Search

Several viruses have been linked to the development of cancer in humans. Epstein-Barr virus (EBV), the first identified human oncovirus, causes a wide range of cancers.

Protein–protein interactions (PPIs) are crucial for various biological processes. Epstein-Barr virus (EBV) proteins typically form complexes, regulating the replication and persistence of the viral genome in human cells. However, the role of EBV protein complexes under physiological conditions remains unclear. In this study, we performed comprehensive analyses of EBV PPIs in living cells using the NanoBiT system. We identified 195 PPIs, many of which have not previously been reported. Computational analyses of these PPIs revealed that BLRF2, which is only found in gammaherpesviruses, is a central protein in the structural network of EBV tegument proteins. To characterize the role of BLRF2, we generated two BLRF2 knockout EBV clones using CRISPR/Cas9. BLRF2 knockout significantly decreased the production of infectious virus particles, which was partially restored by exogenous BLRF2 expression. In addition, self-association of BLRF2 protein was found, and mutation of the residues crucial for the self-association affected stability of the protein. Our data imply that BLRF2 is a tegument network hub that plays important roles in progeny virion maturation. IMPORTANCE EBV remains a significant public health challenge, causing infectious mononucleosis and several cancer types. Therefore, the better understanding of the molecular mechanisms underlying EBV replication is of high clinical importance. As protein–protein interactions (PPIs) are major regulators of virus-associated pathogenesis, comprehensive analyses of PPIs are essential. Previous studies on PPIs in EBV or other herpesviruses have predominantly employed the yeast two-hybrid (Y2H) system, immunoprecipitation, and pulldown assays. Herein, using a novel luminescence-based method, we identified 195 PPIs, most of which have not previously been reported. Computational and functional analyses using knockout viruses revealed that BLRF2 plays a central role in the EBV life cycle, which makes it a valuable target for drug development.

Information regarding the infectivity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in asymptomatic carriers is scarce. In order to determine the duration of infectivity and its correlation with reverse transcription-PCR (RT-PCR) results and time since initial positive PCR test in this population, we evaluated SARS-CoV-2 cell infectivity in nasopharyngeal samples longitudinally obtained from asymptomatic carriers who disembarked from a cruise ship during a COVID-19 outbreak. Of 166 nasopharyngeal samples collected from 39 asymptomatic carriers every 48 h until two consecutive negative PCR test results were obtained, SARS-CoV-2 was successfully isolated from 9 PCR-positive samples which were obtained from 7 persons (18%; 7/39). Viable viruses were isolated predominantly within 7 days after the initial positive PCR test, except for one person who shed viable virus until day 15. The median crossing point (Cp) value of RT-PCR of culture-positive samples was 24.6 (interquartile range [IQR], 20.4 to 25.8; range, 17.9 to 30.3), and Cp values were significantly associated with isolation of viable virus (odds ratio, 0.496; 95% confidence interval [CI], 0.329 to 0.747; P value, 0.001), which was consistent with existing data for symptomatic patients. Genome sequence analysis of SARS-CoV-2 samples consecutively obtained from a person who shed viable virus for 15 days identified the emergence of two novel single nucleotide variants (C8626T transition and C18452T transition) in the sample collected on day 15, with the latter corresponding to an amino acid substitution in nonstructural protein 14. The impact of these mutations on prolonged viable-virus shedding is unclear. These findings underscore the potential role of asymptomatic carriers in transmission. IMPORTANCE A growing number of studies suggest the potential role of asymptomatic SARS-CoV-2 carriers as a major driver of the COVID-19 pandemic; however, virological assessment of asymptomatic infection has largely been limited to reverse transcription-PCR (RT-PCR), which can be persistently positive without necessarily indicating the presence of viable virus (e.g., replication-competent virus). Here, we evaluated the infectivity of asymptomatic SARS-CoV-2 carriers by detecting SARS-CoV-2-induced cytopathic effects on Vero cells using longitudinally obtained nasopharyngeal samples from asymptomatic carriers. We show that asymptomatic carriers can shed viable virus until 7 days after the initial positive PCR test, with one outlier shedding until day 15. The crossing point (Cp) value of RT-PCR was the leading predictive factor for virus viability. These findings provide additional insights into the role of asymptomatic carriers as a source of transmission and highlight the importance of universal source control measures, along with isolation policy for asymptomatic carriers.

