Palmitoylation of KSHV pORF55 is required for Golgi localization and efficient progeny virion production.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a double-stranded DNA virus etiologically associated with multiple malignancies. Both latency and sporadic lytic reactivation contribute to KSHV-associated malignancies, however, the specific roles of many KSHV lytic gene products in KSHV replication remain elusive. In this study, we report that ablation of ORF55, a late gene encoding a tegument protein, does not impact KSHV lytic reactivation but significantly reduces the production of progeny virions. We found that cysteine 10 and 11 (C10 and C11) of pORF55 are palmitoylated, and the palmytoilation is essential for its Golgi localization and secondary envelope formation. Palmitoylation-defective pORF55 mutants are unstable and undergo proteasomal degradation. Notably, introduction of a putative Golgi localization sequence to these palmitoylation-defective pORF55 mutants restores Golgi localization and fully reinstates KSHV progeny virion production. Together, our study provides new insight into the critical role of pORF55 palmitoylation in KSHV progeny virion production and offers potential therapeutic targets for the treatment of related malignancies.

The recruitment of TRiC chaperonin in rotavirus viroplasms correlates with virus replication.

Rotavirus (RV) replication takes place in the viroplasms, cytosolic inclusions that allow the synthesis of virus genome segments and their encapsidation in the core shell, followed by the addition of the second layer of the virion. The viroplasms are composed of several viral proteins, including NSP5, which serves as the main building block. Microtubules, lipid droplets, and miRNA-7 are among the host components recruited in viroplasms. We investigated the interaction between RV proteins and host components of the viroplasms by performing a pull-down assay of lysates from RV-infected cells expressing NSP5-BiolD2. Subsequent tandem mass spectrometry identified all eight subunits of the tailless complex polypeptide I ring complex (TRiC), a cellular chaperonin responsible for folding at least 10% of the cytosolic proteins. Our confirmed findings reveal that TRiC is brought into viroplasms and wraps around newly formed double-layered particles. Chemical inhibition of TRiC and silencing of its subunits drastically reduced virus progeny production. Through direct RNA sequencing, we show that TRiC is critical for RV replication by controlling dsRNA genome segment synthesis, particularly negative-sense single-stranded RNA. Importantly, cryo-electron microscopy analysis shows that TRiC inhibition results in defective virus particles lacking genome segments and polymerase complex (VP1/VP3). Moreover, TRiC associates with VP2 and NSP5 but not with VP1. Also, VP2 is shown to be essential for recruiting TRiC in viroplasms and preserving their globular morphology. This study highlights the essential role of TRiC in viroplasm formation and in facilitating virion assembly during the RV life cycle. IMPORTANCE: The replication of rotavirus takes place in cytosolic inclusions termed viroplasms. In these inclusions, the distinct 11 double-stranded RNA genome segments are co-packaged to complete a genome in newly generated virus particles. In this study, we show for the first time that the tailless complex polypeptide I ring complex (TRiC), a cellular chaperonin responsible for the folding of at least 10% of the cytosolic proteins, is a component of viroplasms and is required for the synthesis of the viral negative-sense single-stranded RNA. Specifically, TRiC associates with NSP5 and VP2, the cofactor involved in RNA replication. Our study adds a new component to the current model of rotavirus replication, where TRiC is recruited to viroplasms to assist replication.

The human papillomavirus late life cycle and links to keratinocyte differentiation.

Regulation of human papillomavirus (HPV) gene expression is tightly linked to differentiation of the keratinocytes the virus infects. HPV late gene expression is confined to the cells in the upper layers of the epithelium where the virus capsid proteins are synthesized. As these proteins are highly immunogenic, and the upper epithelium is an immune-privileged site, this spatial restriction aids immune evasion. Many decades of work have contributed to the current understanding of how this restriction occurs at a molecular level. This review will examine what is known about late gene expression in HPV-infected lesions and will dissect the intricacies of late gene regulation. Future directions for novel antiviral approaches will be highlighted.

IDR-targeting compounds suppress HPV genome replication via disruption of phospho-BRD4 association with DNA damage response factors.

