Late Transcription Research Articles

AcMNPV PKIP is required for hyperexpression of very late genes and involved in the hyperphosphorylation of the viral basic protein P6.9

A previous study showed that a mutation in Autographa californica multiple nucleopolyhedrovirus pkip (ac24) led to severe defects in progeny budded virion production and very late gene transcription at non-permissive temperature. To dissect the underlying mechanism, our early study showed that PKIP is associated with nucleocapsid of budded virion and involved in nucleocapsid assembly. However, how pkip affects very late gene transcription has not been determined. In the present study, double-stranded RNA was used to silence pkip expression during virus infection, resulting in the significant reduction of occlusion body production and polyhedrin expression. To find out whether PKIP regulates polyhedrin expression by affecting the transcription of other viral genes for very late gene expression, a comparative transcriptome analysis of viral genes was performed by RNA sequencing and the result showed that silencing pkip specifically down-regulated transcription of very late genes, while the transcription patterns of the viral genes associated with very late gene transcription were not affected. Since PKIP was reported to interact with and stimulate the activity of virus-encoded protein kinase PK1 and PK1 was involved in the hyperphosphorylation of viral basic protein P6.9, which was required for the maximal hyperexpression of very late genes, we sought to determine the association between PKIP and P6.9. Further experiments showed that PKIP interacted with P6.9 during virus infection, and the deletion of pkip resulted in decreased hyperphosphorylation of P6.9. Taken together, our results indicated that PKIP is involved in hyperphosphorylation of P6.9, which in return maybe required for hyperexpression of very late genes.

Andrias davidianus ranavirus 1R encoding a delayed-early protein promotes cell proliferation by driving cell cycle progression into S phase.

Andrias davidianus ranavirus 1R (ADRV-1R), a core gene of the family Iridoviridae, is predicted to encode a viral transcription factor (vTF) since the protein contains a virus late transcription factor-3 like (VLTF3 like) domain. However, its characteristics and function are still unclear. In this study, the transcription and expression of ADRV-1R were investigated in Chinese giant salamander thymus cells (GSTCs). ADRV-1R transcription starts 6 hours post-infection (hpi), while the protein expression starts 8 hpi. Drug inhibition assay showed that the transcripts are inhibited by cycloheximide (CHX), a de novo protein synthesis inhibitor, indicating that ADRV-1R is a viral delayed-early (DE) gene. Subcellular localization showed that ADRV-1R is distributed in the cell nucleus and cytoplasm. The effect of ADRV-1R overexpression on cell proliferation and virus titer was analyzed. ADRV-1R overexpression significantly promoted the cell proliferation starting at day 2. Flow cytometry analysis further indicated that the protein promotes the GSTC cell cycle progression from G1 phase into S phase (G1/S transition). Moreover, ADRV-1R overexperession significantly increased ADRV titer in GSTCs. The virus titer was 6.3-6.9-fold higher at 36 hpi and further after than the control GSTC lines. These data showed that ADRV-1R is a delayed-early protein promoting cell proliferation and virus titers. Keywords: ranavirus; Andrias davidianus ranavirus; core gene; cell cycle; cell proliferations.

Identification and characterization of Orf viruses isolated from sheep and goats in Southern Italy.

Orf virus (ORFV; Family: Poxviridae) is the causative agent of contagious ecthyma, or Orf disease in sheep, goats and other domestic or wild ruminants with a worldwide distribution. The disease is endemic in Italy, but few data are available about its distribution and epidemiology. In the present study we analysed 32 clinical samples, obtained from crusted scab lesions of 5 goats and 27 sheep, from 19 suspected outbreaks of contagious ecthyma in Apulia and Basilicata regions between 2012 and 2014. Negative staining electron microscopy (EM) and polymerase chain reaction (PCR) targeting the late transcription factor gene (VLTF-1) were used to identify the virus. Isolation was also attempted on BHK-21 cell line. PCR was proved to be more sensitive than EM, as it detected the virus in 28 out of 32 samples, whereas the EM detected it only in 26 out of the 32 samples. The majority of isolated strains forms a monophyletic group; these isolates, according to the VLTF-1 sequencing, are high related to ORFV strains previously shown to circulate in Southern Italy.

