Early Viral Gene Research Articles

8627 Viral Hormones: The Role Of Viral Insulin/IGF-1 Like Peptides In Host-Pathogen Interactions

Abstract Disclosure: A. Chuard: None. Viruses, encode proteins that are similar to host proteins to manipulate the host. Until our recent discovery of viral hormones, examples of viral mimicry were limited to immunomodulatory proteins and growth factors. We recently showed that six viruses in Iridoviridae family encode genes mimicking human insulin and IGF-1. We previously chemically synthesizing these viral insulin/IGF-1 like peptides (VILPs) and showed that VILPs can bind to human insulin and IGF-1 receptor and further stimulate post-receptor signaling. VILPs also stimulate glucose uptake and proliferation in mammalian cells and lower the blood glucose in mice. Originally, VILP-carrying viruses were isolated from fish; however, the role of VILPs in viral pathogenesis is not studied in depth. Here, we examined the effects of Grouper Iridovirus (GIV, one of the VILP carrying viruses) infection and GIV-VILP on host insulin/IGF signaling system using fish cells.To this end, we chemically synthesized GIV-VILP in its single chain (sc, IGF-1 like) and double chain (dc, insulin-like) forms. Stimulation of grouper kidney (GK) and AB9 zebrafish cells with these peptides, insulin and IGF-1, showed that IGF-1 was the most potent ligand in these cells indicating an abundance of IGF-1 receptor (IGF1R). Further, both forms of the VILPs were as potent as insulin in its activity on receptor phosphorylation and post-receptor signaling. Examining the viral kinetics, we also showed that GIV32 gene (encoding GIV VILP) is an early viral gene in both cell types. Virion analysis through mass spectrometry indicated that VILPs are not viral structural proteins. On the other hand, analysis of the supernatant from infected cells indicated that VILPs are secreted peptides during the viral cycle. Our results with supernatant transfer experiments from infected cells to uninfected cells support the same conclusion that VILPs are secreted. The secreted VILPs initiate autophosphorylation of IGF1R and/or insulin receptor (IR), consequently triggering downstream cellular signaling and metabolism. Through the use of specific inhibitors for each receptor, we illustrated that IR signaling is crucial for GIV viral replication. Surprisingly, inhibition of IGF1R increased viral replication and we are currently examining potential inhibitory effects of GIV VILP. Furthermore, VILPs appear to exert a biased effect on the AKT pathway compared to the Erk pathway. Furthermore, the inhibition of the AKT pathway significantly reduces GIV viral replication. In summary, our findings reveal that GIV-VILP is actively produced during infection and contributes to the viral replication. To our knowledge, VILPs are the first hormone-mimics characterized in viral replication. Elucidating the role of VILPs in host-pathogen interactions, we propose a novel viral pathogenesis mechanism where viruses mimic host hormones to manipulate the host's endocrine system. Presentation: 6/3/2024

Bovine papillomavirus gene expression and inflammatory pathway activation vary between equine sarcoid tumour subtypes

Equine sarcoids are common non-metastasising skin tumours in horses, associated with bovine papillomavirus (BPV) infection. Six subtypes are recognised (occult, verrucose, nodular, fibroblastic, mixed and malevolent lesions), with variable clinical behaviour. The pathophysiology underlying varying tumour phenotype is poorly understood, and previous data on associations with viral load have been conflicting. To better understand this clinical variation, we investigated associations between tumour subtype and viral load, viral early protein gene expression, and expression of 10 host genes by quantitative polymerase chain reaction in 27 sarcoids and 5 normal skin samples. Viral DNA copy number did not differ between subtypes but was significantly higher in animals with fewer tumours. Expression of BPV E2 and E6 was higher in occult lesions compared to fibroblastic or nodular lesions, while E5 expression was higher in previously-treated lesions. Of the host genes, only IL6 and IL1B differed between subtypes, with higher expression in fibroblastic lesions, while IL10 and CCL5 were elevated compared to skin in all lesion types, and elevations in TNF and TGFB1 were significant for occult lesions only. Expression of TLR9, ATR, VEGFA and PTGS2 in sarcoids was not significantly different from normal skin, suggesting differences between BPV and human papillomavirus tumorigenesis. Results for BPV viral load and gene expression differed from previous reports and are insufficient to explain the spectrum of tumour phenotypes. Activation of both pro-inflammatory and anti-inflammatory immune pathways in sarcoids could influence tumour growth and effective immune responses, and the contribution of specific infiltrating immune cells requires further investigation.