Compounds binding to the bromodomains of bromodomain and extra-terminal (BET) family proteins, particularly BRD4, are promising anticancer agents. Nevertheless, side effects and drug resistance pose significant obstacles in BET-based therapeutics development. Using high-throughput screening of a 200,000-compound library, we identified small molecules targeting a phosphorylated intrinsically disordered region (IDR) of BRD4 that inhibit phospho-BRD4 (pBRD4)-dependent human papillomavirus (HPV) genome replication in HPV-containing keratinocytes. Proteomic profiling identified two DNA damage response factors-53BP1 and BARD1-crucial for differentiation-associated HPV genome amplification. pBRD4-mediated recruitment of 53BP1 and BARD1 to the HPV origin of replication occurs in a spatiotemporal and BRD4 long (BRD4-L) and short (BRD4-S) isoform-specific manner. This recruitment is disrupted by phospho-IDR-targeting compounds with little perturbation of the global transcriptome and BRD4 chromatin landscape. The discovery of these protein-protein interaction inhibitors (PPIi) not only demonstrates the feasibility of developing PPIi against phospho-IDRs but also uncovers antiviral agents targeting an epigenetic regulator essential for virus-host interaction and cancer development.

Sex and Age Impact CD4+ T Cell Susceptibility to HIV In Vitro through Cell Activation Dynamics.

Cellular composition and the responsiveness of the immune system evolve upon aging and are influenced by biological sex. CD4+ T cells from women living with HIV exhibit a decreased viral replication ex vivo compared to men's. We, thus, hypothesized that these findings could be recapitulated in vitro and infected primary CD4+ T cells with HIV-based vectors pseudotyped with VSV-G or HIV envelopes. We used cells isolated from twenty donors to interrogate the effect of sex and age on permissiveness over a six-day activation kinetics. Our data identified an increased permissiveness to HIV between 24 and 72 h post-stimulation. Sex- and age-based analyses at these time points showed an increased susceptibility to HIV of the cells isolated from males and from donors over 50 years of age, respectively. A parallel assessment of surface markers' expression revealed higher frequencies of activation marker CD69 and of immune checkpoint inhibitors (PD-1 and CTLA-4) in the cells from highly permissive donors. Furthermore, positive correlations were identified between the expression kinetics of CD69, PD-1 and CTLA-4 and HIV expression kinetics. The cell population heterogeneity was assessed using a single-cell RNA-Seq analysis and no cell subtype enrichment was identified according to sex. Finally, transcriptomic analyses further highlighted the role of activation in those differences with enriched activation and cell cycle gene sets in male and older female cells. Altogether, this study brought further evidence about the individual features affecting HIV replication at the cellular level and should be considered in latency reactivation studies for an HIV cure.

Zika virus remodelled ER membranes contain proviral factors involved in redox and methylation pathways.

Zika virus (ZIKV) has emerged as a global health issue, yet neither antiviral therapy nor a vaccine are available. ZIKV is an enveloped RNA virus, replicating in the cytoplasm in close association with ER membranes. Here, we isolate ER membranes from ZIKV-infected cells and determine their proteome. Forty-six host cell factors are enriched in ZIKV remodeled membranes, several of these having a role in redox and methylation pathways. Four proteins are characterized in detail: thioredoxin reductase 1 (TXNRD1) contributing to folding of disulfide bond containing proteins and modulating ZIKV secretion; aldo-keto reductase family 1 member C3 (AKR1C3), regulating capsid protein abundance and thus, ZIKV assembly; biliverdin reductase B (BLVRB) involved in ZIKV induced lipid peroxidation and increasing stability of viral transmembrane proteins; adenosylhomocysteinase (AHCY) indirectly promoting m6A methylation of ZIKV RNA by decreasing the level of S- adenosyl homocysteine and thus, immune evasion. These results highlight the involvement of redox and methylation enzymes in the ZIKV life cycle and their accumulation at virally remodeled ER membranes.

The hypoxia-regulated ectonucleotidase CD73 is a host determinant of HIV latency.

Deciphering the mechanisms underlying viral persistence is critical to achieving a cure for human immunodeficiency virus (HIV) infection. Here, we implement a systems approach to discover molecular signatures of HIV latently infected CD4+ T cells, identifying the immunosuppressive, adenosine-producing ectonucleotidase CD73 as a key surface marker of latent cells. Hypoxic conditioning, reflecting the lymphoid tissue microenvironment, increases the frequency of CD73+ CD4+ T cells and promotes HIV latency. Transcriptomic profiles of CD73+ CD4+ T cells favor viral quiescence, immune evasion, and cell survival. CD73+ CD4+ T cells are capable of harboring a functional HIV reservoir and reinitiating productive infection ex vivo. CD73 or adenosine receptor blockade facilitates latent HIV reactivation in vitro, mechanistically linking adenosine signaling to viral quiescence. Finally, tissue imaging of lymph nodes from HIV-infected individuals on antiretroviral therapy reveals spatial association between CD73 expression and HIV persistence in vivo. Our findings warrant development of HIV-cure strategies targeting the hypoxia-CD73-adenosine axis.