NAD-linked mechanisms of gene de-repression and a novel role for CtBP in persistent adenovirus infection of lymphocytes

BackgroundAdenovirus (AdV) infection is ubiquitous in the human population and causes acute infection in the respiratory and gastrointestinal tracts. In addition to lytic infections in epithelial cells, AdV can persist in a latent form in mucosal lymphocytes, and nearly 80% of children contain viral DNA in the lymphocytes of their tonsils and adenoids. Reactivation of latent AdV is thought to be the source of deadly viremia in pediatric transplant patients. Adenovirus latency and reactivation in lymphocytes is not well studied, though immune cell activation has been reported to promote productive infection from latency. Lymphocyte activation induces global changes in cellular gene expression along with robust changes in metabolic state. The ratio of free cytosolic NAD+/NADH can impact gene expression via modulation of transcriptional repressor complexes. The NAD-dependent transcriptional co-repressor C-terminal Binding Protein (CtBP) was discovered 25 years ago due to its high affinity binding to AdV E1A proteins, however, the role of this interaction in the viral life cycle remains unclear.MethodsThe dynamics of persistently- and lytically-infected cells are evaluated. RT-qPCR is used to evaluate AdV gene expression following lymphocyte activation, treatment with nicotinamide, or disruption of CtBP-E1A binding.ResultsPMA and ionomycin stimulation shifts the NAD+/NADH ratio in lymphocytic cell lines and upregulates viral gene expression. Direct modulation of NAD+/NADH by nicotinamide treatment also upregulates early and late viral transcripts in persistently-infected cells. We found differential expression of the NAD-dependent CtBP protein homologs between lymphocytes and epithelial cells, and inhibition of CtBP complexes upregulates AdV E1A expression in T lymphocyte cell lines but not in lytically-infected epithelial cells.ConclusionsOur data provide novel insight into factors that can regulate AdV infections in activated human lymphocytes and reveal that modulation of cellular NAD+/NADH can de-repress adenovirus gene expression in persistently-infected lymphocytes. In contrast, disrupting the NAD-dependent CtBP repressor complex interaction with PxDLS-containing binding partners paradoxically alters AdV gene expression. Our findings also indicate that CtBP activities on viral gene expression may be distinct from those occurring upon metabolic alterations in cellular NAD+/NADH ratios or those occurring after lymphocyte activation.

Dissecting the Role of DDX21 in Regulating Human Cytomegalovirus Replication.

