC and demonstrated, for the first time, that the NEC of a gammaherpesvirus efficiently induced virion-like vesicle formation from the nuclear membrane in mammalian cells.
Although ORF67 and ORF69 are very important for the nuclear export of herpesviral nucleocapsids, they are not essential. Deficiency in ORF67 or ORF69 expression reduced the number of viral genome copies in culture supernatants more than 1,000-fold (Fig. 1C). The effect on viral lytic replication and transmission was more directly shown by CPE and the immunofluorescence assay. At 40 h posttransfection, the number of cells expressing viral late proteins (e.g., ORF65) in samples transfected with 67S or 69S BAC was far less than that in samples transfected with WT BAC but more than that of the same group at 26 h posttransfection, around which time the first round of viral replication was just completed and progeny viruses were released to infect neighboring cells (Fig. 1D). Previous studies have indicated that herpesviruses may adopt other ways to escape from the nucleus in addition to the primary envelopment-deenvelopment pathway (3134). For example, absence of the PrV NEC caused 1,000-fold less production of virus progenies. However, after serial passage in cell culture, the UL34-negative PrV replicated with wild-type kinetics by inducing nuclear envelope breakdown (31). Consistent with this report, we also observed gaps on nuclear membranes in some cells transfected with 67S or 69S BAC (data not shown). Thus, in the absence of NEC, MHV-68 may also induce nuclear envelope breakdown in order to transport capsids to the cytoplasm.
In the absence of other viral proteins, overexpression of MHV-68 ORF67 resulted in the proliferation of nuclear membrane and cytoplasmic membrane structures (Fig. 6A). The number of membrane layers varies among different structures and even different parts of one structure, indicating that ORF67 alone could induce membrane proliferation but could not control the extent. Coexpression of ORF67 and ORF69 efficiently produced virion-like vesicles from the inner membrane in mammalian cells (Fig. 6D), which was akin to the NEC of PrV (9). In these cells, proliferation of membranes in the cytoplasm was barely observed, and the portion of the nuclear membrane without vesicles was still a bilayer. As judged by the immunofluorescence assay, ORF67 was preferentially located on the nuclear membrane in the presence of ORF69 (Fig. 4A). Thus, ORF69, through its interaction with ORF67, may optimize the localization of ORF67 and restrain its ability to proliferate membranes. It was shown that, when tethered to the membrane artificially, UL31 of PrV (homologue of ORF69) alone was sufficient to induce membrane budding and scission (10). Combined with our results, we speculate that ORF69 of MHV-68 is responsible for bending and scission of the membrane and that its recruitment to the inner nuclear membrane requires ORF67. In previous studies, researchers also found that the NEC of KSHV or EBV could deform the nuclear membrane; however, virion-like vesicles were not observed in mammalian cells transfected with the NEC of KSHV or EBV (17, 35). Several reasons may account for the difference in abilities of NECs from MHV-68, KSHV, and EBV to induce virion-like vesicles: (i) the inherent ability to produce vesicles may vary among NECs of gammaherpesviruses; (ii) another viral factor(s) may be required to assist the NEC of KSHV or EBV to produce vesicles; (iii) mammalian cells have evolved certain strategies to restrict vesicle formation induced by the NEC of KSHV or EBV.
Exogenous expression of the NEC induced numerous vesicles in cells (Fig. 6D), but empty vesicles or primary enveloped virions in PNS were rarely observed during MHV-68 replication (Fig. 2 and 10, and Table 1). One explanation for this discrepancy is the transient nature of nuclear egress, making it difficult to capture primary enveloped virions in the PNS. Alternatively, vesicle formation at the inner nuclear membrane may be suppressed before assembled capsids gain access to the inner nuclear membrane. This would prevent serious damage to the subcellular structure before the primary envelopment process occurs, as seen in Fig. 6D, when only ORF67 and ORF69 were expressed. Regulation of the primary envelopment process is poorly understood. However, both viral and host proteins have been implicated in this process. One such viral protein is ORF69 or its homologues. For example, during HSV-1 replication, phosphorylation of UL31 by US3 inhibits the primary envelopment of capsids (36). ORF69 homologues in HCMV and EBV are also phosphorylated during virus replication (37, 38). In our study, double bands of ORF69 were detected in cells infected with MHV-68 or cotransfected with ORF67 and ORF69 (Fig. 3A and B). The upper band may represent the phosphorylated product of ORF69, although further investigation is required. On the other hand, a host protein TMEM140 was reported to impede the interaction between UL34 and UL31 and the primary envelopment process of HSV-1 (39).
The structures of NECs in gammaherpesviruses have not been determined. We attempted to determine the structure of MHV-68 NEC by the crystallographic method without success (data not shown), possibly because of the nature of MHV-68 NEC. Since structures of NECs in alpha- and betaherpesviruses reported recently showed high similarities and helped to reveal mechanisms of NEC formation and membrane budding in the two subfamilies (2328), we went on to construct the structure model of MHV-68 NEC through homology modeling (Fig. 7). Based on the structure model, we identified five amino acids that paly critical roles in mediating NEC formation (Fig. 8). More importantly, introducing mutations of these amino acids in the context of viral infection severely impaired the translocation of capsids to the cytoplasm (Fig. 10; Table 1), demonstrating the importance of the ORF67-ORF69 interaction in nuclear egress. Furthermore, these binding sites identified in MHV-68 NEC are all conserved in NECs of KSHV and EBV, based on sequence alignment (Fig. 7C), suggesting that they may play important roles in mediating NEC function in KSHV and EBV as well. Indeed, a previous study on KSHV NEC showed that deleting amino acids 61 to 65 of ORF67 abolished its interaction with ORF69 (17). F62 of KSHV ORF67 corresponds to F61 in MHV-68 ORF67, which was shown to be essential for mediating its interaction with ORF69 (Fig. 8) and nuclear egress and viral lytic replication (Fig. 9 and 10; Table 1) in our study. Therefore, our validated structure model of MHV-68 NEC provides a basis for designing anti-gammaherpesviruses drugs.