Cyprinid herpesvirus 3 replication is inhibited via decreased ORF3 promoter activity mediated by multiple cellular transcription factors

Cyprinid herpesvirus 3 (CyHV-3) is an enveloped and double-stranded DNA virus that primarily infects common carp and its variants. The transcriptional regulation program of the viral immediate early (IE) genes plays a crucial role in virus replication. However, the transcriptional regulatory mechanism of CyHV-3 IE genes remains largely unclear. In this study, we found that the IE gene ORF3 promoter, which exhibited the highest activity among the eight CyHV-3 IE genes, was inhibited in the host common carp brain (CCB) cells. Deletions of the ORF3 promoter revealed the presence of multiple regulatory elements. Site-directed deletions of multiple transcription factors binding sites (TFBS), including Yin Yang-1(YY1), TATA-binding protein (TBP), sex-determining region Y-box2 (SOX2), and cyclin-dependent kinase 8 (CDK8) binding sites, markedly inhibited the ORF3 promoter activity. Moreover, overexpression of these transcription factors in CCB cells also led to a noticeable reduction in ORF3 promoter activity. Importantly, YY1, TBP, SOX2, and CDK8 played antiviral roles against CyHV-3 infection, as evidenced by decreased viral transcripts (ORF55, ORF81, and ORF92) and genome copies after overexpression of these TFs followed by CyHV-3 infection. Collectively, CyHV-3 replication was markedly inhibited by reducing the promoter activity of ORF3 mediated by multiple cellular TFs. These findings enhance our understanding of the role of IE gene promoters in CyHV-3 and lays a foundation for in-depth studies on viral gene transcriptional regulation mechanisms.

AcMNPV-miR-2 affects Autographa californica nucleopolyhedrovirus infection by regulating the expression of ac28 and several other viral early genes.

Virus-encoded miRNAs have been extensively studied for their pivotal roles in finetuning viral infections. Baculoviruses, highly pathogenic in insects, remain underexplored concerning their encoded miRNAs. Previous reports outlined three AcMNPV-encoded miRNAs, AcMNPV-miR-1, -miR-3, and -miR-4. This study delves into the functions of another AcMNPV-encoded miRNA, AcMNPV-miR-2 (Ac-miR-2). Through a comprehensive analysis of target gene expression, the impact on larvae, and variations in host immune-related gene expression, we elucidate a functional pathway for Ac-miR-2. This miRNA suppresses viral load and infectivity and prolongs lifespans of infected larva by downregulating specific viral early genes and host immune-related genes. These mechanisms ultimately serve the virus's primary goal of enhanced propagation. Our study significantly contributes to understanding of the intricate regulatory mechanisms of virus-encoded miRNAs in baculovirus infections.

Polyomavirus ALTOs, but not MTs, downregulate viral early gene expression by activating the NF-κB pathway

Polyomaviruses are small, circular dsDNA viruses that can cause cancer. Alternative splicing of polyomavirus early transcripts generates large and small tumor antigens (LT, ST) that play essential roles in viral replication and tumorigenesis. Some polyomaviruses also express middle tumor antigens (MTs) or alternate LT open reading frames (ALTOs), which are evolutionarily related but have distinct gene structures. MTs are a splice variant of the early transcript whereas ALTOs are overprinted on the second exon of the LT transcript in an alternate reading frame and are translated via an alternative start codon. Merkel cell polyomavirus (MCPyV), the only human polyomavirus that causes cancer, encodes an ALTO but its role in the viral lifecycle and tumorigenesis has remained elusive. Here, we show MCPyV ALTO acts as a tumor suppressor and is silenced in Merkel cell carcinoma (MCC). Rescuing ALTO in MCC cells induces growth arrest and activates NF-κB signaling. ALTO activates NF-κB by binding SQSTM1 and TRAF2&3 via two N-Terminal Activating Regions (NTAR1+2), resembling Epstein-Barr virus (EBV) Latent Membrane Protein 1 (LMP1). Following activation, NF-κB dimers bind the MCPyV noncoding control region (NCCR) and downregulate early transcription. Beyond MCPyV, NTAR motifs are conserved in other polyomavirus ALTOs, which activate NF-κB signaling, but are lacking in MTs that do not. Furthermore, polyomavirus ALTOs downregulate their respective viral early transcription in an NF-κB- and NTAR-dependent manner. Our findings suggest that ALTOs evolved to suppress viral replication and promote viral latency and that MCPyV ALTO must be silenced for MCC to develop.