DDX21 regulates the biogenesis of rRNA and transcription of ribonucleoprotein genes. Recently, it has been reported that DDX21 regulates the growth of some RNA viruses through various mechanisms, such as inhibiting viral genome replication, suppressing virion assembly and release, and modulating antiviral immune responses (Chen et al., Cell Host Microbe 15:484-493, 2014, https://doi.org/10.1016/j.chom.2014.03.002; Dong et al., Biophys Res Commun, 473:648-653, 2016, https://doi.org/10.1016/j.bbrc.2016.03.120; and Watanabe et al., PLoS Pathog 5:e1000654, 2009, https://doi.org/10.1371/journal.ppat.1000654). The relationship between DDX21 and DNA viruses has not yet been explored. In this study, we used human cytomegalovirus (HCMV), a large human DNA virus, to investigate the potential role of DDX21 in DNA virus replication. We found that HCMV infection prevented the repression of DDX21 at protein and mRNA levels. Knockdown of DDX21 inhibited HCMV growth in human fibroblast cells (MRC5). Immunofluorescence and quantitative PCR (qPCR) results showed that knockdown of DDX21 did not affect viral DNA replication or the formation of the viral replication compartment but did significantly inhibit viral late gene transcription. Some studies have reported that DDX21 knockdown promotes the accumulation of R-loops that could restrain RNA polymerase II elongation and inhibit the transcription of certain genes. Thus, we used the DNA-RNA hybrid-specific S9.6 antibody to stain R-loops and observed that more R-loops formed in DDX21-knockdown cells than in control cells. Moreover, an DNA-RNA immunoprecipitation assay showed that more R-loops accumulated on a viral late gene in DDX21-knockdown cells. Altogether, these results suggest that DDX21 knockdown promotes the accumulation of R-loops, which prevents viral late gene transcription and consequently results in the suppression of HCMV growth. This finding provides new insight into the relationship between DDX21 and DNA virus replication.IMPORTANCE Previous studies have confirmed that DDX21 is vital for the regulation of various aspects of RNA virus replication. Our research is the first report on the role of DDX21 in HCMV DNA virus replication. We identified that DDX21 knockdown affected HCMV growth and viral late gene transcription. In order to elucidate how DDX21 regulated this transcription, we applied DNA-RNA immunoprecipitation by using the DNA-RNA hybrid-specific S9.6 antibody to test whether more R-loops accumulated on the viral late gene. Consistent with our expectation, more R-loops were detected on the viral late gene at late HCMV infection time points, which demonstrated that the accumulation of R-loops caused by DDX21 knockdown prevented viral late gene transcription and consequently impaired HCMV replication. These results reveal that DDX21 plays an important role in regulating HCMV replication and also provide a basis for investigating the role of DDX21 in regulating other DNA viruses.

S-nitrosoglutathione inhibits adipogenesis in 3T3-L1 preadipocytes by S-nitrosation of CCAAT/enhancer-binding protein \u03b2

Murine 3T3-L1 adipocytes share many similarities with primary fat cells and represent a reliable in vitro model of adipogenesis. The aim of this study was to probe the effect of S-nitrosoglutathione (GSNO) on adipocyte differentiation. Adipogenesis was induced with a mixture of insulin, dexamethasone, and 3-isobutyl-1-methylxanthine in the absence and presence of increasing GSNO concentrations. Biochemical analysis after 7 days of differentiation showed a prominent anti-adipogenic effect of GSNO which was evident as reduced cellular triglycerides and total protein content as well as decreased mRNA and protein expression of late transcription factors (e.g. peroxisome proliferator activated receptor γ) and markers of terminal differentiation (e.g. leptin). By contrast, the nitrosothiol did not affect mRNA and protein expression of CCAAT/enhancer-binding protein β (C/EBPβ), which represents a pivotal early transcription factor of the adipogenic cascade. Differentiation was also inhibited by the NO donor (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate. Biotin switch experiments showed significantly increased S-nitrosation of C/EBPβ variants indicating that posttranslational S-nitrosative modification of this transcription factor accounts for the observed anti-adipogenic effect of NO. Our results suggest that S-nitrosation might represent an important physiological regulatory mechanism of fat cell maturation.

A single phosphoacceptor residue in BGLF3 is essential for transcription of Epstein-Barr virus late genes.

Almost one third of herpesvirus proteins are expressed with late kinetics. Many of these late proteins serve crucial structural functions such as formation of virus particles, attachment to host cells and internalization. Recently, we and others identified a group of Epstein-Barr virus early proteins that form a pre-initiation complex (vPIC) dedicated to transcription of late genes. Currently, there is a fundamental gap in understanding the role of post-translational modifications in regulating assembly and function of the complex. Here, we used mass spectrometry to map potential phosphorylation sites in BGLF3, a core component of the vPIC module that connects the BcRF1 viral TATA box binding protein to other components of the complex. We identified threonine 42 (T42) in BGLF3 as a phosphoacceptor residue. T42 is conserved in BGLF3 orthologs encoded by other gamma herpesviruses. Abolishing phosphorylation at T42 markedly reduced expression of vPIC-dependent late genes and disrupted production of new virus particles, but had no effect on early gene expression, viral DNA replication, or expression of vPIC-independent late genes. We complemented failure of BGLF3(T42A) to activate late gene expression by ectopic expression of other components of vPIC. Only BFRF2 and BVLF1 were sufficient to suppress the defect in late gene expression associated with BGLF3(T42A). These results were corroborated by the ability of wild type BGLF3 but not BGLF3(T42A) to form a trimeric complex with BFRF2 and BVLF1. Our findings suggest that phosphorylation of BGLF3 at threonine 42 serves as a new checkpoint for subsequent formation of BFRF2:BGLF3:BVLF1; a trimeric subcomplex essential for transcription of late genes. Our findings provide evidence that post-translational modifications regulate the function of the vPIC nanomachine that initiates synthesis of late transcripts in herpesviruses.

