Epstein–Barr virus origin of lytic replication mediates association of replicating episomes with promyelocytic leukaemia protein nuclear bodies and replication compartments

We have constructed a recombinant herpes simplex virus type 1 (HSV-1) that simultaneously encodes selected structural proteins from all three virion compartments-capsid, tegument, and envelope-fused with autofluorescent proteins. This triple-fluorescent recombinant, rHSV-RYC, was replication competent, albeit with delayed kinetics, incorporated the fusion proteins into all three virion compartments, and was comparable to wild-type HSV-1 at the ultrastructural level. The VP26 capsid fusion protein (monomeric red fluorescent protein [mRFP]-VP26) was first observed throughout the nucleus and later accumulated in viral replication compartments. In the course of infection, mRFP-VP26 formed small foci in the periphery of the replication compartments that expanded and coalesced over time into much larger foci. The envelope glycoprotein H (gH) fusion protein (enhanced yellow fluorescent protein [EYFP]-gH) was first observed accumulating in a vesicular pattern in the cytoplasm and was then incorporated primarily into the nuclear membrane. The VP16 tegument fusion protein (VP16-enhanced cyan fluorescent protein [ECFP]) was first observed in a diffuse nuclear pattern and then accumulated in viral replication compartments. In addition, it also formed small foci in the periphery of the replication compartments which, however, did not colocalize with the small mRFP-VP26 foci. Later, VP16-ECFP was redistributed out of the nucleus into the cytoplasm, where it accumulated in vesicular foci and in perinuclear clusters reminiscent of the Golgi apparatus. Late in infection, mRFP-VP26, EYFP-gH, and VP16-ECFP were found colocalizing in dots at the plasma membrane, possibly representing mature progeny virus. In summary, this study provides new insights into the dynamics of compartmentalization and interaction among capsid, tegument, and envelope proteins. Similar strategies can also be applied to assess other dynamic events in the virus life cycle, such as entry and trafficking.The herpes simplex virus type 1 (HSV-1) virion consists of three different compartments, capsid, tegument, and envelope. The icosahedral capsid has a diameter of 125 nm and contains the virus genome, a double-stranded DNA of 152 kbp. The structural basis of the capsid are the 162 capsomers, which include 150 hexons and 12 pentons (47). The capsomers are connected in groups of three by a complex formed with two copies of VP23 and one copy of VP19c (47,54,68). The hexons are composed of six molecules of the major capsid protein VP5. Eleven of the 12 pentons are composed of five molecules of VP5, while 1 of the 12, the so-called portal, is a cylindrical structure of 12 molecules of UL6 (46). Also involved in capsid assembly, but not physical components of the capsids, are the scaffold polypeptides VP22a, VP21, and the serine protease, VP24, which is required for capsid maturation (9,26,38,51). Six copies of VP26, a 12-kDa polypeptide encoded by the UL35 gene, occupy the tips of each hexon and thus decorate the surface of the capsid (42, 69). Although not essential...

Section: Discussionmentioning

confidence: 99%

Live Visualization of Herpes Simplex Virus Type 1 Compartment Dynamics

Oliveira

Glauser

Laimbacher

et al. 2008

“…It is known that PML-NBs act as cellular restriction factors that prevent the release of VZV (43). However, in the case of EBV, PML-NBs are important to viral lytic DNA replication (20,50). Although the expression of Zta causes PML-NB dispersion, Adamson and Kenney (30) showed that PML-NBs are only partially dispersed and some structures remain visible under a fluorescence microscope.…”

Section: Discussionmentioning

confidence: 99%

“…EBV virions are commonly quantified using quantitative PCR (qPCR) methods (56). However, these methods could not be utilized in this study, as EBV lytic DNA replication occurs at PML-NBs (35,50); any changes in EBV lytic DNA replication due to a reduction in PML-NBs would affect the production of encapsidated viral particles, ultimately distorting the qPCR results. Accordingly, an ELISA method was developed to determine capsid assembly, on the basis of the principle that on each capsid, VCA interacts with both BFRF3 and BDLF1 (5, 8) but BFRF3 does not interact directly with BDLF1 (8).…”

Section: Localization Of Ebv Capsid Proteins After Lytic Inductionmentioning

confidence: 99%

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Assembly of Epstein-Barr Virus Capsid in Promyelocytic Leukemia Nuclear Bodies