Mitochondrial protein BNIP3 regulates Chikungunya virus replication in the early stages of infection.

Chikungunya virus (CHIKV) is a human pathogen causing outbreaks of febrile illness for which vaccines and specific treatments remain unavailable. Autophagy-related (ATG) proteins and autophagy receptors are a set of host factors that participate in autophagy, but have also shown to function in other unrelated cellular pathways. Although autophagy is reported to both inhibit and enhance CHIKV replication, the specific role of individual ATG proteins remains largely unknown. Here, a siRNA screen was performed to evaluate the importance of the ATG proteome and autophagy receptors in controlling CHIKV infection. We observed that 7 out of 50 ATG proteins impact the replication of CHIKV. Among those, depletion of the mitochondrial protein and autophagy receptor BCL2 Interacting Protein 3 (BNIP3) increased CHIKV infection. Interestingly, BNIP3 controls CHIKV independently of autophagy and cell death. Detailed analysis of the CHIKV viral cycle revealed that BNIP3 interferes with the early stages of infection. Moreover, the antiviral role of BNIP3 was found conserved across two distinct CHIKV genotypes and the closely related Semliki Forest virus. Altogether, this study describes a novel and previously unknown function of the mitochondrial protein BNIP3 in the control of the early stages of the alphavirus viral cycle.

IFIT3 inhibits Epstein-Barr virus reactivation via upregulating innate immunity.

Epstein-Barr virus (EBV), a member of the γ-herpesvirus family, can establish latent infection in B lymphocytes and certain epithelial cells after primary infection. Under certain circumstances, EBV can enter into lytic replication. However, the regulation of EBV latent-lytic infection remains largely unclear. The important immune molecule, interferon-induced protein with tetratricopeptide repeats 3 (IFIT3), was upregulated in EBV latently infected cells. When the lytic replication of EBV was induced, the expression of IFIT3 was further increased. In turn, IFIT3 overexpression dramatically inhibited the lytic replication of EBV, while IFIT3 knockdown facilitated EBV lytic replication. Moreover, upon the lytic induction, the ectopic IFIT3 expression promoted the activation of the interferon (IFN) pathway, including the production of IFN-stimulated genes (ISGs), IFNB1, and the phosphorylation of IFN-regulatory factor 3 (IRF3). In contrast, the depletion of IFIT3 led to decreased ISGs and IFNB1 expression. Mechanically, IFIT3 inhibited EBV lytic replication through IFN signaling. This study revealed that the host innate immune-related factor IFIT3 played an important role in regulating EBV latent-lytic homeostasis. The results implied that EBV has evolved well to utilize host factors to maintain latent infection.

Pioneer factors in viral infection.

Pioneer factors are transcription factors sharing the fascinating ability to bind to compact chromatin and thereby alter its transcriptional fate. Most pioneer factors are known for their importance during embryonic development, for instance, in inducing zygotic genome activation or cell fate decision. Some pioneer factors are actively induced or downregulated by viral infection. With this, viruses are capable to modulate different signaling pathways resulting for example in MHC-receptor up/downregulation which contributes to viral immune evasion. In this article, we review the current state of research on how different viruses (Herpesviruses, Papillomaviruses and Hepatitis B virus) use pioneer factors for their viral replication and persistence in the host, as well as for the development of viral cancer.

ABTB1 facilitates the replication of influenza A virus by counteracting TRIM4-mediated degradation of viral NP protein.

Influenza A viruses (IAVs) continue to cause tremendous economic losses to the global animal industry and respiratory diseases and deaths among humans. The nuclear import of the vRNP complex, composed of polymerase basic protein 1 (PB1), polymerase basic protein 2 (PB2), polymerase acidic protein (PA), nucleoprotein (NP), and viral RNA, is essential for the efficient replication of IAV. Host factors involved in this process can be targeted for the development of countermeasures against IAV infection. Here, we found that Ankyrin Repeat and BTB Domain Containing 1 (ABTB1) promotes the replication of IAV, and positively regulates the nuclear import of the vRNP complex. ABTB1 did not interact directly with NP, indicating that ABTB1 plays an indirect role in facilitating the nuclear import of the vRNP complex. Immunoprecipitation and mass spectrometry revealed that Tripartite Motif Containing 4 (TRIM4) interacts with ABTB1. We found that TRIM4 relies on its E3 ubiquitin ligase activity to inhibit the replication of IAV by targeting and degrading NP within the incoming vRNP complex as well as the newly synthesized NP. ABTB1 interacted with TRIM4, leading to TRIM4 degradation through the proteasome system. Notably, ABTB1-mediated degradation of TRIM4 blocked the effect of TRIM4 on NP stability, and largely counteracted the inhibitory effect of TRIM4 on IAV replication. Our findings define a novel role for ABTB1 in aiding the nuclear import of the vRNP complex of IAV by counteracting the destabilizing effect of TRIM4 on the viral NP protein.