Merkel cell polyomavirus small tumor antigen contributes to immune evasion by interfering with type I interferon signaling.

Merkel cell polyomavirus (MCPyV) is the causative agent of the majority of Merkel cell carcinomas (MCC). The virus has limited coding capacity, with its early viral proteins, large T (LT) and small T (sT), being multifunctional and contributing to infection and transformation. A fundamental difference in early viral gene expression between infection and MCPyV-driven tumorigenesis is the expression of a truncated LT (LTtr) in the tumor. In contrast, sT is expressed in both conditions and contributes significantly to oncogenesis. Here, we identified novel functions of early viral proteins by performing genome-wide transcriptome and chromatin studies in primary human fibroblasts. Due to current limitations in infection and tumorigenesis models, we mimic these conditions by ectopically expressing sT, LT or LTtr, individually or in combination, at different time points. In addition to its known function in cell cycle and inflammation modulation, we reveal a fundamentally new function of sT. We show that sT regulates the type I interferon (IFN) response downstream of the type I interferon receptor (IFNAR) by interfering with the interferon-stimulated gene factor 3 (ISGF3)-induced interferon-stimulated gene (ISG) response. Expression of sT leads to a reduction in the expression of interferon regulatory factor 9 (IRF9) which is a central component of the ISGF3 complex. We further show that this function of sT is conserved in BKPyV. We provide a first mechanistic understanding of which early viral proteins trigger and control the type I IFN response, which may influence MCPyV infection, persistence and, during MCC progression, regulation of the tumor microenvironment.

Dynamic monitoring of viral gene expression reveals rapid antiviral effects of CD8 T cells recognizing the HCMV-pp65 antigen.

Human Cytomegalovirus (HCMV) is a betaherpesvirus that causes severe disease in immunocompromised transplant recipients. Immunotherapy with CD8 T cells specific for HCMV antigens presented on HLA class-I molecules is explored as strategy for long-term relief to such patients, but the antiviral effectiveness of T cell preparations cannot be efficiently predicted by available methods. We developed an Assay for Rapid Measurement of Antiviral T-cell Activity (ARMATA) by real-time automated fluorescent microscopy and used it to study the ability of CD8 T cells to neutralize HCMV and control its spread. As a proof of principle, we used TCR-transgenic T cells specific for the immunodominant HLA-A02-restricted tegumental phosphoprotein pp65. pp65 expression follows an early/late kinetic, but it is not clear at which stage of the virus cycle it acts as an antigen. We measured control of HCMV infection by T cells as early as 6 hours post infection (hpi). The timing of the antigen recognition indicated that it occurred before the late phase of the virus cycle, but also that virion-associated pp65 was not recognized during virus entry into cells. Monitoring of pp65 gene expression dynamics by reporter fluorescent genes revealed that pp65 was detectable as early as 6 hpi, and that a second and much larger bout of expression occurs in the late phase of the virus cycle by 48 hpi. Since transgenic (Tg)-pp65 specific CD8 T cells were activated even when DNA replication was blocked, our data argue that pp65 acts as an early virus gene for immunological purposes. ARMATA does not only allow same day identification of antiviral T-cell activity, but also provides a method to define the timing of antigen recognition in the context of HCMV infection.

Polyomavirus ALTOs, but not MTs, downregulate viral early gene expression by activating the NF-κB pathway.

Polyomaviruses are small, circular dsDNA viruses that can cause cancer. Alternative splicing of polyomavirus early transcripts generates large and small tumor antigens (LT, ST) that play essential roles in viral replication and tumorigenesis. Some polyomaviruses also express middle tumor antigens (MTs) or Alternate LT ORFs (ALTOs), which are evolutionarily related but have distinct gene structures. MTs are a splice variant of the early transcript whereas ALTOs are overprinted on the second exon of the LT transcript in an alternate reading frame and are translated via an alternative start codon. Merkel cell polyomavirus (MCPyV), the only human polyomavirus that causes cancer, encodes an ALTO but its role in the viral lifecycle and tumorigenesis has remained elusive. Here, we show MCPyV ALTO acts as a tumor suppressor and is silenced in Merkel cell carcinoma (MCC). Rescuing ALTO in MCC cells induces growth arrest and activates NF-κB signaling. ALTO activates NF-κB by binding SQSTM1 and TRAF2&3 via two N-Terminal Activating Regions (NTAR1+2), resembling Epstein-Barr virus (EBV) Latent Membrane Protein 1 (LMP1).. Following activation, NF-κB dimers bind the MCPyV non-coding control region (NCCR) and downregulate early transcription. Beyond MCPyV, NTAR motifs are conserved in other polyomavirus ALTOs, which activate NF-κB signaling, but are lacking in MTs that do not. Furthermore, polyomavirus ALTOs downregulate their respective viral early transcription in an NF-κB and NTAR dependent manner. Our findings suggest that ALTOs evolved to suppress viral replication and promote viral latency and that MCPyV ALTO must be silenced for MCC to develop.

