Viral Gene Research Articles

PH-sensitive dual-preventive siRNA-based nanomicrobicide reactivates autophagy and inhibits HIV infection in vaginal CD4+ cells

Women are more susceptible to HIV transmission through unprotected heterosexual intercourse due to biological and social vulnerabilities. Intravaginal delivery of siRNAs targeting viral genes, host genes, or in combination has shown promising outcomes against HSV, HPV and HIV. Therefore, in this study, we designed, developed and evaluated a pH-sensitive RNAi-based combination nanomicrobide for the prevention/reduction of vaginal transmission of HIV. The nanomicrobide was composed of siRNA-PEI encapsulated PLGA-PEG nanoparticles (siRNA NP) loaded in a HEC gel dosage form with siRNA targeting host gene CCR5 and the viral gene Nef as a dual preventive strategy. Knocking down CCR5, a co-receptor for HIV could prevent HIV from attaching to and entering host cells and knocking down Nef could reactivate autophagy that was inhibited by Nef to improve the elimination of intracellular virus that escaped the first line of defense. The siRNA NP showed a desirable particle size and zeta potential for intravaginal delivery and a pH-dependent release profile whereby low amounts of siRNA was released under acidic vaginal conditions (vaginal fluid simulant; VFS, pH 4.2) (6.0 ± 0.4% released over 15 days) but significantly higher amounts of siRNA was released under neutral pH conditions (phosphate buffered saline; PBS, pH 7.4) (22.9 ± 0.4% released over 15 days). The CCR5-Nef-specific siRNA NP efficiently knocked down CCR5 and Nef protein expression by 43% and 63%, respectively, reactivated Nef-blocked autophagy and inhibited the replication of HIV in vitro (71.8% reduction in p24 expression). After being formulated into a gel dosage form, siRNA NP could be readily released from the gel, penetrate the vaginal epithelial layer, get taken up into the target cells and knockdown Nef and CCR5 without causing cytotoxicity in a vaginal mucosal co-culture model. Functionalization of siRNA NP with anti-CD4 antibody and loaded into a 0.5% HEC gel improved vaginal distribution and uptake of siRNA in a mouse model with distribution of siRNA restricted to the reproductive tract without any unwanted systemic uptake. The 0.5% HEC gel loaded with siRNA NP-(m)CD4 significantly downregulated approximately 40% of CCR5 protein in the lower vagina and 36% of CCR5 protein in the upper vaginal and cervical region. In contrast, 0.5% HEC gel loaded with siRNA NP-IgG did not result in significant gene knockdown.

An in vitro antiviral evaluation of punicalagin toward influenza A virus.

Influenza complications are mild to serious, and can cause death in some cases. A great deal of attention has been paid in recent years to the development and use of new antiviral compounds to overcome drug resistance in certain strains of the influenza virus and treat the clinical implications. This study aimed to investigate the antiviral effect of punicalagin and its associated mechanism against influenza A (H1N1) virus in vitro. the ant-influenza activity of punicalagin was studied in Madin-Darby Canine Kidney (MDCK) cells using influenza virus A/Puerto Rico/8/34 (H1N1) (PR8) using Hemagglutinin assay (HA) and 50% tissue culture infective dose (TCID50). Then, the inhibition of haemagglutination, virucidal activity, inhibitory effect at different times, replication of viral RNA and expression of viral genes were investigated. Punicalagin could inhibit influenza virus infection with 50% inhibitory concentration (IC50) of 3.98 μg/ml and selectivity index (SI) value of 6.1. Punicalagin decreased virus titers with an inhibitory effect on virus hemagglutination (p<0.05). Punicalagin also inhibited viral adsorption. The results of virus RNA replication and viral mRNA (NS1 and HA) expression after treatment with punicalagin showed significant suppression of viral mRNA expression but no effect on replication of viral RNA. The results of the present study indicated that punicalagin was effective against influenza infection most probably via inhibition of haemagglutination activity and virus binding.

Swine influenza A virus isolates containing the pandemic H1N1 origin matrix gene elicit greater disease in the murine model.

The 2009 pandemic H1N1 virus rapidly spilled back into North American swine, reassorting with the already genetically diverse swFLUAVs. Notably, the M gene segment quickly replaced the classical M gene segment, suggesting a fitness benefit. Here, using two murine models of infection, we demonstrate that swFLUAV isolates containing the pandemic H1N1 origin M gene caused increased disease compared to isolates containing the classical swine M gene. These results suggest that, in addition to other influenza virus gene segments, the swFLUAV M gene segment contributes to pathogenesis in mammals.