MATERIALS AND METHODS

Cells and viruses.

293T, 293A, BHK-21, and HeLa cells were cultured in complete Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum. Wide-type (WT) or FLAG-ORF67 MHV-68 was propagated by infecting BHK-21 cells at a multiplicity of infection (MOI) of 0.03. To infect BHK-21 cells, the viral inoculum in DMEM was incubated with cells for 1 h with occasional swirling. The inoculum was then removed and replaced with fresh DMEM plus 10% fetal bovine serum. The titer of virus was measured by a plaque assay in BHK-21 cells as previously described (40).

Construction of recombinant MHV-68 (BAC).

Based on the WT bacterial artificial chromosome (BAC) of MHV-68, several mutants were constructed by two-step Red recombination in Escherichia coli, according to procedures described previously (41). For construction of ORF67-null or ORF69-null mutants (67S and 69S), triple-frame stop codons with an introduced SmaI site (5′-CCCGGGTTGATTAATTGA-3′) were inserted 50 nucleotides (nt) downstream of the translation start codon of ORF67 or ORF69 (between nt 96366 and nt 96367 on viral genome for 67S, and nt 98111 and nt 98112 on viral genome for 69S). For construction of FLAG-ORF67 BAC, sequences including the FLAG tag, NaeI site, two glycine codons, and the first nine nucleotides of ORF67 coding region (5′-GACTACAAAGACGATGACGACAAGGCCGGCGGAGGCATGGCTAAC-3′) were inserted after the 9th nucleotide of the ORF67 coding region (between nt 96408 and nt 96409 on the viral genome). For construction of 67-F61A, 69-F52A, or 69-L66A BAC, ORF67-F61, ORF69-F52, or ORF69-L66, respectively, were individually mutated to alanine codons. For each mutant BAC, we constructed the revertant with the same method. Positive clones were selected by restriction enzyme analysis and confirmed by sequencing to ensure that there were no undesired mutations, deletions, or insertions in MHV-68 genome sequences.

