Mono-ubiquitylated ORF45 Mediates Association of KSHV Particles with Internal Lipid Rafts for Viral Assembly and Egress

Wang, Xin; Zhu, Nannan; Li, Wenwei; Zhu, Fanxiu; Wang, Yan; Yuan, Yan

doi:10.1371/journal.ppat.1005332

Cited by 29 publications

(40 citation statements)

References 39 publications

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“…2E, bottom-left panel). Consistent with the mainly cytoplasmic nature of tegument acquisition and envelopment of KSHV virions (52,58), only the cytosolic intron-less circ-vIRF4 ( Fig. 2E) was incorporated into KSHV particles (Fig.…”

Section: Resultssupporting

confidence: 67%

“…Our cytoplasmic/nuclear fractionation experiments showed that circ-vIRF4.IR localizes to the nucleus and was not found in virus particles whereas the exon-only form was cytoplasmic and retained in the virion tegument. This is consistent with herpesvirus tegumentation and the envelopment maturation that occurs in the cytoplasmic compartment (52,58). circPANs and circK7.3s are also packaged into viral particles; however, given the high number and different sizes of circRNAs from these regions, we were unable to determine whether only cytoplasmic forms of these circRNA are incorporated into the KSHV virion.…”

Section: Kshv Circpans Are Inducible Whereas Circ-virf4 Is Constitutisupporting

confidence: 65%

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Kaposi’s Sarcoma-Associated Herpesvirus-Encoded circRNAs Are Expressed in Infected Tumor Tissues and Are Incorporated into Virions

Abere

Zhou

et al. 2020

mBio

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Kaposi's sarcoma-associated herpesvirus (KSHV) has recently been found to generate circular RNAs (circRNAs) from several KSHV genes, most abundantly from K10 (viral interferon regulatory factor 4 [vIRF4]), K7.3, and polyadenylated nuclear (PAN) RNA. To define expression of these circRNAs, KSHV-infected cell lines, patient tissues, and purified virions were examined. KSHV circRNA expression was universally detected in tests of six primary effusion lymphoma (PEL) cell lines but ranged from low-level expression in BC-1 cells dually infected with tightly latent KSHV and Epstein-Barr virus to abundant expression in KSHV-only BCBL-1 cells with spontaneous virus production. Generally, the PAN/K7.3 locus broadly and bidirectionally generated circRNA levels that paralleled the corresponding linear RNA levels. However, RNA corresponding to a particular KSHV circularization site (circ-vIRF4) was minimally induced, despite linear vIRF4 RNA being activated by virus induction. In situ hybridization showed abundant circ-vIRF4 in noninduced PEL cells. All three KSHV circRNAs were isolated as nuclease-protected forms from gradient-purified virions collected from BrK.219 cells infected with a KSHV molecular clone. For circ-vIRF4, the fully processed form that is exported to the cytoplasm was incorporated into virus particles but the nuclear, intron-retaining form was not. The half-life of circ-vIRF4 was twice as long as that of its linear counterpart. The KSHV circRNAs could be detected at a higher rate than their corresponding linear counterparts by in situ hybridization in archival tissues and by reverse transcription-PCR (RT-PCR) in sera stored for over 25 years. In summary, KSHV circRNAs are expressed in infectionassociated diseases, can be regulated depending on virus life cycle, and are incorporated into viral particles for preformed delivery, suggesting a potential function in early infection. IMPORTANCE KSHV has recently been found to encode circRNAs. circRNAs result from back-splicing of an upstream pre-mRNA splice donor exon-intron junction to an acceptor site, generating a covalently closed circle. This study revealed that for one KSHV region, the PAN/K7.3 locus, broadly and bidirectionally generated circRNA levels parallel corresponding linear RNA levels. Another KSHV circularization site (circ-vIRF4), however, showed expression that differed from that of the corresponding linear RNA. All KSHV circRNAs are incorporated into KSHV virions and are potentially expressed as immediate early products in newly infected cells.

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Section: Resultssupporting

confidence: 67%

Section: Kshv Circpans Are Inducible Whereas Circ-virf4 Is Constitutisupporting

confidence: 65%

Kaposi’s Sarcoma-Associated Herpesvirus-Encoded circRNAs Are Expressed in Infected Tumor Tissues and Are Incorporated into Virions

Abere

Zhou

et al. 2020

mBio

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“…ORF45 also interacts with another tegument protein, ORF33, and plays a crucial role in efficient production of KSHV viral particles (15). Most recently, ORF45 was shown to mediate association of KSHV particles with internal lipid rafts for viral assembly and egress (20).…”

mentioning

confidence: 99%

Tegument Protein ORF45 Plays an Essential Role in Virion Morphogenesis of Murine Gammaherpesvirus 68