E3 Ubiquitin Ligases in Gammaherpesviruses and HIV: A Review of Virus Adaptation and Exploitation.

For productive infection and replication to occur, viruses must control cellular machinery and counteract restriction factors and antiviral proteins. Viruses can accomplish this, in part, via the regulation of cellular gene expression and post-transcriptional and post-translational control. Many viruses co-opt and counteract cellular processes via modulation of the host post-translational modification machinery and encoding or hijacking kinases, SUMO ligases, deubiquitinases, and ubiquitin ligases, in addition to other modifiers. In this review, we focus on three oncoviruses, Epstein-Barr virus (EBV), Kaposi's sarcoma herpesvirus (KSHV), and human immunodeficiency virus (HIV) and their interactions with the ubiquitin-proteasome system via viral-encoded or cellular E3 ubiquitin ligase activity.

How host ER membrane chaperones and morphogenic proteins support virus infection.

The multi-functional endoplasmic reticulum (ER) is exploited by viruses to cause infection. Morphologically, this organelle is a highly interconnected membranous network consisting of sheets and tubules whose levels are dynamic, changing in response to cellular conditions. Functionally, the ER is responsible for protein synthesis, folding, secretion and degradation, as well as Ca2+ homeostasis and lipid biosynthesis, with each event catalyzed by defined ER factors. Strikingly, these ER host factors are hijacked by viruses to support different infection steps, including entry, translation, replication, assembly and egress. Although the full repertoire of these ER factors that are hijacked is unknown, recent studies have uncovered several ER membrane machineries that are exploited by viruses - ranging from polyomavirus to flavivirus and coronavirus - to facilitate different steps of their life cycle. These discoveries should provide better understanding of virus infection mechanisms, potentially leading to the development of more effective anti-viral therapies.

All-trans Retinoic Acid Inhibits Hepatitis B Virus Replication by Downregulating HBx Levels via Siah-1-Mediated Proteasomal Degradation.

All-trans retinoic acid (ATRA), the most biologically active metabolite of vitamin A, is known to abolish the potential of HBx to downregulate the levels of p14, p16, and p21 and to stimulate cell growth during hepatitis B virus (HBV) infection, contributing to its chemopreventive and therapeutic effects against HBV-associated hepatocellular carcinoma. Here, we demonstrated that ATRA antagonizes HBx to inhibit HBV replication. For this effect, ATRA individually or in combination with HBx upregulated p53 levels, resulting in upregulation of seven in absentia homolog 1 (Siah-1) levels. Siah-1, an E3 ligase, induces ubiquitination and proteasomal degradation of HBx in the presence of ATRA. The ability of ATRA to induce Siah-1-mediated HBx degradation and the subsequent inhibition of HBV replication was proven in an in vitro HBV replication model. The effects of ATRA became invalid when either p53 or Siah-1 was knocked down by a specific shRNA, providing direct evidence for the role of p53 and Siah-1 in the negative regulation of HBV replication by ATRA.

Hypoxia-inducible factor affects hepatitis B virus transcripts and genome levels as well as the expression and subcellular location of the hepatitis B virus core protein.

Globally, a chronic-hepatitis B virus (HBV) infection is the leading cause of hepatocellular carcinoma (HCC). The transcription factor hypoxia-inducible factor 1 (HIF1) is often elevated in HCC, including HBV-associated HCC. Previous studies have suggested that the expression of the HIF1 subunit, HIF1α, is elevated in HBV-infected hepatocytes; however, whether HIF1 activity affects the HBV lifecycle has not been fully explored. We used a liver-derived cell line and ex vivo cultured primary hepatocytes as models to determine how HIF1 affects the HBV lifecycle. We observed that HIF1 elevates HBV RNA transcript levels, core protein levels, core protein localization to the cytoplasm, and HBV genome replication. Attenuating the transcription activity of HIF1 blocked HIF1-mediated effects on the HBV lifecycle. Our studies show that HIF1 regulates various stages of the HBV lifecycle in hepatocytes and could be a therapeutic target for blocking HBV replication and the development of HBV-associated diseases.