External Guide Sequence Effectively Suppresses the Gene Expression and Replication of Herpes Simplex Virus 2.

Ribonuclease P (RNase P) complexed with an external guide sequence (EGS) represents a promising nucleic acid-based gene targeting approach for gene expression knock-down and modulation. The RNase P-EGS strategy is unique as an EGS can be designed to basepair any mRNA sequence and recruit intracellular RNase P for hydrolysis of the target mRNA. In this study, we provide the first direct evidence that the RNase P-based approach effectively blocks the gene expression and replication of herpes simplex virus 2 (HSV-2), the causative agent of genital herpes. We constructed EGSs to target the mRNA encoding HSV-2 single-stranded DNA binding protein ICP8, which is essential for viral DNA genome replication and growth. In HSV-2 infected cells expressing a functional EGS, ICP8 levels were reduced by 85%, and viral growth decreased by 3000 folds. On the contrary, ICP8 expression and viral growth exhibited no substantial differences between cells expressing no EGS and those expressing a disabled EGS with mutations precluding RNase P recognition. The anti-ICP8 EGS is specific in targeting ICP8 because it only affects ICP8 expression but does not affect the expression of the other viral immediate-early and early genes examined. This study shows the effective and specific anti-HSV-2 activity of the RNase P-EGS approach and demonstrates the potential of EGS RNAs for anti-HSV-2 applications.

HBO1/KAT7/MYST2 HAT complex regulates human adenovirus replicative cycle

Human adenoviruses (HAdV) belong to a small DNA tumor virus family that continues as valuable models in understanding the viral strategies of usurping cell growth regulation. A number of HAdV type 2/5 early viral gene products interact with a variety of cellular proteins to build a conducive environment that promotes viral replication. Here we show that HBO1 (Histone Acetyltransferase Binding to ORC1), a member of the MYST histone acetyltransferase (HAT) complex (also known as KAT7 and MYST2) that acetylates most of the histone H3 lysine 14, is essential for HAdV5 growth. HBO1/MYST2/KAT7 HAT complexes are critical for a variety of cellular processes including control of cell proliferation. In HBO1 downregulated human cells, HAdV5 infection results in reduced expression of E1A and other viral early genes, virus growth is also reduced significantly. Importantly, HBO1 downregulation reduced H3 lysine 14 acetylation at viral promoters during productive infection, likely driving reduced viral gene expression. HBO1 was also associated with viral promoters during infection and co-localized with viral replication centers in the nuclei of infected cells. In transiently transfected cells, overexpression of E1A along with HBO1 stimulated histone acetyltransferase activity of HBO1. E1A also co-immunoprecipitated with HBO1 in transiently transfected cells. In summary, our results demonstrate that HAdV recruits the HBO1 HAT complex to aid in viral replication.

FEAR antiviral response pathway is independent of interferons and countered by poxvirus proteins.

The human facilitates chromatin transcription (FACT) complex is a chromatin remodeller composed of human suppressor of Ty 16 homologue (hSpt16) and structure-specific recognition protein-1 subunits that regulates cellular gene expression. Whether FACT regulates host responses to infection remained unclear. We identify a FACT-mediated, interferon-independent, antiviral pathway that restricts poxvirus replication. Cell culture and bioinformatics approaches suggest that early viral gene expression triggers nuclear accumulation of SUMOylated hSpt16 subunits required for the expression of E26 transformation-specific sequence-1 (ETS-1)-a transcription factor that activates virus restriction programs. However, biochemical studies show that poxvirus-encoded A51R proteins block ETS-1 expression by outcompeting structure-specific recognition protein-1 binding to SUMOylated hSpt16 and by tethering SUMOylated hSpt16 to microtubules. Furthermore, A51R antagonism of FACT enhances poxvirus replication in human cells and virulence in mice. Finally, we show that FACT also restricts rhabdoviruses, flaviviruses and orthomyxoviruses, suggesting broad roles for FACT in antiviral immunity. Our study reveals the FACT-ETS-1 antiviral response (FEAR) pathway to be critical for eukaryotic antiviral immunity and describes a unique mechanism of viral immune evasion.