Plasmid construction.

ORF67 and ORF69 sequences were amplified from WT BAC by PCR. The full-length ORF67 sequence was cloned into the pCMV-HA vector (Clontech) via EcoRI and XhoI sites to construct the HA-ORF67 plasmid. The full-length ORF69 sequence of MHV-68 was cloned into p3×FLAG-CMV-14 (Sigma) via HindIII and BamHI sites to construct the ORF69-3×FLAG plasmid. pTAG-ORF69 was constructed by adding sequences encoding FLAG/calmodulin binding peptide to the N terminus of ORF69 (22) and was a kind gift from Ren Sun (University of California, Los Angeles). Single-site mutant plasmids of ORF67 or ORF69 (HA-ORF67-F61A, HA-ORF67-F65A, HA-ORF67-M180A, ORF69-F52A-3×FLAG, ORF69-F65A-3×FLAG, and ORF69-L66A-3×FLAG) were generated using a two-step oligonucleotide-directed PCR mutagenesis method. To construct prokaryotic expression plasmids of His-SUMO-ORF67Δ40 or His-SUMO-ORF69Δ45, the pET-28a-c(+) vector (Novagen) was first modified to encode a fusion protein with the His tag and SUMO tag in frame with the N terminus of the target protein, and then ORF67Δ40 (1 to 558 nt of ORF67 coding region) or ORF69Δ45 (136 to 879 nt of ORF69 coding region) was cloned into the modified vector via BamHI and XhoI sites.

Expression and purification of MHV-68 ORF67Δ40 and ORF69Δ45.

Escherichia coli BL21-CodonPlus(DE3)-RIPL cells (Stratagene) transformed with His-SUMO-ORF67Δ40 or His-SUMO-ORF69Δ45 plasmids were cultured in lysogeny broth (LB) to an optical density of ∼1.2 at 37°C. Subsequently, ORF67 and ORF69 expression was induced at 16°C for 20 h using 0.5 mM and 0.1 mM isopropyl-β-d-thiogalactopyranoside (IPTG), respectively. Cells expressing ORF67 or ORF69 were then mixed and lysed in buffer A (20 mM HEPES [pH 7.5], 500 mM NaCl, 10% glycerol, 30 mM imidazole) using a high-pressure homogenizing system at 4°C. The cell lysate was clarified by centrifugation at 20,000 × g (Beckman JA-20 rotor) for 1 h. The supernatant was loaded onto a nickel-nitrilotriacetic acid (Ni-NTA) column (Thermo Fisher Scientific) equilibrated with buffer A, and target proteins were eluted with buffer B (20 mM HEPES [pH 7.5], 500 mM NaCl, 10% glycerol, 500 mM imidazole). Subsequently, the His tag and SUMO tag were cut off with Ulp1 protease by overnight digestion at 4°C and then removed along with Ulp1 protease using the Ni-NTA column. The flowthrough from the Ni-NTA column was concentrated and finally loaded onto the Column Superdex 200 10/300 GL (GE Healthcare) equilibrated with buffer C (20 mM HEPES [pH 7.5], 500 mM NaCl, 10% glycerol, and 2 mM dithiothreitol [DTT]) for further purification. Finally, complexes of ORF67Δ40 and ORF69Δ45 from different elution volumes were subjected to SDS-PAGE and Coomassie blue staining.

Antibodies.

