The Contribution of Kaposi’s Sarcoma-Associated Herpesvirus ORF7 and Its Zinc-Finger Motif to Viral Genome Cleavage and Capsid Formation

Iwaisako, Yuki; Watanabe, Tadashi; Futo, Manami; Okabe, Rimiko; Sekine, Yoshifumi; Suzuki, Youichi; Nakano, Takashi; Fujimuro, Masahiro

doi:10.1128/jvi.00684-22

Cited by 7 publications

(30 citation statements)

References 43 publications

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“…However, we found that ORF7 interacted with both ORF29 and ORF67.5 (32). We also showed that ORF7-deficient KSHV formed numerous immature soccer ball-like capsids in the nucleus and failed to cleave the KSHV TR (33). The soccer ball-like capsid was a characteristic immature capsid that contains many particulate structures.…”

Section: Introductionmentioning

confidence: 78%

Kaposi’s sarcoma-associated herpesvirus ORF67.5 functions as a component of the terminase complex

Iwaisako

Watanabe

Suzuki

et al. 2023

Preprint

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Kaposi’s sarcoma-associated herpesvirus (KSHV) is a double-stranded DNA gammaherpesvirus, with a poorly characterized lytic replication cycle. However, the lytic replication cycle of the alpha- and betaherpesviruses are well characterized. During lytic infection of alpha- and betaherpesviruses, the viral genome is replicated as a precursor form, which contains tandem genomes linked via terminal repeats (TRs). One genomic unit of the precursor form is packaged into a capsid and is cleaved at the TR by the terminase complex. While the alpha- and betaherpesvirus terminases are well characterized, the KSHV terminase remains poorly understood. KSHV ORF7, ORF29, and ORF67.5 are presumed to be components of the terminase complex based on their homology to other terminase proteins. We previously reported that ORF7-deficient KSHV formed numerous immature soccer ball-like capsids and failed to cleave the TRs. ORF7 interacted with ORF29 and ORF67.5, however ORF29 and ORF67.5 did not interact with each other. While these results suggested that ORF7 is important for KSHV terminase function and capsid formation, the function of ORF67.5 was completely unknown. Therefore, to analyze the function of ORF67.5, we constructed ORF67.5-deficient BAC16. ORF67.5-deficient KSHV failed to produce infectious virus, failed to cleave the TRs, and numerous soccer ball-like capsids were observed in ORF67.5-deficient KSHV-harboring cells. Furthermore, ORF67.5 promoted the interaction between ORF7 and ORF29, and ORF29 increased the interaction between ORF67.5 and ORF7. Thus, our data indicated that ORF67.5 functions as a component of the KSHV terminase complex by contributing to TR cleavage, terminase complex formation, capsid formation, and virus production.

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Section: Introductionmentioning

confidence: 78%

Kaposi’s sarcoma-associated herpesvirus ORF67.5 functions as a component of the terminase complex

Iwaisako

Watanabe

Suzuki

et al. 2023

Preprint

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“…To establish efficient recombinant virus-producing cells, tetracycline/doxycycline (Dox)-inducible (Tet-On) RTA/ORF50-expressing iSLK cells [ 47 , 48 , 49 ] or iVero cells [ 50 ] were used as recombinant KSHV-producing cells. For maintenance, iSLK cells were cultured in a growth medium containing 1 μg/mL of puromycin (InvivoGen, CA, USA) and 0.25 mg/mL of G418 (Nacalai Tesque, Kyoto, Japan).…”

Section: Methodsmentioning

confidence: 99%

“…The complementation assay was performed as previously described, but with several modifications [ 49 , 52 ]. The iSLK-21del cells (1 × 10 6 cells) were transfected with the 3×Flag-ORF21 plasmid using ScreenFect A plus (Wako) and treated with Dox and NaB.…”

Section: Methodsmentioning

confidence: 99%

Kaposi’s Sarcoma-Associated Herpesvirus ORF21 Enhances the Phosphorylation of MEK and the Infectivity of Progeny Virus

