Biology subjects

Fujimuro, M.

Publications and source records attributed to Fujimuro, M..

6 recordsLinked to original sources

bioRxiv2025

DISC1-PML protein interaction for congenital CMV infection-induced cortical neural progenitor deficit: perturbance of host signaling via viral IE1

Congenital CMV infection is the most common perinatal infection, affecting up to 0.5% of infants. This elicits long-term disabilities that include neuropsychiatric manifestations, such as intellectual disability, microcephaly. Despite its high prevalence, the underlying mechanism of how congenitally acquired CMV infection causes brain pathology remains unknown. Here we discovered the molecular interplay of key host (DISC1 and PML) and viral (IE1) proteins within the neural progenitor cells, which underlay an attenuated neural progenitor proliferation. Abolishing the viral IE1 protein by delivering IE1-targeting CRISPR/Cas9 to fetal brain rescued this progenitor cell deficit, a key pathology in congenital CMV infection. A selective targeting to a viral-specific protein by the CRISPR/Cas9 system is minimal in off-target effects. Therefore, we believe that a pivotal role of IE1 in an attenuated neural progenitor proliferation in the developing cortex through its interfering with interaction between host DISC1 and PML proteins.

developmental biology↗

bioRxiv2024

Characterization of the Kaposi's sarcoma-associated herpesvirus terminase complex component ORF29

Kaposis sarcoma-associated herpesvirus (KSHV) belongs to the gammaherpesvirinae subfamily. In the lytic phase of herpesviruses, viral capsids are formed in the nucleus of the host cell and the replicated viral genome is packaged into capsids. The herpesviral genome is replicated as a precursor head-to-tail concatemer consisting of tandemly repeated genomic units, and each genomic unit is flanked by terminal repeats (TRs). The herpesvirus terminase complex packages one genomic unit into a capsid, by cleavage of the TRs in a precursor genome. Although the terminase complexes of alpha- and beta-herpesviruses are well characterized, in KSHV, the terminase complex is poorly understood. KSHV ORF7, ORF67.5, and ORF29 are thought to be components of the terminase complex. We previously reported that ORF7- or ORF67.5-deficient KSHV formed immature soccer ball-like capsids and failed to cleave the TRs, resulting in decreased virion production. Moreover, ORF7 interacted with ORF29 and ORF67.5. However, ORF29 and ORF67.5 did not interact with each other. Thus, although ORF7 and ORF67.5 are important for KSHV terminase function, the function of ORF29 remains largely unknown. Here, we constructed ORF29-deficient BAC16 and analyzed its virological properties. ORF29 was essential for virion production and TR cleavage. Numerous immature soccer ball-like capsids were observed in ORF29-deficient KSHV-harboring cells. The N-terminal region of ORF29 was important for its interaction with ORF7, although full-length ORF29 was required for effective complexation of the KSHV terminase. Furthermore, ORF29 preferentially interacted with itself rather than with ORF7. Thus, our data shows that ORF29 functions as a fundamental terminase component. IMPORTANCESince the role of ORF29 in the KSHV terminase complex remains unknown, we constructed ORF29-deficient KSHV. Our results demonstrated that ORF29 functions as a component of the KSHV terminase and is critical for capsid formation, TR cleavage, and terminase complexation. Moreover, ORF29 robustly interacted with itself. In HSV-1, a terminase complex containing UL15, UL28, and UL33 forms a trimer, and six trimers assemble into a hexameric ring. The HSV-1 genome passes through this ring and undergoes TR cleavage and genome packaging into a capsid. The self-interaction of ORF29 may be involved in the multimerization of a terminase complex or formation of the KSHV terminase ring. In addition, a novel KSHV protein, which we refer to as ORF29.5, was expressed during the lytic state. ORF29.5 mRNA shares the same stop codon with the ORF29 mRNA. Moreover, a part of the ORF29.5 coding region may overlap with the C-terminal ORF29 coding region.

microbiology↗

bioRxiv2023

Kaposis sarcoma-associated herpesvirus ORF67.5 functions as a component of the terminase complex

