Virus Latent Protein Research Articles

Combination of bortezomib and venetoclax targets the pro-survival function of LMP-1 and EBNA-3C of Epstein-Barr virus in spontaneous lymphoblastoid cell lines.

Epstein-Barr virus (EBV) manipulates the ubiquitin-proteasome system and regulators of Bcl-2 family to enable the persistence of the virus and survival of the host cells through the expression of viral proteins in distinct latency patterns. We postulate that the combination of bortezomib (proteasome inhibitor) and venetoclax (Bcl-2 inhibitor) [bort/venetoclax] will cause synergistic killing of post-transplant lymphoproliferative disorder (PTLD) through targeting the pro-survival function of latent viral proteins such as latent membrane protein-1 (LMP-1) and EBV nuclear antigen-3C (EBNA-3C). Bort/venetoclax could synergistically kill spontaneous lymphoblastoid cell lines (sLCLs) derived from patients with PTLD and EBV-associated hemophagocytic lymphohistiocytosis by inducing DNA damage response, apoptosis and G1-S cell cycle arrest in a ROS-dependent manner. Bortezomib potently induced the expression of Noxa, a pro-apoptotic initiator and when combined with venetoclax, inhibited Mcl-1 and Bcl-2 simultaneously. Bortezomib prevented LMP-1 induced proteasomal degradation of IκBα leading to the suppression of the NF-κB signaling pathway. Bortezomib also rescued Bcl-6 from EBNA-3C mediated proteasomal degradation thus maintaining the repression of cyclin D1 and Bcl-2 causing G1-S arrest and apoptosis. Concurrently, venetoclax inhibited Bcl-2 upregulated by either LMP-1 or EBNA-3C. Bort/venetoclax decreased the expression of phosphorylated p65 and Bcl-2 at serine 70 thereby suppressing the NF-κB signaling pathway and promoting apoptosis, respectively. These data corroborated the marked suppression of the growth of xenograft of sLCL in SCID mice (p<0.001). Taken together, the combination of bortezomib and venetoclax targets the pro-survival function of LMP-1 and EBNA-3C of Epstein-Barr virus in spontaneous lymphoblastoid cell lines.

HPV and RNA Binding Proteins: What We Know and What Remains to Be Discovered

Papillomavirus gene regulation is largely post-transcriptional due to overlapping open reading frames and the use of alternative polyadenylation and alternative splicing to produce the full suite of viral mRNAs. These processes are controlled by a wide range of cellular RNA binding proteins (RPBs), including constitutive splicing factors and cleavage and polyadenylation machinery, but also factors that regulate these processes, for example, SR and hnRNP proteins. Like cellular RNAs, papillomavirus RNAs have been shown to bind many such proteins. The life cycle of papillomaviruses is intimately linked to differentiation of the epithelial tissues the virus infects. For example, viral late mRNAs and proteins are expressed only in the most differentiated epithelial layers to avoid recognition by the host immune response. Papillomavirus genome replication is linked to the DNA damage response and viral chromatin conformation, processes which also link to RNA processing. Challenges with respect to elucidating how RBPs regulate the viral life cycle include consideration of the orchestrated spatial aspect of viral gene expression in an infected epithelium and the epigenetic nature of the viral episomal genome. This review discusses RBPs that control viral gene expression, and how the connectivity of various nuclear processes might contribute to viral mRNA production.

Cardiac glycosides inhibit early and late vaccinia virus protein expression.