Mouse monoclonal anti-hemagglutinin (anti-HA) antibody (H9658) and anti-FLAG antibody (F3165) were purchased from Sigma. Rabbit polyclonal anti-FLAG antibody (HX1819) was purchased from Huaxingbio Science. Mouse monoclonal anti-laminA/C antibody (sc-20681) was purchased from Santa Cruz Biotechnology. Mouse monoclonal anti-ORF33 antibody was described previously (40). Rabbit anti-ORF65 antibody was a kind gift from Ren Sun (University of California, Los Angeles). Purified ORF67 or ORF69 proteins from Escherichia coli were provided to the Experimental Animal Center, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, to generate rabbit polyclonal antibodies against ORF67 or ORF69 of MHV-68. Both antibodies are specific to their target proteins according to Western blotting validation (data not shown).

Immunoprecipitation and immunoblotting.

293T or BHK-21 cells seeded on 6-well plates (∼4 × 105 per well) were transfected with 3 μg of total DNA using jet-PEI (Polyplus transfection) or infected with viruses. At 48 h posttransfection or postinfection, cells were washed once with ice-cold phosphate-buffered saline (PBS) and lysed with 500 μl RIPA buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1% Triton X-100) containing 1 mM phenylmethanesulfonyl fluoride (PSMF) and protease inhibitor cocktail (Roche). Lysate was cleared by centrifugation at 13,000 × g for 15 min. Ten percent of the supernatant was used as the input control, and the rest of it was incubated with 10 μl anti-FLAG M2 agarose (Sigma) equilibrated with RIPA buffer at 4°C for 4 h. Then, the agarose beads were washed with RIPA buffer 5 times, and bound proteins were eluted from beads by heating in SDS sample buffer at 100°C for 5 min. For immunoblotting, samples were separated on 12% SDS-polyacrylamide gels, and proteins on gels were transferred to polyvinylidene difluoride (PVDF) membranes (Millipore). The PVDF membranes were then blocked with 5% nonfat dry milk (diluted with PBS) at room temperature for 1 h and incubated sequentially with primary antibody and secondary antibodies (goat anti-rabbit or anti-mouse IgG conjugated with horseradish peroxidase). Proteins were detected with an enhanced chemiluminescence system (Millipore).

Indirect immunofluorescence assay.

Cells infected with viruses or transfected with BAC or plasmids were fixed with 4% paraformaldehyde for 15 min. After washing with PBS 3 times, cells were permeabilized with 0.2% Triton X-100 in PBS for 10 min. Next, cells were washed with PBS 3 times again and blocked with 5% goat serum for 1 h. After the goat serum was removed, cells were incubated sequentially with primary antibody, secondary antibody (Alexa Fluor 555-conjugated anti-mouse antibody or Alexa Fluor 488-conjugated anti-rabbit antibody; Invitrogen) and DAPI (4′,6-diamidino-2-phenylindole). Then, cells were washed with PBS 3 times and with H2O once. Finally, the cover glass with cells was mounted onto a glass slide with Fluoromount reagent (Sigma), and fluorescence signals were examined using a confocal microscope (Olympus FV1200) or a three-dimensional structured illumination microscope (3D-SIM).

3D-SIM imaging and analysis.

3D-SIM images of immunostained cells were recorded on a DeltaVision OMX V3 imaging system (GE Healthcare) equipped with a 100×/1.40 numerical aperture (NA) oil-immersion lens objective (Olympus), three solid-state lasers (405, 488, and 593 nm), and electron-multiplying charge-coupled-device (CCD) cameras (Photometrics). The microscope is routinely calibrated with 100-nm fluorescent spheres to calculate both the lateral and axial limits of image resolution. Serial Z-stack sectioning was conducted at 125-nm intervals. Images were reconstructed with the SoftWoRx 5.0 software package (GE Healthcare). The following parameters were set: channel-specific optical transfer functions, Wiener filter 0.001000, drift correction with respect to first channel, and custom K0 guess angles for camera positions. Pixel registration was corrected to be less than 1 pixel for all channels using 100-nm TetraSpeck beads (Invitrogen). The reconstructed images were further processed for maximum-intensity projections with SoftWoRx 5.0 software.

Viral DNA extraction and quantification by real-time PCR.