Kaposis 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. IMPORTANCEAlthough the formation and function of the alpha- and betaherpesvirus terminase complexes are well understood, the KSHV terminase complex is still largely uncharacterized. This complex presumably contains KSHV ORF7, ORF29, and ORF67.5. We were the first to report the presence of soccer ball-like capsids in ORF7-deficient KSHV-harboring lytic-induced cells. Here, we demonstrated that ORF67.5-deficient KSHV also formed the soccer ball-like capsids in lytic-induced cells. Moreover, ORF67.5 was required for TR cleavage, infectious virus production, and enhancement of the interaction between ORF7 and ORF29. ORF67.5 has several highly conserved regions among its human herpesviral homologs. These regions were necessary for virus production and for the interaction of ORF67.5 with ORF7, which was supported by the AI-predicted structure model. Importantly, our results provide the first evidence showing that ORF67.5 is essential for terminase complex formation and TR cleavage.

microbiology↗

bioRxiv2023

Conserved cysteine residues in Kaposis sarcoma herpesvirus ORF34 are necessary for viral production and viral pre-initiation complex formation.

Kaposis sarcoma herpesvirus (KSHV) ORF34 plays a significant role as a component of the viral pre-initiation complex (vPIC), which is indispensable for late gene expression across beta and gamma herpesviruses. Although the key role of ORF34 within the vPIC and its function as a hub protein have been recognized, further clarification regarding its specific contribution to vPIC functionality and interactions with other components is required. This study employed a deep-learning algorithm-assisted structural model of ORF34, revealing highly conserved amino acid residues across human beta- and gamma-herpesviruses localized in structured domains. Thus, we engineered ORF34 alanine-scanning mutants by substituting conserved residues with alanine. These mutants were evaluated for their ability to interact with other vPIC factors and restore viral production in cells harboring the ORF34-deficient KSHV-BAC. Our experimental results highlight the crucial role of the 4 cysteine residues conserved in ORF34: a tetrahedral arrangement consisting of a pair of C-Xn-C consensus motifs. This suggests the potential incorporation of metal cations in interacting with ORF24 and ORF66 vPIC components, facilitating late gene transcription, and promoting overall virus production by capturing metal cations. In summary, our findings underline the essential role of conserved cysteines in KSHV ORF34 for effective vPIC assembly and viral replication, thereby enhancing our understanding of the complex interplay between the vPIC components. IMPORTANCEThe initiation of late gene transcription is universally conserved across the gamma- and beta-herpesvirus families. This process employs a viral pre-initiation complex (vPIC), which is analogous to a cellular PIC. Although KSHV ORF34 is a critical factor for viral replication and is a component of the vPIC, the specifics of vPIC formation and the essential domains crucial for its function remain unclear. Structural predictions suggest that the 4 conserved cysteines (C170, C175, C256, and C259) form a tetrahedron that coordinates the metal cation. We further investigated the role of these conserved amino acids in interactions with other vPIC components, late gene expression, and virus production, to demonstrate for the first time that these cysteines are pivotal for these functions. This discovery not only deepens our comprehensive understanding of ORF34 and vPIC dynamics but also lays the groundwork for more detailed studies on herpesvirus replication mechanisms in future research.

microbiology↗

bioRxiv2022

The contribution of Kaposis sarcoma-associated herpesvirus ORF7 and its zinc-finger motif to viral genome cleavage and capsid formation