Cardiac glycosides (CGs) are natural steroid glycosides, which act as inhibitors of the cellular sodium-potassium ATPase pump. Although traditionally considered toxic to human cells, CGs are widely used as drugs for the treatment of cardiovascular-related medical conditions. More recently, CGs have been explored as potential anti-viral drugs and inhibit replication of a range of RNA and DNA viruses. Previously, a compound screen identified CGs that inhibited vaccinia virus (VACV) infection. However, no further investigation of the inhibitory potential of these compounds was performed, nor was there investigation of the stage(s) of the poxvirus lifecycle they impacted. Here, we investigated the anti-poxvirus activity of a broad panel of CGs. We found that all CGs tested were potent inhibitors of VACV replication. Our virological experiments showed that CGs did not impact virus infectivity, binding, or entry. Rather, experiments using recombinant viruses expressing reporter proteins controlled by VACV promoters and arabinoside release assays demonstrated that CGs inhibited early and late VACV protein expression at different concentrations. Lack of virus assembly in the presence of CGs was confirmed using electron microscopy. Thus, we expand our understanding of compounds with anti-poxvirus activity and highlight a yet unrecognized mechanism by which poxvirus replication can be inhibited.

Vaccinia Virus Defective Particles Lacking the F17 Protein Do Not Inhibit Protein Synthesis: F17, a Double-Edged Sword for Protein Synthesis?

Vaccinia virus (Orthopoxvirus) F17 protein is a major virion structural phosphoprotein having a molecular weight of 11 kDa. Recently, it was shown that F17 synthesised in infected cells interacts with mTOR subunits to evade cell immunity and stimulate late viral protein synthesis. Several years back, we purified an 11 kDa protein that inhibited protein synthesis in reticulocyte lysate from virions, and that possesses all physico-chemical properties of F17 protein. To investigate this discrepancy, we used defective vaccinia virus particles devoid of the F17 protein (designated iF17- particles) to assess their ability to inhibit protein synthesis. To this aim, we purified iF17- particles from cells infected with a vaccinia virus mutant which expresses F17 only in the presence of IPTG. The SDS-PAGE protein profiles of iF17- particles or derived particles, obtained by solubilisation of the viral membrane, were similar to that of infectious iF17 particles. As expected, the profiles of full iF17- particles and those lacking the viral membrane were missing the 11 kDa F17 band. The iF17- particles did attach to cells and injected their viral DNA into the cytoplasm. Co-infection of the non-permissive BSC40 cells with a modified vaccinia Ankara (MVA) virus, expressing an mCherry protein, and iF17- particles, induced a strong mCherry fluorescence. Altogether, these experiments confirmed that the iF17- particles can inject their content into cells. We measured the rate of protein synthesis as a function of the multiplicity of infection (MOI), in the presence of puromycin as a label. We showed that iF17- particles did not inhibit protein synthesis at high MOI, by contrast to the infectious iF17 mutant. Furthermore, the measured efficiency to inhibit protein synthesis by the iF17 mutant virus generated in the presence of IPTG, was threefold to eightfold lower than that of the wild-type WR virus. The iF17 mutant contained about threefold less F17 protein than wild-type WR. Altogether these results strongly suggest that virion-associated F17 protein is essential to mediate a stoichiometric inhibition of protein synthesis, in contrast to the late synthesised F17. It is possible that this discrepancy is due to different phosphorylation states of the free and virion-associated F17 protein.

Wilms' tumor 1 (WT1) antigen is overexpressed in Kaposi Sarcoma and is regulated by KSHV vFLIP.

