Fluorescent Tagging and Cellular Distribution of the Kaposi's Sarcoma-Associated Herpesvirus ORF45 Tegument Protein

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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“…1A). Such distribution is consistent with the subcellular distribution of its homologue in KSHV (27,28).…”

supporting

confidence: 71%

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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“…GFP expression was used as a marker for infection in the context of BAC16, yet neither this gene nor its product are necessary for the maintenance of KSHV episome. Similar results were obtained upon targeting of orf45, an immediate-early lytic gene, which is not expressed during latent infection and does not have a role during latency in SLK cells (25,(28)(29)(30). Therefore, it appears that the reduction in the burden of KSHV DNA upon targeting of these genes is due to incomplete repair of cleaved DNA, and is not associated with the functions of the protein products of these genes.…”

Section: Quantitative Analysis Of Kshv Dnasupporting

confidence: 71%

“…After blastocidine selection, the resulting cells were infected with BAC16-mCherry-ORF45 recombinant KSHV, and selected with hygromycin. The BAC16-mCherry-ORF45 clone was previously generated based on the recombinant bacterial artificial chromosome 16 (BAC) containing the entire KSHV genome and a GFP-IREShygromycin expression cassette under the control of the EF-1α promoter; it also encodes the immediate-early lytic gene product ORF45 protein fused at its N-terminus to monomeric Cherry fluorescent protein (mCherry) (24,25). BAC16-based recombinant KSHV can be manipulated in E. coli and has become a valuable tool to enable characterization of the different genes and functional domains of KSHV (24).…”

Section: Resultsmentioning

confidence: 99%

Targeting the Kaposi’s Sarcoma-associated Herpesvirus Genome With the CRISPR-Cas9 Platform in Latently Infected Cells

Haddad

Kalt

Shovman

et al. 2020

Preprint

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Background: Kaposi’s sarcoma-associated herpesvirus (KSHV) is a transforming gammaherpes. Like other herpesviruses, KSHV infection is for life long and there is no treatment that can cure of patients from the virus. In addition, there is urgent need to target viral genes to study their role during the infection cycle. The CRISPR-Cas9 technology offers a means to target viral genomes and thus may offer a novel strategy for viral cure as well for better understanding of the infection process. We evaluated the suitability of this platform for the targeting of KSHV. Methods: We have used BAC16 genome, which contains an expression cassette encoding hygromycin-resistance and a GFP marker gene. Three genes were targeted: gfp which serves as a marker for infection; orf45 encoding a lytic viral protein; and orf73, encoding LANA which is crucial for latent infection. The fraction of cells expressing GFP as well as viral DNA levels and LANA expression were monitored and viral genomes were sequenced. Results: We found that KSHV episomes can be targeted by CRISPR-Cas9. Interestingly, the quantity of KSHV DNA declined, even when target sites were not functionally important for latency. In addition, we show that antibiotic selection, used to maintain infection, interferes with the outcome of targeting.Conclusions: Our study provides insights to the use of this fundamental approach for the study and manipulation of KSHV. It provides guidelines for the targeting CRISPR-Cas9 to the viral genome and for outcomes interpretation.

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“…The inserted DNA fragment was sequenced to confirm the stop mutation. To construct a virus expressing the mCherry protein fused to the 5= end of vBcl-2 (BAC16-mCherry-vBcl-2) and the mCherry protein inserted into BAC16-vBcl-2-stop (BAC16-mCherry-vBcl-2-stop), we employed the universal transfer plasmid pEP-mCherry (38). This plasmid was used as a PCR template to obtain a linear DNA fragment encompassing the kanamycin resistance cassette and an I-SceI restriction site, flanked by mCherry containing a small internal duplication, and flanking 47 bp of viral sequences from nucleotides 29914 to 29961 and 30011 to 29965, located upstream and downstream of the KSHV orf16 start codon, respectively.…”

Section: Methodsmentioning

confidence: 99%

Viral Bcl-2 Encoded by the Kaposi's Sarcoma-Associated Herpesvirus Is Vital for Virus Reactivation

Gelgor

Kalt

Bergson

et al. 2015

J Virol

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The Kaposi's sarcoma-associated herpesvirus (KSHV) open reading frame 16 (orf16) encodes a viral Bcl-2 (vBcl-2) protein which shares sequence and functional homology with the Bcl-2 family. Like its cellular homologs, vBcl-2 protects various cell types from apoptosis and can also negatively regulate autophagy. vBcl-2 is transcribed during lytic infection; however, its exact function has not been determined to date. By using bacterial artificial chromosome 16 (BAC16) clone carrying the full-length KSHV genome, we have generated recombinant KSHV mutants that fail to express vBcl-2 or express mCherry-tagged vBcl-2. We show that the vBcl-2 protein is expressed at relatively low levels during lytic induction and that a lack of vBcl-2 largely reduces the efficiency of KSHV reactivation in terms of lytic gene expression, viral DNA replication, and production of infectious particles. In contrast, the establishment of latency was not affected by the absence of vBcl-2. Our findings suggest an important role for vBcl-2 during initial phases of lytic reactivation and/or during subsequent viral propagation. Given the known functions of vBcl-2 in regulating apoptosis and autophagy, which involve its direct interaction with cellular proteins and thus require high levels of protein expression, it appears that vBcl-2 may have additional regulatory functions that do not depend on high levels of protein expression. IMPORTANCEThe present study shows for the first time the expression of endogenous vBcl-2 protein in KSHV-infected cell lines and demonstrates the importance of vBcl-2 during the initial phases of lytic reactivation and/or during its subsequent propagation. It is suggested that vBcl-2 has additional regulatory functions beyond apoptosis and autophagy repression that do not depend on high levels of protein expression. K aposi's sarcoma-associated herpesvirus (KSHV), also referred to as human herpesvirus 8 (HHV-8), is a gamma-2 herpesvirus. KSHV is causally linked to particular human cancers, including Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), and plasmablastic multicentric Castleman's disease (1-7). Among human viruses, KSHV is most closely related to the Epstein-Barr virus (EBV), a tumorigenic gamma-1 herpesvirus known to be associated with lymphomas and nasopharyngeal carcinoma. Like all other herpesviruses, the infectious cycle of KSHV includes two alternative infection phases, latent and lytic. Latent infection allows the virus to establish long-term persistent infection and involves the presence of viral episomes and expression of a small set of viral genes. Lytic infection is needed for the maintenance of viral reservoirs and for virus spread and involves a temporally regulated cascade of viral gene expression and DNA replication, leading to the production and release of new virions. KSHV exists mainly in its latent form in KS lesions and in PEL, yet a small subpopulation of cells undergoes lytic replication. Analogously, most KSHV-infected cultured cells are latently infected. Lytic virus react...

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Fluorescent Tagging and Cellular Distribution of the Kaposi's Sarcoma-Associated Herpesvirus ORF45 Tegument Protein

Cited by 16 publications

References 43 publications

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

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

Targeting the Kaposi’s Sarcoma-associated Herpesvirus Genome With the CRISPR-Cas9 Platform in Latently Infected Cells

Viral Bcl-2 Encoded by the Kaposi's Sarcoma-Associated Herpesvirus Is Vital for Virus Reactivation

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