Yamaguchi

Watanabe

Iwaisako

et al. 2023

IJMS

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Kaposi’s sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus-8, is the causative agent of Kaposi’s sarcoma, Castleman’s disease, and primary effusion lymphoma. Although the functions of the viral thymidine kinases (vTK) of herpes simplex virus-1/2 are well understood, that of KSHV ORF21 (an ortholog of vTK) is largely unknown. Here, we investigated the role of ORF21 in lytic replication and infection by generating two ORF21-mutated KSHV BAC clones: ORF21-kinase activity deficient KSHV (21KD) and stop codon-induced ORF21-deleted KSHV (21del). The results showed that both ORF21 mutations did not affect viral genome replication, lytic gene transcription, or the production of viral genome-encapsidated particles. The ORF21 molecule-dependent function, other than the kinase function of ORF21, was involved in the infectivity of the progeny virus. ORF21 was expressed 36 h after the induction of lytic replication, and endogenously expressed ORF21 was localized in the whole cytoplasm. Moreover, ORF21 upregulated the MEK phosphorylation and anchorage-independent cell growth. The inhibition of MEK signaling by U0126 in recipient target cells suppressed the number of progeny virus-infected cells. These suggest that ORF21 transmitted as a tegument protein in the progeny virus enhances the new infection through MEK up-regulation in the recipient cell. Our findings indicate that ORF21 plays key roles in the infection of KSHV through the manipulation of the cellular function.

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“…To establish efficient recombinant virus-producing cells, tetracycline/doxycycline (Dox)-inducible (Tet-On) RTA/ORF50-expressing iSLK cells [47][48][49] or iVero cells [50] were used as recombinant KSHV-producing cells. For maintenance, iSLK cells were cultured in growth medium containing 1 μg/ml of puromycin (InvivoGen, CA, USA) and 0.25 mg/ml of G418 (Nacalai Tesque, Kyoto, Japan).…”

Section: Establishment Of Doxycycline-inducible Recombinant Kshv-expr...mentioning

confidence: 99%

“…The complementation assay was performed as previously described but with several modifications [49,52]. The iSLK-21del cells (1 × 106 cells) were transfected with 3xFlag-ORF21 plasmid using ScreenFect A plus (Fujifilm Wako Pure Chemical Corporation, Osaka, Japan) and treated with Dox and NaB.…”

Section: Complementation Assaymentioning

confidence: 99%

Kaposi’s Sarcoma-associated Herpesvirus ORF21 Enhances the Phosphorylation of MEK and the Infectivity of Progeny Virus

Yamaguchi¹,

Watanabe²,

Iwaisako³

et al. 2022

Preprint

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Kaposi’s sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus-8, is the causative agent of Kaposi’s sarcoma, Castleman’s disease, and primary effusion lymphoma. Although the functions of the viral thymidine kinases (vTK) of herpes simplex virus-1/2 and varicella zoster virus are well understood, that of KSHV ORF21 (an ortholog of vTK) is largely unknown. Here, we investigated the role of ORF21 in lytic replication and infection by generating two ORF21-mutated KSHV BAC clones: ORF21-kinase activity deficient-KSHV (21KD) and stop codon-induced ORF21-deleted-KSHV (21del). The results showed that both ORF21-mutations did not affect viral genome replication, lytic genes transcription, or the production of viral genome-encapsidated particles. ORF21 molecule-dependent function, other than the kinase function of ORF21, was involved in the infectivity of progeny virus. ORF21 was expressed at 36 h after induction of lytic replication, and endogenously expressed ORF21 was localized in the whole cytoplasm and decreased on the cell surface area. Moreover, the effects of ORF21 expression on signaling pathways and proliferation were analyzed. The results showed that ORF21 upregulated the MEK phosphorylation and anchorage-independent cell growth. These findings indicate that ORF21 plays key roles in both infection and oncogenesis of KSHV through the manipulation of cellular function.

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The Contribution of Kaposi’s Sarcoma-Associated Herpesvirus ORF7 and Its Zinc-Finger Motif to Viral Genome Cleavage and Capsid Formation

Cited by 7 publications

References 43 publications

Kaposi’s sarcoma-associated herpesvirus ORF67.5 functions as a component of the terminase complex

Kaposi’s sarcoma-associated herpesvirus ORF67.5 functions as a component of the terminase complex

Kaposi’s Sarcoma-Associated Herpesvirus ORF21 Enhances the Phosphorylation of MEK and the Infectivity of Progeny Virus

Kaposi’s Sarcoma-associated Herpesvirus ORF21 Enhances the Phosphorylation of MEK and the Infectivity of Progeny Virus

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