In people living with HIV, Kaposi Sarcoma (KS), a vascular neoplasm caused by KS herpesvirus (KSHV/HHV-8), remains one of the most common malignancies worldwide. Individuals living with HIV, receiving otherwise effective antiretroviral therapy, may present with extensive disease requiring chemotherapy. Hence, new therapeutic approaches are needed. The Wilms' tumor 1 (WT1) protein is overexpressed and associated with poor prognosis in several hematologic and solid malignancies and has shown promise as an immunotherapeutic target. We found that WT1 was overexpressed in >90% of a total 333 KS biopsies, as determined by immunohistochemistry and image analysis. Our largest cohort from ACTG, consisting of 294 cases was further analyzed demonstrating higher WT1 expression was associated with more advanced histopathologic subtypes. There was a positive correlation between the proportion of infected cells within KS tissues, assessed by expression of the KSHV-encoded latency-associated nuclear antigen (LANA), and WT1 positivity. Areas with high WT1 expression showed sparse T-cell infiltrates, consistent with an immune evasive tumor microenvironment. We show that major oncogenic isoforms of WT1 are overexpressed in primary KS tissue and observed WT1 upregulation upon de novo infection of endothelial cells with KSHV. KSHV latent viral FLICE-inhibitory protein (vFLIP) upregulated total and major isoforms of WT1, but upregulation was not seen after expression of mutant vFLIP that is unable to bind IKKƴ and induce NFκB. siRNA targeting of WT1 in latent KSHV infection resulted in decreased total cell number and pAKT, BCL2 and LANA protein expression. Finally, we show that ESK-1, a T cell receptor-like monoclonal antibody that recognizes WT1 peptides presented on MHC HLA-A0201, demonstrates increased binding to endothelial cells after KSHV infection or induction of vFLIP expression. We propose that oncogenic isoforms of WT1 are upregulated by KSHV to promote tumorigenesis and immunotherapy directed against WT1 may be an approach for KS treatment.

MRNA-based Vaccines Targeting the T-cell Epitope-rich Domain of Epstein Barr Virus Latent Proteins Elicit Robust Anti-Tumor Immunity in Mice.

Epstein-Barr virus (EBV) is associated with various malignancies and infects >90% of the global population. EBV latent proteins are expressed in numerous EBV-associated cancers and contribute to carcinogenesis, making them critical therapeutic targets for these cancers. Thus, this study aims to develop mRNA-based therapeutic vaccines that express the T-cell-epitope-rich domain of truncated latent proteins of EBV, including truncatedlatent membrane protein 2A (Trunc-LMP2A), truncated EBV nuclear antigen 1 (Trunc-EBNA1), and Trunc-EBNA3A. The vaccines effectively activate both cellular and humoral immunity in mice and show promising results in suppressing tumor progression and improving survival time in tumor-bearing mice. Furthermore, it is observed that the truncated forms of the antigens, Trunc-LMP2A, Trunc-EBNA1, and Trunc-EBNA3A, are more effective than full-length antigens in activating antigen-specific immune responses. In summary, the findings demonstrate the effectiveness of mRNA-based therapeutic vaccines targeting the T-cell-epitope-rich domain of EBV latent proteins and providing new treatment options for EBV-associated cancers.

EBV Impact in Peripheral Macrophages' Polarization Cytokines in Pediatric Patients.

Macrophages are exceptionally flexible cells. The presence of inflammatory cytokines such as IFN-γ and TNF-α results in an M1 (CD68) activation, while cytokines such as IL-10 or TGF-β induce the M2 (CD163) activation. Our aim was to study the behavior of peripheral cytokines involved in macrophage polarization and relate them with tissue findings to further comprehend the role of macrophages in EBV pediatric infection. We studied cytokine expression in tonsils and peripheral blood samples of children in different stages of infection. Peripheral cytokines were compared with macrophage polarization markers and viral protein expression in tonsils. Only IL-10 showed a negative correlation between compartments, exclusively in patients undergoing viral reactivation (R). Higher expressions of peripheral IL-1β, IL-23, and IL-12p40 in R children were observed. Lower expressions of local and peripheral TNF-α in patients with broader expressions of latent and lytic viral proteins were demonstrated. In healthy carrier (HC) patients, IL-23 positively correlated with CD163, and IP-10 positively correlated with CD68. Our results indicated that EBV might modulate antigen expression in the presence of TNF-α and influence peripheral cytokine expression differently in each stage of infection. Moreover, peripheral cytokines might have a particular role in macrophage polarization in HC.

Epstein-Barr virus infectious particles initiate B cell transformation and modulate cytokine response.