Broad-Range Papillomavirus Transcriptome as a Biomarker of Papillomavirus-Associated Cervical High-Grade Cytology

Human papillomaviruses (HPVs) are responsible for >99% of cervical cancers. Molecular diagnostic tests based on the detection of viral DNA or RNA have low positive predictive values for the identification of cancer or precancerous lesions. Triage with the Papanicolaou test lacks sensitivity; and even when combined with molecular detection of high-risk HPV, this results in a significant number of unnecessary colposcopies. We have developed a broad-range detection test of HPV transcripts to take a snapshot of the transcriptome of 16 high-risk or putative high-risk HPVs in cervical lesions (HPVs 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, 73, and 82). The purpose of this novel molecular assay, named HPV RNA-Seq, is to detect and type HPV-positive samples and to determine a combination of HPV reads at certain specific viral spliced junctions that can better correlate with high-grade cytology, reflecting the presence of precancerous cells. In a proof-of-concept study conducted on 55 patients, starting from cervical smears, we have shown that HPV RNA-Seq can detect papillomaviruses with performances comparable to a widely used HPV reference molecular diagnostic kit; and a combination of the number of sequencing reads at specific early versus late HPV transcripts can be used as a marker of high-grade cytology, with encouraging diagnostic performances as a triage test.

HIV-1 is rarely detected in blood and colon myeloid cells during viral-suppressive antiretroviral therapy.

The aim of this study was to explore the contribution of blood and colon myeloid cells to HIV persistence during antiretroviral therapy (ART). Leukapheresis was collected from HIV-infected individuals with undetectable plasma viral load during ART (HIV + ART; n = 15) and viremics untreated (HIV+; n = 6). Rectal sigmoid biopsies were collected from n = 8 HIV+ART. Myeloid cells (total monocytes (Mo), CD16/CD16 Mo, CD1c dendritic cells) and CD4 T cells were isolated by magnetic-activated cell sorting (MACS) and/or fluorescence-activated cell sorting (FACS) from peripheral blood. Matched myeloid and CCR6CD4 T cells were isolated from blood and rectal biopsies by FACS. Levels of early (RU5 primers), late (Gag primers) and/or integrated HIV-DNA (Alu/HIV primers) were quantified by nested real-time PCR. Replication-competent HIV was amplified by co-culturing cells from HIV-positive individuals with CD3/CD28-activated CD4 T cells from uninfected donors. Early/late but not integrated HIV reverse transcripts were detected in blood myeloid subsets of four out of 10 HIV+ART; in contrast, integrated HIV-DNA was exclusively detected in CD4 T cells. In rectal biopsies, late HIV reverse transcripts were detected in myeloid cells and CCR6CD4 T cells from one out of eight and seven out of eight HIV+ART individuals, respectively. Replication-competent HIV was outgrown from CD4 T cells but not from myeloid of untreated/ART-treated HIV-positive individuals. In contrast to CD4 T cells, blood and colon myeloid cells carry detectable HIV only in a small fraction of HIV+ART individuals. This is consistent with the documented resistance of Mo to HIV infection and the rapid turnover of Mo-derived macrophages in the colon. Future assessment of multiple lymphoid and nonlymphoid tissues is required to include/exclude myeloid cells as relevant HIV reservoirs during ART.