Kaposis sarcoma-associated herpesvirus (KSHV) is the causative agent of endothelial and B cell malignancies. During KSHV lytic infection, lytic-related proteins are synthesized, viral genomes are replicated as a tandemly repeated form, and subsequently, capsids are assembled. The herpesvirus terminase complex is proposed to package an appropriate genome unit into an immature capsid, by cleavage of terminal repeats (TRs) flanking tandemly linked viral genomes. Although the mechanism of capsid formation in - and {beta}-herpesviruses are well-studied, in KSHV, it remains largely unknown. It has been proposed that KSHV ORF7 is a terminase subunit, and ORF7 harbors a zinc-finger motif, which is conserved among other herpesviral terminases. However, the biological significance of ORF7 is unknown. We previously reported that KSHV ORF17 is essential for the cleavage of inner scaffold proteins in capsid maturation, and ORF17 knockout (KO) induced capsid formation arrest between the procapsid and B-capsid stages. However, it remains unknown if ORF7-mediated viral DNA cleavage occurs before or after ORF17-mediated scaffold collapse. We analyzed the role of ORF7 during capsid formation using ORF7-KO-, ORF7&17-double-KO (DKO)-, and ORF7-zinc-finger motif mutant-KSHVs. We found that ORF7 acted after ORF17 in the capsid formation process, and ORF7-KO-KSHV produced incomplete capsids harboring non-spherical internal structures, which resembled soccer balls. This soccer ball-like capsid was formed after ORF17-mediated B-capsid formation. Moreover, ORF7-KO- and zinc-finger motif KO-KSHV failed to appropriately cleave the TR on replicated genome and had a defect in virion production. Thus, our data revealed that ORF7 contributes to terminase-mediated viral genome cleavage and capsid formation. IMPORTANCEIn herpesviral capsid formation, the viral terminase complex cleaves the TR sites on newly synthesized tandemly repeating genomes and inserts an appropriate genomic unit into an immature capsid. Herpes simplex virus 1 (HSV-1) UL28 is a subunit of the terminase complex that cleaves the replicated viral genome. However, the physiological importance of the UL28 homolog, KSHV ORF7, remains poorly understood. Here, using several ORF7-deficent KSHVs, we found that ORF7 acted after ORF17-mediated scaffold collapse in the capsid maturation process. Moreover, ORF7 and its zinc-finger motif were essential for both cleavage of TR sites on the KSHV genome and virus production. ORF7-deficient KSHVs produced incomplete capsids that resembled a soccer ball. To our knowledge, this is the first report showing ORF7-KO-induced soccer ball-like capsids production and ORF7 function in the KSHV capsid assembly process. Our findings provide insights into the role of ORF7 in KSHV capsid formation.

microbiology↗

bioRxiv2019

Kaposi’s sarcoma-associated herpesvirus ORF66 is essential for late gene expression and virus production via interaction with ORF34

Kaposis sarcoma-associated herpesvirus (KSHV) is closely associated with B-cell and endothelial cell malignancies. After the initial infection, KSHV retains its viral genome in the nucleus of the host cell and establishes a lifelong latency. During lytic infection, KSHV encoded lytic-related proteins are expressed in a sequential manner and are classified as immediate early, early, and late gene transcripts. The transcriptional initiation of KSHV late genes is thought to require the complex formation of the virus specific pre-initiation complex (vPIC), which may consist of at least 6 transcription factors (ORF18, 24, 30, 31, 34, and 66). However, the functional role of ORF66 in vPIC during KSHV replication remains largely unclear. Here, we generated ORF66-deficient KSHV using a BAC system to evaluate its role during viral replication. While ORF66-deficient KSHV demonstrated mainly attenuated late gene expression and decreased viral production, viral DNA replication was unaffected. CHIP analysis showed that ORF66 bound to the promoters of late gene (K8.1), but did not to those of latent gene (ORF72), immediate early gene (ORF16) and early gene (ORF46/47). Furthermore, we found that three highly conserved C-X-X-C sequences and a conserved leucine-repeat in the C-terminal region of ORF66 were essential for interaction with ORF34 and viral production. The interaction between ORF66 and ORF34 occurred in a zinc-dependent manner. Our data support a model, in which ORF66 serves as a critical vPIC component to promote late viral gene expression and viral production.\n\nIMPORTANCEKSHV ORF66, a late gene product, and vPIC are thought to contribute significantly to late gene expression during the lytic replication. However, the physiological importance of ORF66 in terms of viral replication and vPIC formation remains poorly understood. Therfore, we generated a ORF66-deficient BAC clone and evaluated its viral replication. Results showed that ORF66 played a critical role in virus production and the transcription of L genes. To our knowledge, this is the first report showing ORF66 function in virus replication using ORF66-deficient KSHV. We also clarified that ORF66 interacted with the transcription start site of K8.1 gene, a late gene. Furthermore, we identified the ORF34-binding motifs in the ORF66 C-terminus: three C-X-X-C sequences and a leucine-repeat sequence, which are highly conserved among {beta}- and {gamma}-herpesviruses. Our study provides insights into the regulatory mechanisms of not only the late gene expression of KSHV but also those of other herpesviruses.

microbiology↗