The Epstein-Barr virus efficiently infects and transforms B lymphocytes. During this process, infectious viral particles transport the viral genome to the nucleus of target cells. We show here that these complex viral structures serve additional crucial roles by activating transcription of the transforming genes encoded by the virus. We show that components of the infectious particle sequentially activate proinflammatory B lymphocyte signaling pathways that, in turn, activate viral gene expression but also cause cytokine release. However, virus infection activates expression of ZFP36L1, an RNA-binding stress protein that limits the length and the intensity of the cytokine response. Thus, the infectious particles can activate viral gene expression and initiate cellular transformation at the price of a limited immune response.

Retrospective study on the association of human herpesvirus reactivation with severe DRESS: A description of blood and skin reactivations.

Drug Reaction with Eosinophilia and Systemic Symptoms (DRESS) is a severe adverse event (mortality of 10%). Its pathophysiology involves herpesviruses, particularly HHV-6, but the exact mechanisms are still poorly understood. To describe severe cases of DRESS and especially their association with herpesvirus reactivation. This study was a multicentre case series conducted between 2007 and 2021 at five University Hospital Centres in France. The study included patients who had severe DRESS, which was defined as death, transfer to the intensive care unit (ICU), or severe damage to internal organs. We excluded patients without blood PCR sample, without a drug formally attributed or with RegiSCAR score < 6. We collected data on severity, causative drug, associated visceral damage and results of viral blood PCRs. HHV-6 reactivation was studied in skin biopsies by detection of small non-coding transcripts (HHV-6 miR-aU14) and a late viral protein (GP82/105). Fifty-two patients were included (29 female, median age 62, interquartile range (IQR) [37;72]). Eight patients (15%) died, 13 (27%) were admitted to ICU. Most patients (n = 34; 65%) had multisystem involvement: most frequent was liver (n = 46; 88%), then renal failure (n = 24; 46%). Forty patients (77%) had at least one blood viral reactivation among HHV-6, EBV or CMV, of which 21 (53%) had at least two. Median time of blood HHV-6 reactivation was 24 days (IQR [20;35]). HHV-6 reactivation was demonstrated in 15 out of 20 skin biopsies, with a median time of 11 days [9;17]. We confirmed the high rate of HHV-6 reactivation in severe DRESS and demonstrated cutaneous HHV-6 reactivation using small non-coding transcripts (HHV-6 miR-aU14), which preceded viral PCR positivity in blood. These results suggest that HHV-6 reactivation during DRESS may start in skin. Furthermore, search for miR-aU14 in skin biopsy could become a useful diagnostic tool for early detection of HHV-6 reactivation.

Identification and characterization of a polyomavirus in the thornback skate (Raja clavata)

Members of the family Polyomaviridae have a circular double-stranded DNA genome that have been identified in various hosts ranging from mammals to arachnids. Here we report the identification and analysis of a complete genome sequence of a novel polyomavirus, Raja clavata polyomavirus (RcPyV1), from a cartilaginous fish, the thornback skate (Raja clavata). The genome sequence was determined using a metagenomics approach with an aim to provide baseline viral data in cartilaginous fish in different ecosystems. The RcPyV1 genome (4,195 nucleotides) had typical organization of polyomavirus, including early antigens (small T; Large T) encoded on one strand and late viral proteins (VP1; VP2) on the complementary strand. Maximum-likelihood phylogenetic analysis of the large T-antigen revealed that RcPyV1 clusters with a polyomavirus obtained from another cartilaginous fish, the guitarfish polyomavirus 1 (GfPyV1). These two share ~ 56% pairwise identity in LT and VP1 protein sequences. These analyses support the hypothesis that cartilaginous fishes have a specific lineage of polyomaviruses.

Human cytomegalovirus mediates APOBEC3B relocalization early during infection through a ribonucleotide reductase-independent mechanism.