293T cells were transfected with WT or mutant BAC using jet-PEI (Polyplus transfection). At 60 h posttransfection, extracellular virion DNA was extracted and examined. Two hundred microliters of supernatant from each sample was first treated with 4 units of DNase I (NEB) in reaction buffer (10 mM Tris-HCl [pH 7.6], 2.5 mM MgCl2, and 0.5 mM CaCl2) at 37°C for 30 min and subsequently heated at 75°C for 10 min to inactivate DNase I. Viral DNA was then extracted with the TIANamp Virus DNA/RNA kit (catalog number DP315; Tiangen Biotech) according to the manufacturer’s instructions. Briefly, supernatant was incubated with proteinase K, carrier RNA, and lysis buffer at 56°C for 15 min. Viral DNA/carrier RNA was isolated and purified with a DNA/RNA adsorption column and dissolved in 30 μl double-distilled water (ddH2O). Triplicate real-time PCR was performed using 1 μl sample, SYBR green, and primers specific to the ORF24 coding region of MHV-68 (sense, 5′-ATACAAGTTCATCAACATCTC-3′; antisense, 5′-TACATCAGCGACATCTAC-3′) on a QuantStudio 7 Flex System (Thermo Fisher Scientific). Viral genome copies were determined with a standard curve obtained by measuring 5-fold gradient dilution of MHV-68 BAC DNA (10 ng to 1.28 × 10−6 ng).

Transmission electron microscopy analysis.

293T cells transfected with plasmids or BAC were fixed with 2.5% glutaraldehyde (Sigma) at 4°C for 12 h and with 1% OsO4 at room temperature for 2 h, dehydrated through a graded series of ethanols (30%, 50%, 70%, 90%, 95%, and 100%), and embedded in Epon. Ultrathin sections (∼70 nm) were stained with 2% uranylacetate and 0.3% lead citrate and examined with a 120-kV transmission electron microscope (Tecnai Spirit; FEI).

ACKNOWLEDGMENTS

We thank Can Peng, Lei Sun, Shufeng Sun, Shuoguo Li, Yan Teng, and Chunli Jiang at the Center for Biological Imaging (CBI), Institute of Biophysics, for technical help with EM sample preparation, SIM image analysis, and confocal image analysis, and the Experimental Animal Center, Institute of Genetics and Developmental Biology, for generating the rabbit polyclonal antibodies against MHV-68 ORF67 and ORF69 used in the study. We also thank members of the Deng laboratory for helpful discussions.
This work was supported by grant 2016YFA0502101 from the Ministry of Science and Technology of the People’s Republic of China and grants from the National Natural Science Foundation of China (numbers 81630059 and 81325012).