Wang

Kuo

Chang

et al. 2015

The Epstein-Barr virus (EBV) capsid contains a major capsid protein, VCA; two minor capsid proteins, BDLF1 and BORF1; and a small capsid protein, BFRF3. During the lytic cycle, these capsid proteins are synthesized and imported into the host nucleus for capsid assembly. This study finds that EBV capsid proteins colocalize with promyelocytic leukemia (PML) nuclear bodies (NBs) in P3HR1 cells during the viral lytic cycle, appearing as nuclear speckles under a confocal laser scanning microscope. In a glutathione S-transferase pulldown study, we show that BORF1 interacts with PML-NBs in vitro. BORF1 also colocalizes with PML-NBs in EBV-negative Akata cells after transfection and is responsible for bringing VCA and the VCA-BFRF3 complex from the cytoplasm to PML-NBs in the nucleus. Furthermore, BDLF1 is dispersed throughout the cell when expressed alone but colocalizes with PML-NBs when BORF1 is also present in the cell. In addition, this study finds that knockdown of PML expression by short hairpin RNA does not influence the intracellular levels of capsid proteins but reduces the number of viral particles produced by P3HR1 cells. Together, these results demonstrate that BORF1 plays a critical role in bringing capsid proteins to PMLNBs, which may likely be the assembly sites of EBV capsids. The mechanisms elucidated in this study are critical to understanding the process of EBV capsid assembly. IMPORTANCECapsid assembly is an important event during the Epstein-Barr virus (EBV) lytic cycle, as this process is required for the production of virions. In this study, confocal microscopy revealed that the EBV capsid protein BORF1 interacts with promyelocytic leukemia (PML) nuclear bodies (NBs) in the host nucleus and is responsible for transporting the other EBV capsid proteins, including VCA, BDLF1, and BFRF3, to these subnuclear locations prior to initiation of capsid assembly. This study also found that knockdown of PML expression by short hairpin RNA significantly reduces EBV capsid assembly capabilities. This enhanced understanding of capsid assembly offers potential for the development of novel antiviral strategies and therapies that can prevent the propagation and spread of EBV.

“…ICP8 (17) and BALF2 (18) are abundantly expressed throughout lytic replication and are highly concentrated in nuclear replication bodies where viral DNA replication occurs. These bodies contain many cellular replication and repair factors (19).…”

mentioning

confidence: 99%

Interaction of Kaposi's Sarcoma-Associated Herpesvirus ORF6 Protein with Single-Stranded DNA

Özgür

Griffith

2014

Kaposi's sarcoma-associated herpesvirus (KSHV) ORF6 is homologous to the herpes simplex virus 1 (HSV-1) ICP8 and EpsteinBarr virus (EBV) BALF2 proteins. Here, we describe its single-stranded DNA (ssDNA) binding properties. Based on previous findings with ICP8 and BALF2, a 60-amino-acid C-terminal deletion mutant of Orf6 was generated, and the protein was purified to explore the function of the C terminus in ssDNA binding. We showed that full-length ORF6 binds cooperatively to M13 ssDNA, disrupting its secondary structure and extending it to a length equivalent to that of duplex M13 DNA. The width of the ORF6-ssDNA filament is 9 nm, and a 7.3-nm repeat can be distinguished along the filament axis. Fluorescence polarization analysis revealed that the wild-type and C-terminal mutant ORF6 proteins bind equally well to short ssDNA substrates, with dissociation constant (K d ) values of 2.2 ؋ 10 ؊7 M and 1.5 ؋ 10 ؊7 M, respectively. These values were confirmed by electrophoretic mobility shift assay (EMSA) analysis, which also suggested that binding by the full-length protein may involve both monomers and small multimers. While no significant difference in affinities of binding between full-length ORF6 and the C-terminal deletion mutant were observed with the short DNAs, binding of the C-terminal mutant protein to M13 ssDNA showed a clear lack of cooperativity as seen by electron microscopy (EM). Incubation of a duplex DNA containing a long single-stranded tail with doublehelical ORF6 protein filaments revealed that the ssDNA segment can be enveloped within the protein filament without disrupting the filament structure. IMPORTANCEThis work describes the biochemical characterization of the single-stranded DNA binding protein of KSHV, ORF6, central to viral DNA replication in infected cells. A C-terminal deletion mutant protein was generated to aid in understanding the role of the C terminus in DNA binding. Here we analyze the binding of the wild-type and mutant proteins to short oligomeric and longer genomic ssDNA substrates. Although it is capable of interacting with the short substrates, the inability of mutant ORF6 to form oligomers in solution hindered it from fully covering the long genomic substrates. We previously showed that ORF6 forms long filaments in solution, and we showed here that these can absorb ssDNA without disruption of the filament structure. This work will provide an important basis for future studies by us and/or others.