The APOBEC3 family of DNA cytosine deaminases comprises an important arm of the innate antiviral defense system. The gamma-herpesviruses Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus and the alpha-herpesviruses herpes simplex virus (HSV)-1 and HSV-2 have evolved an efficient mechanism to avoid APOBEC3 restriction by directly binding to APOBEC3B and facilitating its exclusion from the nuclear compartment. The only viral protein required for APOBEC3B relocalization is the large subunit of the ribonucleotide reductase (RNR). Here, we ask whether this APOBEC3B relocalization mechanism is conserved with the beta-herpesvirus human cytomegalovirus (HCMV). Although HCMV infection causes APOBEC3B relocalization from the nucleus to the cytoplasm in multiple cell types, the viral RNR (UL45) is not required. APOBEC3B relocalization occurs rapidly following infection suggesting the involvement of an immediate early or early (IE/E) viral protein. In support of this possibility, genetic (IE1 mutant) and pharmacologic (cycloheximide) strategies that prevent the expression of IE/E viral proteins also block APOBEC3B relocalization. In comparison, the treatment of infected cells with phosphonoacetic acid, which interferes with viral late protein expression, still permits A3B relocalization. These results combine to indicate that the beta-herpesvirus HCMV uses an RNR-independent, yet phenotypically similar, molecular mechanism to antagonize APOBEC3B. IMPORTANCE Human cytomegalovirus (HCMV) infections can range from asymptomatic to severe, particularly in neonates and immunocompromised patients. HCMV has evolved strategies to overcome host-encoded antiviral defenses to achieve lytic viral DNA replication and dissemination and, under some conditions, latency and long-term persistence. Here, we show that HCMV infection causes the antiviral factor, APOBEC3B, to relocalize from the nuclear compartment to the cytoplasm. This overall strategy resembles that used by related herpesviruses. However, the HCMV relocalization mechanism utilizes a different viral factor(s) and available evidence suggests the involvement of at least one protein expressed at the early stages of infection. This knowledge is important because a greater understanding of this mechanism could lead to novel antiviral strategies that enable APOBEC3B to naturally restrict HCMV infection.

Epstein–Barr virus microRNA miR-BART2-5p accelerates nasopharyngeal carcinoma metastasis by suppressing RNase Ⅲ endonuclease DICER1

The development and progression of nasopharyngeal carcinoma (NPC) is closely associated with Epstein-Barr virus (EBV) infection. NPC is usually asymptomatic until it spreads to other sites, and more than 70% of cases are classified as locally advanced disease at diagnosis. EBV-positive nasopharyngeal cancer tissues express only limited viral latent proteins, but express high levels of the EBV-encoded BamHI-A rightward transcript (BART) miRNA molecules. Here, we report that EBV-miRNA-BART2-5p (BART2-5p) promotes NPC cell invasion and metastasis invivo and invitro but has no effect on NPC cell proliferation and apoptosis. In addition, BART2-5p altered the mRNA and miRNA expression profiles of NPC cells. The development of human tumors has been reported to be associated with altered miRNAs expression, and overall miRNAs expression is reduced in many types of tumors. We found that BART2-5p downregulated the expression of several miRNAs that could exert oncogenic functions. Mechanistically, BART2-5p directly targets the RNase III endonuclease DICER1, inhibiting its function of cleaving double-stranded stem-loop RNA into short double-stranded RNA, which in turn causes altered expression of a series of key epithelial-mesenchymal transition molecules, and reverting DICER1 expression can rescue this phenotype. Furthermore, analysis from clinical samples showed a negative correlation between BART2-5p and DICER1 expression. According to our study, high expression of BART2-5p in tissues and plasma of patients with NPC is associated with poor prognosis. Our results suggest that, BART2-5p can accelerate NPC metastasis through modulating miRNA profiles which are mediated by DICER1, implying a novel role of EBV miRNAs in the pathogenesis of NPC.