Thrombin cleavage of the hepatitis E virus polyprotein at multiple conserved locations is required for genome replication.

The genomes of positive-sense RNA viruses encode polyproteins that are essential for mediating viral replication. These viral polyproteins must undergo proteolysis (also termed polyprotein processing) to generate functional protein units. This proteolysis can be performed by virally-encoded proteases as well as host cellular proteases, and is generally believed to be a key step in regulating viral replication. Hepatitis E virus (HEV) is a leading cause of acute viral hepatitis. The positive-sense RNA genome is translated to generate a polyprotein, termed pORF1, which is necessary and sufficient for viral genome replication. However, the mechanism of polyprotein processing in HEV remains to be determined. In this study, we aimed to understand processing of this polyprotein and its role in viral replication using a combination of in vitro translation experiments and HEV sub-genomic replicons. Our data suggest no evidence for a virally-encoded protease or auto-proteolytic activity, as in vitro translation predominantly generates unprocessed viral polyprotein precursors. However, seven cleavage sites within the polyprotein (suggested by bioinformatic analysis) are susceptible to the host cellular protease, thrombin. Using two sub-genomic replicon systems, we demonstrate that mutagenesis of these sites prevents replication, as does pharmacological inhibition of serine proteases including thrombin. Overall, our data supports a model where HEV uses host proteases to support replication and could have evolved to be independent of a virally-encoded protease for polyprotein processing.

Lack of strong innate immune reactivity renders macrophages alone unable to control productive Varicella-Zoster Virus infection in an isogenic human iPSC-derived neuronal co-culture model.

With Varicella-Zoster Virus (VZV) being an exclusive human pathogen, human induced pluripotent stem cell (hiPSC)-derived neural cell culture models are an emerging tool to investigate VZV neuro-immune interactions. Using a compartmentalized hiPSC-derived neuronal model allowing axonal VZV infection, we previously demonstrated that paracrine interferon (IFN)-α2 signalling is required to activate a broad spectrum of interferon-stimulated genes able to counteract a productive VZV infection in hiPSC-neurons. In this new study, we now investigated whether innate immune signalling by VZV-challenged macrophages was able to orchestrate an antiviral immune response in VZV-infected hiPSC-neurons. In order to establish an isogenic hiPSC-neuron/hiPSC-macrophage co-culture model, hiPSC-macrophages were generated and characterised for phenotype, gene expression, cytokine production and phagocytic capacity. Even though immunological competence of hiPSC-macrophages was shown following stimulation with the poly(dA:dT) or treatment with IFN-α2, hiPSC-macrophages in co-culture with VZV-infected hiPSC-neurons were unable to mount an antiviral immune response capable of suppressing a productive neuronal VZV infection. Subsequently, a comprehensive RNA-Seq analysis confirmed the lack of strong immune responsiveness by hiPSC-neurons and hiPSC-macrophages upon, respectively, VZV infection or challenge. This may suggest the need of other cell types, like T-cells or other innate immune cells, to (co-)orchestrate an efficient antiviral immune response against VZV-infected neurons.

Inhibition of AIM2 inflammasome activation by SOX/ORF37 promotes lytic replication of Kaposi's sarcoma-associated herpesvirus.

Inflammasomes are one kind of important innate immune defense against viral and bacterial infections. Several inflammasome-forming sensors detect molecular patterns of invading pathogens and then trigger inflammasome activation and/or pyroptosis in infected cells, and viruses employ unique strategies to hijack or subvert inflammasome activation. Infection with herpesviruses induces the activation of diverse inflammasomes, including AIM2 and IFI16 inflammasomes; however, how Kaposi's sarcoma-associated herpesvirus (KSHV) counteracts inflammasome activation largely remains unclear. Here, we reveal that the KSHV ORF37-encoded SOX protein suppresses AIM2 inflammasome activation independent of its viral DNA exonuclease activity and host mRNA turnover. SOX interacts with the AIM2 HIN domain through the C-terminal Motif VII region and disrupts AIM2:dsDNA polymerization and ASC recruitment and oligomerization. The Y443A or F444A mutation of SOX abolishes the inhibition of AIM2 inflammasome without disrupting SOX nuclease activity, and a short SOX peptide is capable of inhibiting AIM2 inflammasome activation; consequently, infection with SOX-null, Y443A, or F444A Bac16 recombinant viruses results in robust inflammasome activation, suppressed lytic replication, and increased pyroptosis in human lymphatic endothelial cells in an AIM2-dependent manner. These results reveal that KSHV SOX suppresses AIM2 inflammasome activation to promote KSHV lytic replication and inhibit pyroptosis, representing a unique mechanism for evasion of inflammasome activation during KSHV lytic cycle.