Temporally controlled gene expression is necessary for the propagation of herpesviruses. To achieve this, herpesviruses encode several transcriptional regulators. In Epstein-Barr virus, BcRF1 associates with five viral proteins (BDLF4, BGLF3, BFRF2, BVLF1, and BDLF3.5) to form the viral late (L) gene regulatory complex, which is called the viral preinitiation complex (vPIC), on TATT-containing promoters. However, regulation of the vPIC has been largely unexplored. In this study, we performed two screens using a kinase inhibitor library and identified a series of cyclin-dependent kinase (CDK) inhibitors that downregulated the expression of L genes without any impact on viral DNA replication through destabilization of the BDLF4 protein. Knockdown of CDK2 by short hairpin RNA (shRNA) and proteasome inhibitor treatment showed that phosphorylation of the BDLF4 protein prevented ubiquitin-mediated degradation. Moreover, we demonstrated that cyclin A- and E-associated CDK2 complexes phosphorylated BDLF4 in vitro, and we identified several serine/threonine phosphorylation sites in BDLF4. Phosphoinactive and phosphomimic mutants revealed that phosphorylation at threonine 91 plays a role in stabilizing BDLF4. Therefore, our findings indicate that S-like-phase CDKs mediate the regulation of L gene expression through stabilization of the BDLF4 protein, which makes the temporal L gene expression system more robust. IMPORTANCE Late (L) genes represent more than one-third of the herpesvirus genome, suggesting that many of these genes are indispensable for the life cycle of the virus. With the exception of BCRF1, BDLF2, and BDLF3, Epstein-Barr virus L genes are transcribed by viral regulators, which are known as the viral preinitiation complex (vPIC) and the host RNA polymerase II complex. Because the vPIC is conserved in beta- and gammaherpesviruses, studying the control of viral L gene expression by the vPIC contributes to the development of drugs that specifically inhibit these processes in beta- and gammaherpesvirus infections/diseases. In this study, we demonstrated that CDK inhibitors induced destabilization of the vPIC component BDLF4, leading to a reduction in L gene expression and subsequent progeny production. Our findings suggest that CDK inhibitors may be a therapeutic option against beta- and gammaherpesviruses in combination with existing inhibitors of herpesvirus lytic replication, such as ganciclovir.

Latent membrane protein 1 (LMP1) is a major oncogene essential for primary B cell transformation by Epstein-Barr virus (EBV). Previous studies suggested that some transcription factors, such as PU.1, RBP-Jκ, NF-κB, and STAT, are involved in this expression, but the underlying mechanism is unclear. Here, we identified binding sites for PAX5, AP-2, and EBF in the proximal LMP1 promoter (ED-L1p). We first confirmed the significance of PU.1 and POU domain transcription factor binding for activation of the promoter in latency III. We then focused on the transcription factors AP-2 and early B cell factor (EBF). Interestingly, among the three AP-2-binding sites in the LMP1 promoter, two motifs were also bound by EBF. Overexpression, knockdown, and mutagenesis in the context of the viral genome indicated that AP-2 plays an important role in LMP1 expression in latency II in epithelial cells. In latency III B cells, on the other hand, the B cell-specific transcription factor EBF binds to the ED-L1p and activates LMP1 transcription from the promoter. IMPORTANCE Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) is crucial for B cell transformation and oncogenesis of other EBV-related malignancies, such as nasopharyngeal carcinoma and T/NK lymphoma. Its expression is largely dependent on the cell type or condition, and some transcription factors have been implicated in its regulation. However, these previous reports evaluated the significance of specific factors mostly by reporter assay. In this study, we prepared point-mutated EBV at the binding sites of such transcription factors and confirmed the importance of AP-2, EBF, PU.1, and POU domain factors. Our results will provide insight into the transcriptional regulation of the major oncogene LMP1.

An entirely plasmid-based reverse genetics system for rotaviruses was established very recently. We improved the reverse genetics system to generate recombinant rotavirus by transfecting only 11 cDNA plasmids for its 11 gene segments under the condition of increasing the ratio of the cDNA plasmids for NSP2 and NSP5 genes. Utilizing this highly efficient system, we then engineered infectious recombinant rotaviruses expressing bioluminescent (NanoLuc luciferase) and fluorescent (enhanced green fluorescent protein [EGFP] and mCherry) reporters. These recombinant rotaviruses expressing reporters remained genetically stable during serial passages. Our reverse genetics approach and recombinant rotaviruses carrying reporter genes will be great additions to the tool kit for studying the molecular virology of rotavirus and for developing future next-generation vaccines and expression vectors. IMPORTANCE Rotavirus is one of the most important pathogens causing severe gastroenteritis in young children worldwide. In this paper, we describe a robust and simple reverse genetics system based on only rotavirus cDNAs and its application for engineering infectious recombinant rotaviruses harboring bioluminescent (NanoLuc) and fluorescent (EGFP and mCherry) protein genes. This highly efficient reverse genetics system and recombinant group A rotaviruses expressing reporters could be powerful tools for the study of different aspects of rotavirus replication. Furthermore, they may be useful for next-generation vaccine production for this medically important virus.