Method for the growth of Herpes Simplex Virus (HSV-1 and HSV-2) in an oxygen-free environment

Vaginal herpes infections occur under low oxygen conditions, including anoxia. Using an anaerobic (0% O2) cell system, the infection rate, early protein expression, and progeny viability were determined. Anaerobic HeLa 229 cells (0% O2; anaerobic DMEM; 24h) were infected with MOI 1 of HSV1/2, then incubated under aerobic (control) and anaerobic conditions. Infectivity (6h post-infection (pi)) was assessed by ONPG activity and enumerating X-gal-stained cells. HSV1/2 viral immediate-early entry, early entry, and late entry protein expression were determined by fluorescent antibody staining followed by HSV1/2 glycoprotein B mouse monoclonal antibody and DAPI. Statistical analysis was performed using t-test and data were considered significant when p ≤ 0.05 (GraphPad Prism). HSV1/2 infection under standard aerobic conditions was approximately twice that measured for anaerobically infected cells. In addition, the level of HSV-2 infectivity was significantly (p &lt; 0.05) higher (~8-fold) than that of HSV-1, regardless of oxygen level. HSV entry stage protein expression (ICP8, ICP27, VP5) was detected for anaerobic HSV1/2. A similar pattern was observed for anaerobically grown progeny of HSV1/2, i.e., the number of progeny was higher when anaerobic progenies were grown under aerobic conditions (anaerobic to aerobic) as compared to those grown under anaerobic conditions (anaerobic to anaerobic). In addition, the progeny levels grown solely under anaerobic conditions trended higher for HSV-1 than HSV-2 progeny but were not significantly different. These findings show that HSV1/2 can infect and replicate in anaerobically growing cells and that oxygen is not essential for productive replication.

African swine fever virus pA104R protein acts as a suppressor of type I interferon signaling.

This study evaluates the role of the late viral protein, pA104R, in African swine fever virus immunosuppression. ASFV-encoded pA104R is a putative histone-like protein that is highly conserved throughout different virulent and non-virulent isolates. Previous studies have demonstrated that pA104R plays a vital role in the ASFV replication cycle and is a potential target for antiviral therapy. Here, we demonstrated that pA104R is a potent antagonist of type I interferon signaling. IFN-stimulated response element activity and subsequent transcription of co-transfected and endogenous interferon-stimulated genes were attenuated by pA104R treatment in HEK-293 T cells. Immunoprecipitation assay and reciprocal pull-down showed that pA104R does not interact directly with STAT1, STAT2, or IRF9. However, pA104R could inhibit IFN signaling by attenuating STAT1 phosphorylation, and we identified the critical amino acid residues (R/H69,72 and K/R92,94,97) involved through the targeted mutation functional assays. Although pA104R is a histone-like protein localized to the nucleus, it did not inhibit IFN signaling through its DNA-binding capacity. In addition, activation of the ISRE promoter by IRF9-Stat2(TA), a STAT1-independent pathway, was inhibited by pA104R. Further results revealed that both the transcriptional activation and recruitment of transcriptional stimulators by interferon-stimulated gene factor 3 were not impaired. Although we failed to determine a mechanism for pA104R-mediated IFN signaling inhibition other than attenuating the phosphorylation of STAT1, these results might imply a possible involvement of epigenetic modification by ASFV pA104R. Taken together, these findings support that pA104R is an antagonist of type I interferon signaling, which may interfere with multiple signaling pathways.

Deregulation of host gene expression by HPV16 E8^E2 knock-out genomes is due to increased productive replication

Productive replication of human papillomaviruses (HPV) only takes place in differentiating keratinocytes. The HPV16 E8^E2 protein acts as a repressor of viral gene expression and genome replication and HPV16 E8^E2 knock-out (E8-) genomes display enhanced viral late protein expression in differentiated cells. Global transcriptome analysis of differentiated HPV16 wild-type and E8-cell lines revealed a small number of differentially expressed genes which are not related to cell cycle, DNA metabolism or keratinocyte differentiation. The analysis of selected genes suggested that deregulation requires cell differentiation and positively correlated with the expression of viral late, not early transcripts. Consistent with this, the additional knock-out of the viral E4 and E5 genes, which are known to enhance productive replication, attenuated the deregulation of these host cell genes. In summary, these data reveal that productive HPV16 replication modulates host cell transcription.