Insect cells of Spodoptera frugiperda support WSSV gene replication but not progeny virion assembly

The lacking of stable and susceptible cell lines has hampered research on pathogenic mechanism of crustacean white spot syndrome virus (WSSV). To look for the suitable cell line which can sustain WSSV infection, we performed the studies on WSSV infection in the Spodoptera frugiperda (Sf9) insect cells. In consistent with our previous study in vitro in crayfish hematopoietic tissue cells, the WSSV envelope was detached from nucleocapsid around 2 hpi in Sf9 cells, which was accompanied with the cytoplasmic transport of nucleocapsid toward the cell nucleus within 3 hpi. Furthermore, the expression profile of both gene and protein of WSSV was determined in Sf9 cells after viral infection, in which a viral immediate early gene IE1 and an envelope protein VP28 exhibited gradually increased presence from 3 to 24 hpi. Similarly, the significant increase of WSSV genome replication was found at 3–48 hpi in Sf9 cells after infection with WSSV, indicating that Sf9 cells supported WSSV genome replication. Unfortunately, no assembled progeny virion was observed at 24 and 48 hpi in Sf9 cell nuclei as determined by transmission electron microscope, suggesting that WSSV progeny could not be assembled in Sf9 cell line as the viral structural proteins could not be transported into cell nuclei. Collectively, these findings provide a cell model for comparative analysis of WSSV infection mechanism with crustacean cells.

BmNPV Bm60 is a key target gene used by a resistant strain of Bombyx mori to inhibit BmNPV proliferation

Bombyx mori nucleopolyhedrovirus (BmNPV) is a pathogen that causes significant losses to the silkworm industry. Numerous antiviral genes and proteins have been identified by studying silkworm resistance to BmNPV. However, the molecular mechanism of silkworm resistance to BmNPV is unclear. We analyzed the differences between the susceptible strain 871 and a near-isogenic resistant strain 871C. The survival of strain 871C was significantly greater than that of 871 after oral and subcutaneous exposure to BmNPV. Strain 871C exhibited a nearly 10,000-fold higher LD50 for BmNPV compared to 871. BmNPV proliferation was significantly inhibited in all tested tissues of strain 871C using HE strain and fluorescence analysis. Strain 871C exhibited cellular resistance to BmNPV rather than peritrophic membrane or serum resistance. Strain 871C suppressed the expression of the viral early gene Bm60. This led to the inhibition of BmNPV DNA replication and late structural gene transcription based on the cascade regulation of baculovirus gene expression. Bm60 could also interact with the viral DNA binding protein and alkaline nuclease, as well as host proteins Methylcrotonoyl-CoA carboxylase subunit alpha, mucin-2-like protein, and 30 K-8. Overexpression of 30 K-8 significantly inhibited BmNPV proliferation. These results increase understanding of the molecular mechanism behind silkworm resistance to BmNPV and suggest targets for the breeding of resistant silkworm strains and the controlling pest of Lepidoptera.

A Revision of Herpes Simplex Virus Type 1 Transcription: First, Repress; Then, Express.

The herpes virus genome bears more than 80 strong transcriptional promoters. Upon entry into the host cell nucleus, these genes are transcribed in an orderly manner, producing five immediate-early (IE) gene products, including ICP0, ICP4, and ICP22, while non-IE genes are mostly silent. The IE gene products are necessary for the transcription of temporal classes following sequentially as early, leaky late, and true late. A recent analysis using precision nuclear run-on followed by deep sequencing (PRO-seq) has revealed an important step preceding all HSV-1 transcription. Specifically, the immediate-early proteins ICP4 and ICP0 enter the cell with the incoming genome to help preclude the nascent antisense, intergenic, and sense transcription of all viral genes. VP16, which is also delivered into the nucleus upon entry, almost immediately reverses this repression on IE genes. The resulting de novo expression of ICP4 and ICP22 further repress antisense, intergenic, and early and late viral gene transcription through different mechanisms before the sequential de-repression of these gene classes later in infection. This early repression, termed transient immediate-early protein-mediated repression (TIEMR), precludes unproductive, antisense, intergenic, and late gene transcription early in infection to ensure the efficient and orderly progression of the viral cascade.