Jia

Shen

et al. 2016

J Virol

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cTegument proteins play critical roles in herpesvirus morphogenesis. ORF45 is a conserved tegument protein of gammaherpesviruses; however, its role in virion morphogenesis is largely unknown. In this work, we determined the ultrastructural localization of murine gammaherpesvirus 68 (MHV-68) ORF45 and found that this protein was incorporated into virions around the site of host-derived vesicles. Notably, the absence of ORF45 inhibited nucleocapsid egress and blocked cytoplasmic virion maturation, demonstrating that ORF45 is essential for MHV-68 virion morphogenesis.A s a unique structure of herpesviruses, tegument functions in multiple aspects of the viral life cycle, including modulation of the host cellular environment and initiation of viral gene transcription at the immediate-early phase of virus infection, transport of nucleocapsids to the nuclear pore, and virion morphogenesis and egress (1-10). Virion morphogenesis is a complicated event that takes place in the late phase of the herpesvirus lytic life cycle. After acquisition of viral genomes in the nucleus, nucleocapsids bud at the inner nuclear membrane to form primary virions in the perinuclear space, followed by deenvelopment at the outer nuclear membrane, releasing capsids into the cytoplasm. Capsids then undergo tegumentation and reenvelopment to obtain tegument proteins and envelope glycoproteins (11,12,13). Finally, mature virions are released from the cell.ORF45 is a virion-associated protein that is conserved in gammaherpesviruses but has no homologue in alpha-or betaherpesviruses. The ORF45 homologues in Kaposi's sarcoma-associated herpesvirus (KSHV), murine gammaherpesvirus 68 (MHV-68), Epstein-Barr virus (EBV), and rhesus rhadinovirus (RRV) are 407 amino acids (aa), 206 aa, 217 aa, and 353 aa, respectively (14, 15). The homology mainly lies in the C-terminal region of the protein. Among these homologues, KSHV ORF45 is the best studied. It is virion associated (16,17) and is a multifunctional protein. It interacts with interferon regulatory factor 7 and inhibits virus-induced interferon production, leading to viral immune evasion (2, 18). Characterization of a KSHV ORF45-null bacterial artificial chromosome (BAC) showed that although ORF45 is not essential for KSHV lytic replication, it plays roles in both early and late stages of viral infection (19). Indeed, ORF45 was shown to interact with kinesin-2 to mediate the transport of viral particles along microtubules (4). ORF45 also interacts with another tegument protein, ORF33, and plays a crucial role in efficient production of KSHV viral particles (15). Most recently, ORF45 was shown to mediate association of KSHV particles with internal lipid rafts for viral assembly and egress (20).MHV-68 ORF45 has also been identified as a virion-associated protein (21,22,23). Biochemical analysis demonstrated that ORF45 is a true tegument protein that is packaged into virions (23). By construction and analysis of an ORF45-null MHV-68 mutant using a BAC system, Jia et al. demonstrated that the ORF45-null MH...

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“…It can associate with the ubiquitin-specific protease USP7 and stabilize ORF33 and ORF36 (15,16). Monoubiquitinated ORF45 mediates the association between KSHV virus particles and lipid rafts for virus assembly and egress (17); it also interacts with kinesin-2 (13). However, with low sequence homology between EBV BKRF4 and KSHV ORF45, the amino acid sequences in the interacting domains with those partners are not well conserved in EBV BKRF4.…”

Section: Discussionmentioning

confidence: 99%

Epstein-Barr Virus BKRF4 Gene Product Is Required for Efficient Progeny Production

Masud

Watanabe

Yoshida

et al. 2017

J Virol

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Epstein-Barr virus (EBV), a member of human gammaherpesvirus, infects mainly B cells. EBV has two alternative life cycles, latent and lytic, and is reactivated occasionally from the latent stage to the lytic cycle. To combat EBV-associated disorders, understanding the molecular mechanisms of the EBV lytic replication cycle is also important. Here, we focused on an EBV lytic gene, BKRF4. Using our anti-BKRF4 antibody, we revealed that the BKRF4 gene product is expressed during the lytic cycle with late kinetics. To characterize the role of BKRF4, we constructed BKRF4-knockout mutants using the bacterial artificial chromosome (BAC) and CRISPR/Cas9 systems. Although disruption of the BKRF4 gene had almost no effect on viral protein expression and DNA synthesis, it significantly decreased progeny virion levels in HEK293 and Akata cells. Furthermore, we show that BKRF4 is involved not only in production of progeny virions but also in increasing the infectivity of the virus particles. Immunoprecipitation assays revealed that BKRF4 interacted with a virion protein, BGLF2. We showed that the C-terminal region of BKRF4 was critical for this interaction and for efficient progeny production. Immunofluorescence analysis revealed that BKRF4 partially colocalized with BGLF2 in the nucleus and perinuclear region. Finally, we showed that BKRF4 is a phosphorylated, possible tegument protein and that the EBV protein kinase BGLF4 may be important for this phosphorylation. Taken together, our data suggest that BKRF4 is involved in the production of infectious virions. Although the latent genes of EBV have been studied extensively, the lytic genes are less well characterized. This study focused on one such lytic gene, BKRF4, which is conserved only among gammaherpesviruses (ORF45 of Kaposi's sarcoma-associated herpesvirus or murine herpesvirus 68). After preparing the BKRF4 knockout virus using B95-8 EBV-BAC, we demonstrated that the BKRF4 gene was involved in infectious progeny particle production. Importantly, we successfully generated a BKRF4 knockout virus of Akata using CRISPR/Cas9 technology, confirming the phenotype in this separate strain. We further showed that BKRF4 interacted with another virion protein, BGLF2, and demonstrated the importance of this interaction in infectious virion production. These results shed light on the elusive process of EBV progeny maturation in the lytic cycle. Notably, this study describes a successful example of the generation and characterization of an EBV construct with a disrupted lytic gene using CRISPR/Cas9 technology.

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Mono-ubiquitylated ORF45 Mediates Association of KSHV Particles with Internal Lipid Rafts for Viral Assembly and Egress

Cited by 29 publications

References 39 publications

Kaposi’s Sarcoma-Associated Herpesvirus-Encoded circRNAs Are Expressed in Infected Tumor Tissues and Are Incorporated into Virions

Kaposi’s Sarcoma-Associated Herpesvirus-Encoded circRNAs Are Expressed in Infected Tumor Tissues and Are Incorporated into Virions

Tegument Protein ORF45 Plays an Essential Role in Virion Morphogenesis of Murine Gammaherpesvirus 68

Epstein-Barr Virus BKRF4 Gene Product Is Required for Efficient Progeny Production

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