PD-L1 is upregulated in CD163+ tonsillar macrophages from children undergoing EBV primary infection.

Epstein-Barr Virus (EBV) is a tumor associated virus that modulates not only the infected cells but also innate and adaptive immunity. Macrophages play a key role in tumor development and progression. Particularly, the M2 phenotype (CD163) with anti-inflammatory activity contributes to a favorable microenvironment for tumor development while the M1 (CD68) proinflammatory phenotype contributes to a restrictive one. In the context of pediatric EBV infection, little is known about macrophage contribution to PD-L1 expression, a molecule involved in immune exhaustion. We studied tonsils of primary infected (PI), healthy carriers (HC), reactivated (R), and not infected (NI) pediatric patients. Positive correlations were demonstrated for CD68+PD-L1+ in R and for CD163+PD-L1+ only in PI. Furthermore, CD163+PD-L1+ cell numbers were higher than PD-L1+CD68+ in PI patients. In addition, a positive correlation between PD-L1+CD163+ cells and LMP1 viral latent protein was observed in PI patients, and a positive correlation between PD-L1+CD68+ cells and BMRF1 lytic antigen was demonstrated. A positive correlation between TGF-β and PD-L1 expression was demonstrated in HC patients. Our findings indicate that EBV's lytic and latent antigens might be regulating macrophages' PD-L1 expression, particularly in PI patients, whereas, surprisingly, only TGF-β could be related to total PD-L1 upregulation. Given the relevance of macrophages and the PD-1/PD-L1 pathway in tumor progression and survival, more studies in early EBV infection could help to develop EBV-associated tumor therapies.

EBNA2-EBF1 complexes promote MYC expression and metabolic processes driving S-phase progression of Epstein-Barr virus–infected B cells

Epstein-Barr virus (EBV) is a human tumor virus which preferentially infects resting human B cells. Upon infection invitro, EBV activates and immortalizes these cells. The viral latent protein EBV nuclear antigen 2 (EBNA2) is essential for B cell activation and immortalization; it targets and binds the cellular and ubiquitously expressed DNA-binding protein CBF1, thereby transactivating a plethora of viral and cellular genes. In addition, EBNA2 uses its N-terminal dimerization (END) domain to bind early B cell factor 1 (EBF1), a pioneer transcription factor specifying the B cell lineage. We found that EBNA2 exploits EBF1 to support key metabolic processes and to foster cell cycle progression of infected B cells in their first cell cycles upon activation. The α1-helix within the END domain was found to promote EBF1 binding. EBV mutants lacking the α1-helix in EBNA2 can infect and activate B cells efficiently, but activated cells fail to complete the early S phase of their initial cell cycle. Expression of MYC, target genes of MYC and E2F, as well as multiple metabolic processes linked to cell cycle progression are impaired in EBVΔα1-infected B cells. Our findings indicate that EBF1 controls B cell activation via EBNA2 and, thus, has a critical role in regulating the cell cycle of EBV-infected B cells. This is a function of EBF1 going beyond its well-known contribution to B cell lineage specification.