Epstein-Barr virus (EBV) is a human gammaherpesvirus that causes infectious mononucleosis and several malignancies, such as endemic Burkitt lymphoma and nasopharyngeal carcinoma. Herpesviruses carry genes that can modify cell functions, including transcription and ubiquitination, thereby facilitating viral growth and survival in infected cells. Using a reporter screening system, we revealed the involvement of several EBV gene products in such processes. Of these, BGLF2 activated the AP-1 signaling pathway through phosphorylation of p38 and c-Jun N-terminal kinase (JNK). Knockout of the BGLF2 gene did not affect viral gene expression and viral genome DNA replication, but resulted in marked reduction of progeny titer. We also found that the BGLF2 disruption resulted in significant loss of infectivity upon de novo infection. Interestingly, expression of a binding partner, BKRF4, repressed the activation of AP-1 by BGLF2. These results shed light on the physiological role of the tegument protein BGLF2. IMPORTANCE Epstein-Barr virus (EBV), an oncogenic gammaherpesvirus, carries ~80 genes. While several genes have been investigated extensively, most lytic genes remain largely unexplored. Therefore, we cloned 71 EBV lytic genes into an expression vector and used reporter assays to screen for factors that activate signal transduction pathways, viral and cellular promoters. BGLF2 activated the AP-1 signaling pathway, likely by interacting with p38 and c-Jun N-terminal kinase (JNK), and increased infectivity of the virus. We also revealed that BKRF4 can negatively regulate AP-1 activity. Therefore, it is suggested that EBV exploits and modifies the AP-1 signaling pathway for its replication and survival.

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. IMPORTANCE 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.

Reactivation of Epstein-Barr virus (EBV) from latency is dependent on expression of the viral transactivator BZLF1 protein, whose promoter (Zp) normally exhibits only low basal activity but is activated in response to chemical or biological inducers. Using a reporter assay system, we screened for factors that can activate Zp and isolated genes, including those encoding MEF2B, KLF4, and some cellular b-Zip family transcription factors. After confirming their importance and functional binding sites in reporter assays, we prepared recombinant EBV-BAC, in which the binding sites were mutated. Interestingly, the MEF2 mutant virus produced very low levels of BRLF1, another transactivator of EBV, in addition to BZLF1 in HEK293 cells. The virus failed to induce a subset of early genes, such as that encoding BALF5, upon lytic induction, and accordingly, could not replicate to produce progeny viruses in HEK293 cells, but this restriction could be completely lifted by exogenous supply of BRLF1, together with BZLF1. In B cells, induction of BZLF1 by chemical inducers was inhibited by point mutations in the ZII or the three SP1/KLF binding sites of EBV-BAC Zp, while leaky BZLF1 expression was less affected. Mutation of MEF2 sites severely impaired both spontaneous and induced expression of not only BZLF1, but also BRLF1 in comparison to wild-type or revertant virus cases. We also observed that MEF2 mutant EBV featured relatively high repressive histone methylation, such as H3K27me3, but CpG DNA methylation levels were comparable around Zp and the BRLF1 promoter (Rp). These findings shed light on BZLF1 expression and EBV reactivation from latency.

Productive replication of the Epstein-Barr virus (EBV) occurs in discrete sites in nuclei, called replication compartments, where viral genome DNA synthesis and transcription take place. The replication compartments include subnuclear domains, designated BMRF1 cores, which are highly enriched in the BMRF1 protein. During viral lytic replication, newly synthesized viral DNA genomes are organized around and then stored inside BMRF1 cores. Here, we examined spatial distribution of viral early and late gene mRNAs within replication compartments using confocal laser scanning microscopy and three-dimensional surface reconstruction imaging. EBV early mRNAs were mainly located outside the BMRF1 cores, while viral late mRNAs were identified inside, corresponding well with the fact that late gene transcription is dependent on viral DNA replication. From these results, we speculate that sites for viral early and late gene transcription are separated with reference to BMRF1 cores.