AcMNPV PKIP is associated with nucleocapsid of budded virions and involved in nucleocapsid assembly

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) orf24 (pkip) is a unique Alphabaculovirus gene. A previous study showed that a temperature-sensitive mutant of AcMNPV with a mutation in pkip displayed severe defects in progeny budded virion (BV) production and very late gene transcription, however, the underlying mechanism has not been determined. To investigate the function of pkip in the baculovirus replication cycle, we constructed a pkip-knockout AcMNPV bacmid in this study. Our results showed that deletion of pkip led to significant reduction of BV production, while the synthesis of viral DNA and the transcription of early and late genes were not affected. Further examination by transmission electron microscopy analysis showed that deletion of pkip resulted in the formation of massive electron-lucent tubular structures in the nucleus of the infected cells, along with some normal electron‐dense nucleocapsids. The pkip-encoded protein PKIP could be detected at late phase during infection and was distributed in both the cytoplasm and nuclei of viruses-infected cells, with a ring pattern near the inner nuclear membrane and punctate distribution in the virogenic stroma area. Biochemical fractionation of virions into nucleocapsid and envelop components showed that PKIP was associated with the nucleocapsid fraction of BV. Taken together, our results indicated that PKIP is associated with nucleocapsids of BV and involved in nucleocapsid assembly, which contributes to the optimal production of BV.

An integrative approach identifies direct targets of the late viral transcription complex and an expanded promoter recognition motif in Kaposi's sarcoma-associated herpesvirus.

The structural proteins of DNA viruses are generally encoded by late genes, whose expression relies on recruitment of the host transcriptional machinery only after the onset of viral genome replication. β and γ-herpesviruses encode a unique six-member viral pre-initiation complex (vPIC) for this purpose, although how the vPIC directs specific activation of late genes remains largely unknown. The specificity underlying late transcription is particularly notable given that late gene promoters are unusually small, with a modified TATA-box being the only recognizable element. Here, we explored the basis for this specificity using an integrative approach to evaluate vPIC-dependent gene expression combined with promoter occupancy during Kaposi’s sarcoma-associated herpesvirus (KSHV) infection. This approach distinguished the direct and indirect targets of the vPIC, ultimately revealing a novel promoter motif critical for KSHV vPIC binding. Additionally, we found that the KSHV vPIC component ORF24 is required for efficient viral DNA replication and identified a ORF24 binding element in the origin of replication that is necessary for late gene promoter activation. Together, these results identify an elusive element that contributes to vPIC specificity and suggest novel links between KSHV DNA replication and late transcription.

SV40 virion formation functions as a novel epigenetic switch controlling early and late transcription

Simian Virus 40 (SV40) is a small double‐strand DNA virus, which exists in the form of chromatin throughout its lifecycle. Because of its chromatin structure, the virus is expected to undergo the typical forms of epigenetic regulation, including differential nucleosome location and histone modifications. In order to characterize the epigenetic changes occurring during the formation of virions from late‐stage SV40 minichromosomes (encapsidation), we have compared the location of nucleosomes containing acetylated or methylated lysines from the histone tails of H3 and H4 present in the chromatin from late‐stage minichromosomes, encapsidation intermediates, and disrupted virions. In late‐stage minichromosomes, we found that nucleosomes carrying histone modifications were primarily found in the SV40 regulatory region within the enhancer and at the early and late transcription start sites. Significantly, the nucleosome containing modified histones within the enhancer would be expected to repress early transcription by overlying regulatory sequences in the SP1 and enhancer regions required for early transcription while still allowing for late transcription. In contrast, in chromatin from virions the nucleosome found in the enhancer has been shifted approximately 70 base‐pairs toward the late region uncovering the regulatory sequences necessary for early transcription and likely repressing late transcription. This encapsidation‐dependent epigenetic switch was associated with the final stages of virion formation.Support or Funding InformationThis work was funded by grants from the National Institutes of Health, AI094441 (to B.M.) and GM098328 (to L.B.) and P20 GM113123 (to Epigenetics Core)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Decidualization of Human Endometrial Stromal Fibroblasts is a Multiphasic Process Involving Distinct Transcriptional Programs.