Antiviral activity of brequinar against African swine fever virus infection in vitro

African swine fever virus (ASFV) is a double-stranded DNA virus that causes an acute and hemorrhagic disease in domestic swine, resulting in significant economic losses to the global porcine industry. The lack of vaccines and antiviral drugs highlights the urgent need for antiviral studies against ASFV. Here, we report that brequinar (BQR), which is a specific inhibitor of dihydroorotate dehydrogenase, robustly inhibits ASFV replication in Vero cells, as well as in porcine macrophages. We demonstrate that BQR exerts its antiviral activity in a dose-dependent manner through the depletion of pyrimidine pool. Although BQR does not affect the synthesis of an early viral protein, pI215L, the synthesis of late viral proteins, p17 and p72, is suppressed in the presence of BQR. We also show that BQR is able to induce cellular antiviral response in ASFV-infected macrophages by enhancing the expression of interferon-stimulated genes. Taken together, our study reveals that targeting nucleotide biosynthesis represents a promising strategy for developing antiviral agents against ASFV.

Mathematical Modeling of Protracted HCMV Replication using Genome Substrates and Protein Temporal Profiles

RationaleClinical manifestations of human cytomegalovirus (HCMV) infection, a major cause of morbidity and mortality in the immunocompromised, are associated with the lytic replication cycle. Lytic replication occurs over 96 hrs and is marked by viral DNA (vDNA) synthesis and expression of viral proteins, the latter of which can be generalized into distinct temporal classifications. Given the complexity of the lytic replication cycle, mathematical modeling can be used to probe this elaborate system. Recently, mathematical modeling has been used to study the early lytic replication cycle and the transition between lytic and latent cycles. To date, there appears to be few modeling studies focusing on the late phases of the lytic replication cycle.MethodvDNA replication kinetics were probed using qPCR assays with absolute quantification over varying multiplicities of infection (MOIs) in fibroblasts. An empiric model of vDNA replication was generated and parameterized using these data and then compared to qPCR data obtained post hoc. The vDNA model was used to drive a model of late viral infection that incorporated protein temporal class expression and culminated in virion production. This model was parameterized using data from immunoblots of representative proteins from the late temporal profile and viral titering data at three different MOIs. Predictions from this model were then compared to post hoc high‐temporal‐resolution, fluorescence‐based concentration measurements of one of the representative proteins over a 96‐hr replication cycle.ResultsA MOI‐dependent model of vDNA and late viral replication was developed from the experimental data. The model of late viral replication is mechanistic. It predicts feedback inhibition of late viral protein production and an increase in late viral protein degradation following an increase in vDNA. Both the vDNA and late replication models yielded 3 primary predictions: (1) presence of saturation kinetics at high MOIs, (2) inefficient replication at low MOIs, and (3) a range of MOIs in which vDNA replication and virus production are maximized. Saturation kinetics at high MOIs likely represents physical limitations of cellular machinery. Inefficient replication at low MOIs likely indicates a minimum MOI required to sustain infection. Identification of a maximal range of MOIs likely represents a set of conditions most favorable to HCMV lytic replication where extraneous viral material is minimized, while both the number of cells and the amount of viral material delivered to each cell is maximized.ConclusionA mathematical model of HCMV vDNA synthesis and late protein expression was generated and predicts a range of MOIs where maximal replication occurs, which can be extended to predict protein dynamics during infection.

Rifampicin for COVID-19.

Vaccinations for coronavirus disease-2019 (COVID-19) have begun more than a year before, yet without specific treatments available. Rifampicin, critically important for human medicine (World Health Organization’s list of essential medicines), may prove pharmacologically effective for treatment and chemoprophylaxis of healthcare personnel and those at higher risk. It has been known since 1969 that rifampicin has a direct selective antiviral effect on viruses which have their own RNA polymerase (severe acute respiratory syndrome coronavirus 2), like the main mechanism of action of remdesivir. This involves inhibition of late viral protein synthesis, the virion assembly, and the viral polymerase itself. This antiviral effect is dependent on the administration route, with local application resulting in higher drug concentrations at the site of viral replication. This would suggest also trying lung administration of rifampicin by nebulization to increase the drug’s concentration at infection sites while minimizing systemic side effects. Recent in silico studies with a computer-aided approach, found rifampicin among the most promising existing drugs that could be repurposed for the treatment of COVID-19.