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REFERENCES

1.
Mettenleiter TC, Muller F, Granzow H, Klupp BG. 2013. The way out: what we know and do not know about herpesvirus nuclear egress. Cell Microbiol 15:170–178.
2.
Lee CP, Chen MR. 2010. Escape of herpesviruses from the nucleus. Rev Med Virol 20:214–230.
3.
Lv Y, Zhou S, Gao S, Deng H. 2019. Remodeling of host membranes during herpesvirus assembly and egress. Protein Cell 10:315–326.
4.
Speese SD, Ashley J, Jokhi V, Nunnari J, Barria R, Li Y, Ataman B, Koon A, Chang YT, Li Q, Moore MJ, Budnik V. 2012. Nuclear envelope budding enables large ribonucleoprotein particle export during synaptic Wnt signaling. Cell 149:832–846.
5.
Reynolds AE, Wills EG, Roller RJ, Ryckman BJ, Baines JD. 2002. Ultrastructural localization of the herpes simplex virus type 1 UL31, UL34, and US3 proteins suggests specific roles in primary envelopment and egress of nucleocapsids. J Virol 76:8939–8952.
6.
Muranyi W, Haas J, Wagner M, Krohne G, Koszinowski UH. 2002. Cytomegalovirus recruitment of cellular kinases to dissolve the nuclear lamina. Science 297:854–857.
7.
Fuchs W, Klupp BG, Granzow H, Osterrieder N, Mettenleiter TC. 2002. The interacting UL31 and UL34 gene products of pseudorabies virus are involved in egress from the host-cell nucleus and represent components of primary enveloped but not mature virions. J Virol 76:364–378.
8.
Sharma M, Kamil JP, Coughlin M, Reim NI, Coen DM. 2014. Human cytomegalovirus UL50 and UL53 recruit viral protein kinase UL97, not protein kinase C, for disruption of nuclear lamina and nuclear egress in infected cells. J Virol 88:249–262.
9.
Klupp BG, Granzow H, Fuchs W, Keil GM, Finke S, Mettenleiter TC. 2007. Vesicle formation from the nuclear membrane is induced by coexpression of two conserved herpesvirus proteins. Proc Natl Acad Sci U S A 104:7241–7246.
10.
Lorenz M, Vollmer B, Unsay JD, Klupp BG, Garcia-Saez AJ, Mettenleiter TC, Antonin W. 2015. A single herpesvirus protein can mediate vesicle formation in the nuclear envelope. J Biol Chem 290:6962–6974.
11.
Bigalke JM, Heuser T, Nicastro D, Heldwein EE. 2014. Membrane deformation and scission by the HSV-1 nuclear egress complex. Nat Commun 5:4131.
12.
Farina A, Feederle R, Raffa S, Gonnella R, Santarelli R, Frati L, Angeloni A, Torrisi MR, Faggioni A, Delecluse HJ. 2005. BFRF1 of Epstein-Barr virus is essential for efficient primary viral envelopment and egress. J Virol 79:3703–3712.
13.
Granato M, Feederle R, Farina A, Gonnella R, Santarelli R, Hub B, Faggioni A, Delecluse HJ. 2008. Deletion of Epstein-Barr virus BFLF2 leads to impaired viral DNA packaging and primary egress as well as to the production of defective viral particles. J Virol 82:4042–4051.
14.
Lee CP, Liu PT, Kung HN, Su MT, Chua HH, Chang YH, Chang CW, Tsai CH, Liu FT, Chen MR. 2012. The ESCRT machinery is recruited by the viral BFRF1 protein to the nucleus-associated membrane for the maturation of Epstein-Barr virus. PLoS Pathog 8:e1002904.
15.
Lee C-P, Liu G-T, Kung H-N, Liu P-T, Liao Y-T, Chow L-P, Chang L-S, Chang Y-H, Chang C-W, Shu W-C, Angers A, Farina A, Lin S-F, Tsai C-H, Bouamr F, Chen M-R. 2016. The ubiquitin ligase Itch and ubiquitination regulate BFRF1-mediated nuclear envelope modification for Epstein-Barr virus maturation. J Virol 90:8994–9007.
16.
Desai PJ, Pryce EN, Henson BW, Luitweiler EM, Cothran J. 2012. 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. J Virol 86:594–598.
17.
Luitweiler EM, Henson BW, Pryce EN, Patel V, Coombs G, McCaffery JM, Desai PJ. 2013. Interactions of the Kaposi’s Sarcoma-associated herpesvirus nuclear egress complex: ORF69 is a potent factor for remodeling cellular membranes. J Virol 87:3915–3929.
18.
Rajcani J, Kudelova M. 2005. Murine herpesvirus pathogenesis: a model for the analysis of molecular mechanisms of human gamma herpesvirus infections. Acta Microbiol Immunol Hung 52:41–71.
19.
Peng L, Ryazantsev S, Sun R, Zhou ZH. 2010. Three-dimensional visualization of gammaherpesvirus life cycle in host cells by electron tomography. Structure 18:47–58.
20.
Guo H, Wang L, Peng L, Zhou ZH, Deng H. 2009. Open reading frame 33 of a gammaherpesvirus encodes a tegument protein essential for virion morphogenesis and egress. J Virol 83:10582–10595.
21.
Virgin HW, IV, Latreille P, Wamsley P, Hallsworth K, Weck KE, Dal Canto AJ, Speck SH. 1997. Complete sequence and genomic analysis of murine gammaherpesvirus 68. J Virol 71:5894–5904.
22.
Lee S, Salwinski L, Zhang C, Chu D, Sampankanpanich C, Reyes NA, Vangeloff A, Xing F, Li X, Wu TT, Sahasrabudhe S, Deng H, Lacount DJ, Sun R. 2011. An integrated approach to elucidate the intra-viral and viral-cellular protein interaction networks of a gamma-herpesvirus. PLoS Pathog 7:e1002297.