Rewiring of the Host Cell Metabolome and Lipidome during Lytic Gammaherpesvirus Infection Is Essential for Infectious-Virus Production.

Oncogenic virus infections are estimated to cause ~15% of all cancers. Two prevalent human oncogenic viruses are members of the gammaherpesvirus family: Epstein-Barr virus (EBV) and Kaposi's sarcoma herpesvirus (KSHV). We use murine herpesvirus 68 (MHV-68), which shares significant homology with KSHV and EBV, as a model system to study gammaherpesvirus lytic replication. Viruses implement distinct metabolic programs to support their life cycle, such as increasing the supply of lipids, amino acids, and nucleotide materials necessary to replicate. Our data define the global changes in the host cell metabolome and lipidome during gammaherpesvirus lytic replication. Our metabolomics analysis found that MHV-68 lytic infection induces glycolysis, glutaminolysis, lipid metabolism, and nucleotide metabolism. We additionally observed an increase in glutamine consumption and glutamine dehydrogenase protein expression. While both glucose and glutamine starvation of host cells decreased viral titers, glutamine starvation led to a greater loss in virion production. Our lipidomics analysis revealed a peak in triacylglycerides early during infection and an increase in free fatty acids and diacylglyceride later in the viral life cycle. Furthermore, we observed an increase in the protein expression of multiple lipogenic enzymes during infection. Interestingly, pharmacological inhibitors of glycolysis or lipogenesis resulted in decreased infectious virus production. Taken together, these results illustrate the global alterations in host cell metabolism during lytic gammaherpesvirus infection, establish essential pathways for viral production, and recommend targeted mechanisms to block viral spread and treat viral induced tumors. IMPORTANCE Viruses are intracellular parasites which lack their own metabolism, so they must hijack host cell metabolic machinery in order to increase the production of energy, proteins, fats, and genetic material necessary to replicate. Using murine herpesvirus 68 (MHV-68) as a model system to understand how similar human gammaherpesviruses cause cancer, we profiled the metabolic changes that occur during lytic MHV-68 infection and replication. We found that MHV-68 infection of host cells increases glucose, glutamine, lipid, and nucleotide metabolic pathways. We also showed inhibition or starvation of glucose, glutamine, or lipid metabolic pathways results in an inhibition of virus production. Ultimately, targeting changes in host cell metabolism due to viral infection can be used to treat gammaherpesvirus-induced cancers and infections in humans.

Epstein-Barr Virus BBLF1 Mediates Secretory Vesicle Transport to Facilitate Mature Virion Release.

Enveloped viruses undergo a complex multistep process of assembly, maturation, and release into the extracellular space utilizing host secretory machinery. Several studies of the herpesvirus subfamily have shown that secretory vesicles derived from the trans-Golgi network (TGN) or endosomes transport virions into the extracellular space. However, the regulatory mechanism underlying the release of Epstein-Barr virus, a human oncovirus, remains unclear. We demonstrate that disruption of BBLF1, a tegument component, suppressed viral release and resulted in the accumulation of viral particles on the inner side of the vesicular membrane. Organelle separation revealed the accumulation of infectious viruses in fractions containing vesicles derived from the TGN and late endosomes. Deficiency of an acidic amino acid cluster in BBLF1 reduced viral secretion. Moreover, truncational deletion of the C-terminal region of BBLF1 increased infectious virus production. These findings suggest that BBLF1 regulates the viral release pathway and reveal a new aspect of tegument protein function. IMPORTANCE 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. Accumulating literature has demonstrated the role of viral reactivation in tumorigenesis. Elucidating the functions of viral lytic genes induced by reactivation, and the mechanisms of lytic infection, is essential to understanding pathogenesis. Progeny viral particles synthesized during lytic infection are released outside the cell after the assembly, maturation, and release steps, leading to further infection. Through functional analysis using BBLF1-knockout viruses, we demonstrated that BBLF1 promotes viral release. The acidic amino acid cluster in BBLF1 was also important for viral release. Conversely, mutants lacking the C terminus exhibited more efficient virus production, suggesting that BBLF1 is involved in the fine-tuning of progeny release during the EBV life cycle.