Decidual stromal cells differentiate from endometrial stromal fibroblasts (ESFs) under the influence of progesterone and cyclic adenosine monophosphate (cAMP) and are essential for implantation and the maintenance of pregnancy. They evolved in the stem lineage of placental (eutherian) mammals coincidental with the evolution of implantation. Here we use the well-established in vitro decidualization protocol to compare early (3 days) and late (8 days) gene transcription patterns in immortalized human ESF. We document extensive, dynamic changes in the early and late decidual cell transcriptomes. The data suggest the existence of an early signal transducer and activator of transcription (STAT) pathway dominated state and a later nuclear factor κB (NFKB) pathway regulated state. Transcription factor expression in both phases is characterized by putative or known progesterone receptor ( PGR) target genes, suggesting that both phases are under progesterone control. Decidualization leads to proliferative quiescence, which is reversible by progesterone withdrawal after 3 days but to a lesser extent after 8 days of decidualization. In contrast, progesterone withdrawal induces cell death at comparable levels after short or long exposure to progestins and cAMP. We conclude that decidualization is characterized by a biphasic gene expression dynamic that likely corresponds to different phases in the establishment of the fetal-maternal interface.

Genome replication affects transcription factor binding mediating the cascade of herpes simplex virus transcription

In herpes simplex virus type 1 (HSV-1) infection, the coupling of genome replication and transcription regulation has been known for many years; however, the underlying mechanism has not been elucidated. We performed a comprehensive transcriptomic assessment and factor-binding analysis for Pol II, TBP, TAF1, and Sp1 to assess the effect genome replication has on viral transcription initiation and elongation. The onset of genome replication resulted in the binding of TBP, TAF1, and Pol II to previously silent late promoters. The viral transcription factor, ICP4, was continuously needed in addition to DNA replication for activation of late gene transcription initiation. Furthermore, late promoters contain a motif that closely matches the consensus initiator element (Inr), which robustly bound TAF1 postreplication. Continued DNA replication resulted in reduced binding of Sp1, TBP, and Pol II to early promoters. Therefore, the initiation of early gene transcription is attenuated following DNA replication. Herein, we propose a model for how viral DNA replication results in the differential utilization of cellular factors that function in transcription initiation, leading to the delineation of kinetic class in HSV-productive infection.

Directed Nucleosome Sliding during the Formation of the Simian Virus 40 Particle Exposes DNA Sequences Required for Early Transcription.

Simian virus 40 (SV40) exists as chromatin throughout its life cycle and undergoes typical epigenetic regulation mediated by changes in nucleosome location and associated histone modifications. In order to investigate the role of epigenetic regulation during the encapsidation of late-stage minichromosomes into virions, we mapped the locations of nucleosomes containing acetylated or methylated lysines in the histone tails of H3 and H4 present in the chromatin from 48-h-postinfection minichromosomes and disrupted virions. In minichromosomes obtained late in infection, nucleosomes were found carrying various histone modifications primarily in the regulatory region, with a major nucleosome located within the enhancer and other nucleosomes at the early and late transcriptional start sites. The nucleosome found in the enhancer would be expected to repress early transcription by blocking access to part of the SP1 binding sites and the left side of the enhancer in late-stage minichromosomes while also allowing late transcription. In chromatin from virions, the principal nucleosome located in the enhancer was shifted ∼70 bases in the late direction from what was found in minichromosomes, and the level of modified histones was increased throughout the genome. The shifting of the enhancer-associated nucleosome to the late side would effectively serve as a switch to relieve the repression of early transcription found in late minichromosomes while likely also repressing late transcription by blocking access to necessary regulatory sequences. This epigenetic switch appeared to occur during the final stage of virion formation.IMPORTANCE For a virus to complete infection, it must produce a new virus particle in which the genome is able to support a new infection. This is particularly important for viruses like simian virus 40 (SV40), which exist as chromatin throughout their life cycles, since chromatin structure plays a major role in the regulation of the life cycle. In order to determine the role of SV40 chromatin structure late in infection, we mapped the locations of nucleosomes and their histone tail modifications in SV40 minichromosomes and in the SV40 chromatin found in virions using chromatin immunoprecipitation-DNA sequencing (ChIP-Seq). We have identified a novel viral transcriptional control mechanism in which a nucleosome found in the regulatory region of the SV40 minichromosome is directed to slide during the formation of the virus particle, exposing transcription factor binding sites required for early transcription that were previously blocked by the presence of the nucleosome.