A distinct isoform of lymphoid enhancer binding factor 1 (LEF1) epigenetically restricts EBV reactivation to maintain viral latency.

As a human tumor virus, EBV is present as a latent infection in its associated malignancies where genetic and epigenetic changes have been shown to impede cellular differentiation and viral reactivation. We reported previously that levels of the Wnt signaling effector, lymphoid enhancer binding factor 1 (LEF1) increased following EBV epithelial infection and an epigenetic reprogramming event was maintained even after loss of the viral genome. Elevated LEF1 levels are also observed in nasopharyngeal carcinoma and Burkitt lymphoma. To determine the role played by LEF1 in the EBV life cycle, we used in silico analysis of EBV type 1 and 2 genomes to identify over 20 Wnt-response elements, which suggests that LEF1 may bind directly to the EBV genome and regulate the viral life cycle. Using CUT&RUN-seq, LEF1 was shown to bind the latent EBV genome at various sites encoding viral lytic products that included the immediate early transactivator BZLF1 and viral primase BSLF1 genes. The LEF1 gene encodes various long and short protein isoforms. siRNA depletion of specific LEF1 isoforms revealed that the alternative-promoter derived isoform with an N-terminal truncation (ΔN LEF1) transcriptionally repressed lytic genes associated with LEF1 binding. In addition, forced expression of the ΔN LEF1 isoform antagonized EBV reactivation. As LEF1 repression requires histone deacetylase activity through either recruitment of or direct intrinsic histone deacetylase activity, siRNA depletion of LEF1 resulted in increased histone 3 lysine 9 and lysine 27 acetylation at LEF1 binding sites and across the EBV genome. Taken together, these results indicate a novel role for LEF1 in maintaining EBV latency and restriction viral reactivation via repressive chromatin remodeling of critical lytic cycle factors.

Anti-Herpes Simplex Virus Type 1 Activity Evaluation of Natural Derived Phloroglucinol Derivatives and Their Molecular Mechanisms Study.

HSV-1 is a common infection that can cause cold sores. In this study, the anti-HSV-1 virus activity of three series compounds A1-A9, B1-B12, C1-C22 was screened by MTT assay, qRT-PCR assay, Western blot assay and viruses' plaque assays. The results of MTT assay disclosed that phloroglucinol derivatives C2 and C3 effectively inhibited the death of HSV-1 infected vero cells with the CC50 values of C2 and C3 were 72.64 μmol/L and 32.62 μmol/L in HaCaT cells, 137.6 μmol/L and 48.55 μmol/L in Hela cells. The IC50 values of C3 in vero cells and Hela cells were 19.26 μmol/L and 22.98 μmol/L, respectively. In the qRT-PCR experiments, it showed that C2 and C3 effectively reduced the synthesis of HSV-1 early viral gene VP16 and late viral gene gD. The Western blot results showed that both C2 and C3 inhibited the expression of HSV-1 gD protein in a concentration-dependent manner. Lastly, viruses' plaque assay results showed that C2 and C3 inhibited the production of HSV-1 progeny virus in Hela cells and HaCaT cells in a concentration-dependent manner. Taken together, these results suggest that C2 and C3 are promising candidate that warrants further attention in the development of anti-HSV-1 drugs.

White spot syndrome virus hijacks host PP2A-FOXO axes to promote its propagation

Viruses have developed superior strategies to escape host defenses or exploit host components and enable their infection. The forkhead box transcription factor O family proteins (FOXOs) are reportedly utilized by human cytomegalovirus during their reactivation in mammals, but if FOXOs are exploited by viruses during their infection remains unclear. In the present study, we found that the FOXO of kuruma shrimp (Marsupenaeus japonicus) was hijacked by white spot syndrome virus (WSSV) during infection. Mechanistically, the expression of leucine carboxyl methyl transferase 1 (LCMT1) was up-regulated during the early stages of WSSV infection, which activated the protein phosphatase 2A (PP2A) by methylation, leading to dephosphorylation of FOXO and translocation into the nucleus. The FOXO directly promoted transcription of the immediate early gene, wsv079 of WSSV, which functioned as a transcriptional activator to initiate the expression of viral early and late genes. Thus, WSSV utilized the host LCMT1-PP2A-FOXO axis to promote its replication during the early infection stage. We also found that, during the late stages of WSSV infection, the envelope protein of WSSV (VP26) promoted PP2A activity by directly binding to FOXO and the regulatory subunit of PP2A (B55), which further facilitated FOXO dephosphorylation and WSSV replication via the VP26-PP2A-FOXO axis in shrimp. Overall, this study reveals novel viral strategies by which WSSV hijacks host LCMT1-PP2A-FOXO or VP26-PP2A-FOXO axes to promote its propagation, and provides clinical targets for WSSV control in shrimp aquaculture.