The regulation of human cytomegalovirus (HCMV) late gene expression by viral proteins is poorly understood, and these viral proteins could be targets for novel antivirals. HCMV open reading frames (ORFs) UL79, -87, and -95 encode proteins with homology to late gene transcription factors of murine gammaherpesvirus 68 ORFs 18, 24, and 34, respectively. To determine whether these HCMV proteins are also essential for late gene transcription of a betaherpesvirus, we mutated HCMV ORFs UL79, -87, and -95. Cells were infected with the recombinant viruses at high and low multiplicities of infection (MOIs). While viral DNA was detected with the recombinant viruses, infectious virus was not detected unless the wild-type viral proteins were expressed in trans . At a high MOI, mutation of ORF UL79, -87, or -95 had no effect on the level of major immediate-early (MIE) gene expression or viral DNA replication, but late viral gene expression from the UL44, -75, and -99 ORFs was not detected. At a low MOI, preexpression of UL79 or -87, but not UL95, in human fibroblast cells negatively affected the level of MIE viral gene expression and viral DNA replication. The products of ORFs UL79, -87, and -95 were expressed as early viral proteins and recruited to prereplication complexes (pre-RCs), along with UL44, before the initiation of viral DNA replication. All three HCMV ORFs are indispensable for late viral gene expression and viral growth. The roles of UL79, -87, and -95 in pre-RCs for late viral gene expression are discussed.

The promoter of the major immediate-early (MIE) genes of human cytomegalovirus (HCMV), also referred to as the CMV promoter, possesses a cis -acting element positioned downstream of the TATA box between positions −14 and −1 relative to the transcription start site (+1). We determined the role of the cis -acting element in viral replication by comparing recombinant viruses with the cis -acting element replaced with other sequences. Recombinant virus with the simian CMV counterpart replicated efficiently in human foreskin fibroblasts, as well as wild-type virus. In contrast, replacement with the murine CMV counterpart caused inefficient MIE gene transcription, RNA splicing, MIE and early viral gene expression, and viral DNA replication. To determine which nucleotides in the cis -acting element are required for efficient MIE gene transcription and splicing, we constructed mutations within the cis -acting element in the context of a recombinant virus. While mutations in the cis -acting element have only a minor effect on in vitro transcription, the effects on viral replication are major. The nucleotides at −10 and −9 in the cis -acting element relative to the transcription start site (+1) affect efficient MIE gene transcription and splicing at early times after infection. The cis -acting element also acts as a cis -repression sequence when the viral IE86 protein accumulates in the infected cell. We demonstrate that the cis -acting element has an essential role in viral replication.

Epstein-Barr virus (EBV) is a gammaherpesvirus, associated with infectious mononucleosis and various types of malignancy. We focused here on the BDLF4 gene of EBV and identified it as a lytic gene, expressed with early kinetics. Viral late gene expression of the BDLF4 knockout strain was severely restricted; this could be restored by an exogenous supply of BDLF4. These results indicate that BDLF4 is important for the EBV lytic replication cycle, especially in late gene expression.

We previously reported that nucleolin, a representative nucleolar marker, interacts with nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) through two independent regions of NS5B, amino acids 208 to 214 and 500 to 506. We also showed that truncated nucleolin that harbors the NS5B-binding region inhibited the RNA-dependent RNA polymerase activity of NS5B in vitro, suggesting that nucleolin may be involved in HCV replication. To address this question, we focused on NS5B amino acids 208 to 214. We constructed one alanine-substituted clustered mutant (CM) replicon, in which all the amino acids in this region were changed to alanine, as well as seven different point mutant (PM) replicons, each of which harbored an alanine substitution at one of the amino acids in the region. After transfection into Huh7 cells, the CM replicon and the PM replicon containing NS5B W208A could not replicate, whereas the remaining PM replicons were able to replicate. In vivo immunoprecipitation also showed that the W208 residue of NS5B was essential for its interaction with nucleolin, strongly suggesting that this interaction is essential for HCV replication. To gain further insight into the role of nucleolin in HCV replication, we utilized the small interfering RNA (siRNA) technique to investigate the knockdown effect of nucleolin on HCV replication. Cotransfection of replicon RNA and nucleolin siRNA into Huh7 cells moderately inhibited HCV replication, although suppression of nucleolin did not affect cell proliferation. Taken together, our findings strongly suggest that nucleolin is a host component that interacts with HCV NS5B and is indispensable for HCV replication.

Leptomycin B (LMB) is a specific inhibitor of Crm1-dependent nuclear export of proteins. The replication of herpes simplex virus (HSV) is normally highly sensitive to LMB; a resistant HSV variant, however, was isolated by serial passages of the virus. Analysis of marker transfer and viral DNA sequences revealed that a single amino acid substitution within the ICP27 gene is responsible for conferring this resistance.