Gardenia jasminoides Ellis inhibit white spot syndrome virus replication in red swamp crayfish Procambarus clarkii

As one of the most dangerous pathogens in the aquaculture of crustaceans, white spot syndrome virus (WSSV) induces a high mortality rate and huge economic losses in crustacean industries. Up to now, there are no commercially available drugs to control the virus. Thus, it is important to find an antiviral drug to combat highly lethal WSSV outbreaks. In the present study, the anti-WSSV activities of 23 medicinal plant extracts were investigated in crayfish Procambarus clarkii. In order to better evaluate the antiviral activities of plant extracts, it was important to build a WSSV infection model in crayfish first. The results of WSSV infection showed that WSSV could infect most tissues of crayfish, and the heaviest infected tissue is the gills. The time course curve of WSSV amount change in crayfish assumed a very similar shape with the typical viral one-step growth curve. On the basis of virus infection model, the anti-WSSV activities of 23 plant extracts were investigated. Among these plants, Gardenia jasminoides showed the highest inhibition on WSSV replication, with 92.31%. A further study showed that G. jasminoides improved survival rate of WSSV-challenged crayfish. Mechanistically, G. jasminoides reduced Hsp70 expression and virus immediate-early gene ie1, early gene DNApol, late gene VP28 transcript, indicating that G. jasminoides inhibited WSSV replication via blocking the immediate-early stage gene ie1 transcript. Furthermore, the expression of antioxidants (cMnSOD, mMnSOD, CAT and GST) and apoptosis-related factors (Bax and Bax-inhibitor-1) were significantly increased in G. jasminoides-treated crayfish, suggesting that G. jasminoides could modulate apoptosis-related factors and possessed anti-oxidative activity in crayfish. Taken together, G. jasminoides could inhibit WSSV replication and improve survival rate of WSSV-challenged crayfish. This outcome offers a scientific basis for the use of extracts of G. jasminoides against WSSV, and it is worth focusing on the isolation and identification of effective active compounds from G. jasminoides.

Usability of reference-free transcriptome assemblies for detection of differential expression: a case study on Aethionema arabicum dimorphic seeds

BackgroundRNA-sequencing analysis is increasingly utilized to study gene expression in non-model organisms without sequenced genomes. Aethionema arabicum (Brassicaceae) exhibits seed dimorphism as a bet-hedging strategy – producing both a less dormant mucilaginous (M+) seed morph and a more dormant non-mucilaginous (NM) seed morph. Here, we compared de novo and reference-genome based transcriptome assemblies to investigate Ae. arabicum seed dimorphism and to evaluate the reference-free versus -dependent approach for identifying differentially expressed genes (DEGs).ResultsA de novo transcriptome assembly was generated using sequences from M+ and NM Ae. arabicum dry seed morphs. The transcripts of the de novo assembly contained 63.1% complete Benchmarking Universal Single-Copy Orthologs (BUSCO) compared to 90.9% for the transcripts of the reference genome. DEG detection used the strict consensus of three methods (DESeq2, edgeR and NOISeq). Only 37% of 1533 differentially expressed de novo assembled transcripts paired with 1876 genome-derived DEGs. Gene Ontology (GO) terms distinguished the seed morphs: the terms translation and nucleosome assembly were overrepresented in DEGs higher in abundance in M+ dry seeds, whereas terms related to mRNA processing and transcription were overrepresented in DEGs higher in abundance in NM dry seeds. DEGs amongst these GO terms included ribosomal proteins and histones (higher in M+), RNA polymerase II subunits and related transcription and elongation factors (higher in NM). Expression of the inferred DEGs and other genes associated with seed maturation (e.g. those encoding late embryogenesis abundant proteins and transcription factors regulating seed development and maturation such as ABI3, FUS3, LEC1 and WRI1 homologs) were put in context with Arabidopsis thaliana seed maturation and indicated that M+ seeds may desiccate and mature faster than NM. The 1901 transcriptomic DEG set GO-terms had almost 90% overlap with the 2191 genome-derived DEG GO-terms.ConclusionsWhilst there was only modest overlap of DEGs identified in reference-free versus -dependent approaches, the resulting GO analysis was concordant in both approaches. The identified differences in dry seed transcriptomes suggest mechanisms underpinning previously identified contrasts between morphology and germination behaviour of M+ and NM seeds.