Engineered Human Adenoviruses of Species B and C Report Early, Intermediate Early, and Late Viral Gene Expression.

Adenoviruses (AdVs) are being developed for oncolytic or vaccination therapy against existing and emerging conditions. Well-characterized replication-competent human and human primate AdVs expressing multiple payloads are desirable, but their replication in rodent models is limited. To score the timing of adenoviral gene expression in cell cultures, we developed fully replication-competent transcriptional reporter viruses for HAdV-C5, -B3, and -B35. The picornavirus-derived 2A sequence, which induces cotranslational peptide splitting and reinitiation (skipping), was linked to GFP and the fused sequence was inserted C-terminal of the early gene E1A, the intermediate early gene protein IX and the late fiber gene. The 2A peptide induced ribosomal skipping during translation of the messenger RNA (mRNA) and gave rise to GFP from the corresponding viral promoters, as shown by immunoblotting and flow cytometry analyses of human and rodent cells. In human cells, both species B and C AdV exhibited highest reporter expression for fiber, followed by protein IX and lowest for E1A. Inoculation with either HAdV-C5 or -B3/35 viruses encoding protein IX- or fiber-GFP gave rise to higher GFP levels in hamster than mouse cells. Remarkably, despite rather low 2A ribosomal skipping efficiency of ∼50% for E1A-2A-GFP, protein IX-2A-GFP, and fiber-2A-GFP, unprocessed protein IX-2A-GFP and fiber-2A-GFP fusion proteins were efficiently incorporated into HAdV-B3 virions, respectively. These data indicate that the B3 C-termini of protein IX and fiber can be considered for retargeting engineered oncolytic or vaccination vectors, or for antigen display. The variable expression levels of transgenes from different subviral promoters may be used to improve oncolytic AdV vectors expressing therapeutic genes.

The Dynamics of Synthesis and Localization of Jumbo Phage RNA Polymerases inside Infected Cells.

A nucleus-like structure composed of phage-encoded proteins and containing replicating viral DNA is formed in Pseudomonas aeruginosa cells infected by jumbo bacteriophage phiKZ. The PhiKZ genes are transcribed independently from host RNA polymerase (RNAP) by two RNAPs encoded by the phage. The virion RNAP (vRNAP) transcribes early viral genes and must be injected into the cell with phage DNA. The non-virion RNAP (nvRNAP) is composed of early gene products and transcribes late viral genes. In this work, the dynamics of phage RNAPs localization during phage phiKZ infection were studied. We provide direct evidence of PhiKZ vRNAP injection in infected cells and show that it is excluded from the phage nucleus. The nvRNAP is synthesized shortly after the onset of infection and localizes in the nucleus. We propose that spatial separation of two phage RNAPs allows coordinated expression of phage genes belonging to different temporal classes.

Ginkgolic acid inhibits the replication of pseudorabies virus in vitro and in vivo by suppressing the transcription of viral late genes

Pseudorabies virus (PRV) belongs to the species of alphaherpesvirus that can cause substantial economic losses to the world swine industry. Therefore, research on anti-PRV compounds is of great value. In this study, it was found that ginkgolic acid could efficiently inhibit the replication of PRV, and the IC50 and CC50 were 3.407 μM and 102.3 μM, respectively. Moreover, it was discovered that ginkgolic acid had no effect on the adsorption, entry, and release stages of the PRV replication cycle. Importantly, it was found that ginkgolic acid could significantly suppress the transcription of PRV late genes, while the transcription of viral immediate early and early genes was not affected. Finally, in vivo experiments showed that ginkgolic acid could significantly reduce the viral load of PRV in multiple tissues and increase 30% survival rate of mice upon the challenge of PRV. Taken together, a novel PRV replication inhibitor, ginkgolic acid, which worked through suppressing the transcription of the late genes, was found in this study. This study provides a potential therapy method for the infection of PRV.