23.
Bigalke JM, Heldwein EE. 2015. Structural basis of membrane budding by the nuclear egress complex of herpesviruses. EMBO J 34:2921–2936.
24.
Lye MF, Sharma M, El Omari K, Filman DJ, Schuermann JP, Hogle JM, Coen DM. 2015. Unexpected features and mechanism of heterodimer formation of a herpesvirus nuclear egress complex. EMBO J 34:2937–2952.
25.
Walzer SA, Egerer-Sieber C, Sticht H, Sevvana M, Hohl K, Milbradt J, Muller YA, Marschall M. 2015. Crystal structure of the human cytomegalovirus pUL50-pUL53 core nuclear egress complex provides insight into a unique assembly scaffold for virus-host protein interactions. J Biol Chem 290:27452–27458.
26.
Leigh KE, Sharma M, Mansueto MS, Boeszoermenyi A, Filman DJ, Hogle JM, Wagner G, Coen DM, Arthanari H. 2015. Structure of a herpesvirus nuclear egress complex subunit reveals an interaction groove that is essential for viral replication. Proc Natl Acad Sci U S A 112:9010–9015.
27.
Hagen C, Dent KC, Zeev-Ben-Mordehai T, Grange M, Bosse JB, Whittle C, Klupp BG, Siebert CA, Vasishtan D, Bäuerlein FJB, Cheleski J, Werner S, Guttmann P, Rehbein S, Henzler K, Demmerle J, Adler B, Koszinowski U, Schermelleh L, Schneider G, Enquist LW, Plitzko JM, Mettenleiter TC, Grünewald K. 2015. Structural basis of vesicle formation at the inner nuclear membrane. Cell 163:1692–1701.
28.
Zeev-Ben-Mordehai T, Weberruß M, Lorenz M, Cheleski J, Hellberg T, Whittle C, El Omari K, Vasishtan D, Dent KC, Harlos K, Franzke K, Hagen C, Klupp BG, Antonin W, Mettenleiter TC, Grünewald K. 2015. Crystal structure of the herpesvirus nuclear egress complex provides insights into inner nuclear membrane remodeling. Cell Rep 13:2645–2652.
29.
Waterhouse A, Bertoni M, Bienert S, Studer G, Tauriello G, Gumienny R, Heer FT, de Beer TAP, Rempfer C, Bordoli L, Lepore R, Schwede T. 2018. SWISS-MODEL: homology modelling of protein structures and complexes. Nucleic Acids Res 46:W296–W303.
30.
Benkert P, Biasini M, Schwede T. 2011. Toward the estimation of the absolute quality of individual protein structure models. Bioinformatics 27:343–350.
31.
Grimm KS, Klupp BG, Granzow H, Muller FM, Fuchs W, Mettenleiter TC. 2012. Analysis of viral and cellular factors influencing herpesvirus-induced nuclear envelope breakdown. J Virol 86:6512–6521.
32.
Maric M, Haugo AC, Dauer W, Johnson D, Roller RJ. 2014. Nuclear envelope breakdown induced by herpes simplex virus type 1 involves the activity of viral fusion proteins. Virology 460–461:128–137.
33.
Schulz KS, Klupp BG, Granzow H, Passvogel L, Mettenleiter TC. 2015. Herpesvirus nuclear egress: pseudorabies virus can simultaneously induce nuclear envelope breakdown and exit the nucleus via the envelopment-deenvelopment-pathway. Virus Res 209:76–86.
34.
Wild P, Senn C, Manera CL, Sutter E, Schraner EM, Tobler K, Ackermann M, Ziegler U, Lucas MS, Kaech A. 2009. Exploring the nuclear envelope of herpes simplex virus 1-infected cells by high-resolution microscopy. J Virol 83:408–419.
35.
Gonnella R, Farina A, Santarelli R, Raffa S, Feederle R, Bei R, Granato M, Modesti A, Frati L, Delecluse HJ, Torrisi MR, Angeloni A, Faggioni A. 2005. Characterization and intracellular localization of the Epstein-Barr virus protein BFLF2: interactions with BFRF1 and with the nuclear lamina. J Virol 79:3713–3727.
36.
Mou F, Wills E, Baines JD. 2009. Phosphorylation of the U(L)31 protein of herpes simplex virus 1 by the U(S)3-encoded kinase regulates localization of the nuclear envelopment complex and egress of nucleocapsids. J Virol 83:5181–5191.
37.
Sharma M, Bender BJ, Kamil JP, Lye MF, Pesola JM, Reim NI, Hogle JM, Coen DM. 2015. Human cytomegalovirus UL97 phosphorylates the viral nuclear egress complex. J Virol 89:523–534.
38.
Wu TT, Liao HI, Tong L, Leang RS, Smith G, Sun R. 2011. Construction and characterization of an infectious murine gammaherpesvirus-68 bacterial artificial chromosome. J Biomed Biotechnol 2011:926258.
39.
Guan Y, Guo L, Yang E, Liao Y, Liu L, Che Y, Zhang Y, Wang L, Wang J, Li Q. 2014. HSV-1 nucleocapsid egress mediated by UL31 in association with UL34 is impeded by cellular transmembrane protein 140. Virology 464–465:1–10.
40.
Shen S, Jia X, Guo H, Deng H. 2015. Gammaherpesvirus tegument protein ORF33 is associated with intranuclear capsids at an early stage of the tegumentation process. J Virol 89:5288–5297.
41.
Tischer BK, von Einem J, Kaufer B, Osterrieder N. 2006. Two-step red-mediated recombination for versatile high-efficiency markerless DNA manipulation in Escherichia coli. Biotechniques 40:191–197.