Analysis of Early Phase HIV-1 Replication and Integration Events by Using Real-time PCR.

Upon entry into a host cell, the HIV-1 virus undergoes a series of critical early replication events including reverse transcription, nuclear import, and integration of its cDNA into the host genome. Molecular assays used to detect and analyze changes in HIV-1 early phase replication events are valuable tools in developing potential antiretroviral drugs, as well as studying the pathogenesis of HIV. Described here are the molecular assays utilized to detect and quantify HIV-1 early, intermediate, and late reverse transcription (RT) products. In addition to this, protocols for quantifying HIV-1 2-LTR circle DNA and proviral DNA after integration are also included. In these protocols, the optimized TaqMan Real-time PCR reagent is used to increase assay sensitivity and reproducibility. Furthermore, a nested PCR is applied to HIV-1 integration quantification with increased accuracy.

The QP509L and Q706L superfamily II RNA helicases of African swine fever virus are required for viral replication, having non-redundant activities

ABSTRACT African swine fever virus is complex DNA virus that infects pigs with mortality rates up to 100% leading to devastating socioeconomic effected in the affected countries. There is neither a vaccine nor a treatment to control ASF. African swine fever virus genome encodes two putative SF2 RNA helicases (QP509L and Q706L). In the present study, we found that these two RNA helicases do not share a common ancestral besides sharing a sequence overlap. Although, our phylogenetic studies revealed that they are conserved among virulent and non-virulent isolates, it was possible to observe a degree of variation between isolates corresponding to different genotypes occurring in distinct geographic regions. Further experiments showed that QP509L and Q706L are actively transcribed from 4 h post infection. The immunoblot analysis revealed that both protein co-localized in the viral factories at 12 h post infection, however, QP509L was also detected in the cell nucleus. Finally, siRNA assays uncover the relevant role of these proteins during viral cycle progression, in particular, for the late transcription, genome replication, and viral progeny (a reduction of infectious particles up to 99.4% when siRNA against QP509L was used and 98.4% for siRNA against Q706L). Thus, our results suggest that both helicases are essential during viral infection, highlighting the potential use of these enzymes as target for drug and vaccine development against African swine fever.

Synthesis and Anti-HIV Profile of a Novel Tetrahydroindazolylbenzamide Derivative Obtained by Oxazolone Chemistry.

A new tetrahydroindazolylbenzamide derivative has been synthesized, characterized, and evaluated as HIV-inhibitor. The biological data revealed the ability to inhibit HIV proliferation with low cytotoxicity allowing for significant selectivity (EC50 2.77 μM; CC50 118.7 μM; SI = 68). The compound did not inhibit the viral integrase as demonstrated by in vitro studies. QPCR experiments showed that the block of viral replication occurred at early replication steps, prior to integration, profiling it as a late reverse transcription inhibitor. An efficient multistep strategy was adopted for the synthesis of the scaffold, consisting of a sequential ring-opening reaction of oxazol-5-(4H)-one with 1,3-diketone, followed by cyclocondensation with hydrazine and hydrolysis of the nitrile to the desired carboxamide.