Information & Contributors

Information

Published In

cover image Journal of Virology
Journal of Virology
Volume 93Number 24December 2019
eLocator: 10.1128/jvi.01422-19
Editor: Richard M. Longnecker, Northwestern University

History

Received: 22 August 2019
Accepted: 17 September 2019
Published online: 26 November 2019

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Keywords

  1. herpesvirus
  2. murine gammaherpesvirus 68
  3. nuclear egress complex

Contributors

Authors

Ying Lv
CAS Key Laboratory of Infection and Immunity, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China
University of Chinese Academy of Sciences, Beijing, China
Sheng Shen
CAS Key Laboratory of Infection and Immunity, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China
Lingjiao Xiang
CAS Key Laboratory of Infection and Immunity, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China
University of Chinese Academy of Sciences, Beijing, China
Xing Jia
CAS Key Laboratory of Infection and Immunity, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China
Yanjie Hou
National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China
Dacheng Wang
University of Chinese Academy of Sciences, Beijing, China
CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China
National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China
Hongyu Deng
CAS Key Laboratory of Infection and Immunity, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China
University of Chinese Academy of Sciences, Beijing, China
CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China

Editor

Richard M. Longnecker
Editor
Northwestern University

Notes

Address correspondence to Hongyu Deng, [email protected].
Y.L. and S.S. contributed equally to this work.

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