Community ecology and functional potential of bacteria, archaea, eukarya and viruses in Guerrero Negro microbial mat

In this study, the microbial ecology, potential environmental adaptive mechanisms, and the potential evolutionary interlinking of genes between bacterial, archaeal and viral lineages in Guerrero Negro (GN) microbial mat were investigated using metagenomic sequencing across a vertical transect at millimeter scale. The community composition based on unique genes comprised bacteria (98.01%), archaea (1.81%), eukarya (0.07%) and viruses (0.11%). A gene-focused analysis of bacteria archaea, eukarya and viruses showed a vertical partition of the community. The greatest coverages of genes of bacteria and eukarya were detected in first layers, while the highest coverages of genes of archaea and viruses were found in deeper layers. Many genes potentially related to adaptation to the local environment were detected, such as UV radiation, multidrug resistance, oxidative stress, heavy metals, salinity and desiccation. Those genes were found in bacterial, archaeal and viral lineages with 6477, 44, and 1 genes, respectively. The evolutionary histories of those genes were studied using phylogenetic analysis, showing an interlinking between domains in GN mat.

Adeno-associated viral gene therapy for sialidosis using small and large animal models

Adeno-associated viral gene therapy for sialidosis using small and large animal models

Exploring virus-host-environment interactions in a chemotrophic-based underground estuary

BackgroundViruses play important roles in modulating microbial communities and influencing global biogeochemistry. There is now growing interest in characterising their ecological roles across diverse biomes. However, little is known about viral ecology in low-nutrient, chemotrophic-based environments. In such ecosystems, virus-driven manipulation of nutrient cycles might have profound impacts across trophic levels. In particular, anchialine environments, which are low-energy underground estuaries sustained by chemotrophic processes, represent ideal model systems to study novel virus-host-environment interactions.ResultsHere, we employ metagenomic sequencing to investigate the viral community in Bundera Sinkhole, an anchialine ecosystem rich in endemic species supported by microbial chemosynthesis. We find that the viruses are highly novel, with less than 2% representing described viruses, and are hugely abundant, making up as much as 12% of microbial intracellular DNA. These highly abundant viruses largely infect important prokaryotic taxa that drive key metabolic processes in the sinkhole. Further, the abundance of viral auxiliary metabolic genes (AMGs) involved in nucleotide and protein synthesis was strongly correlated with declines in environmental phosphate and sulphate concentrations. These AMGs encoded key enzymes needed to produce sulphur-containing amino acids, and phosphorus metabolic enzymes involved in purine and pyrimidine nucleotide synthesis. We hypothesise that this correlation is either due to selection of these AMGs under low phosphate and sulphate concentrations, highlighting the dynamic interactions between viruses, their hosts, and the environment; or, that these AMGs are driving increased viral nucleotide and protein synthesis via manipulation of host phosphorus and sulphur metabolism, consequently driving nutrient depletion in the surrounding water.ConclusionThis study represents the first metagenomic investigation of viruses in anchialine ecosystems, and provides new hypotheses and insights into virus-host-environment interactions in such ‘dark’, low-energy environments. This is particularly important since anchialine ecosystems are characterised by diverse endemic species, both in their microbial and faunal assemblages, which are primarily supported by microbial chemosynthesis. Thus, virus-host-environment interactions could have profound effects cascading through all trophic levels.

Re-expression of epigenetically silenced PTPRR by histone acetylation sensitizes RAS-mutant lung adenocarcinoma to SHP2 inhibition.

Silenced protein tyrosine phosphatase receptor type R (PTPRR) participates in mitogen-activated protein kinase (MAPK) signaling cascades during the genesis and development of tumors. Rat sarcoma virus (Ras) genes are frequently mutated in lung adenocarcinoma, thereby resulting in hyperactivation of downstream MAPK signaling. However, the molecular mechanism manipulating the regulation and function of PTPRR in RAS-mutant lung adenocarcinoma is not known. Patient records collected from the Cancer Genome Atlas and Gene Expression Omnibus showed that silenced PTPRR was positively correlated with the prognosis. Exogenous expression of PTPRR suppressed the proliferation and migration of lung cancer cells. PTPRR expression and Src homology 2 containing protein tyrosine phosphatase 2 (SHP2) inhibition acted synergistically to control ERK1/2 phosphorylation in RAS-driven lung cancer cells. Chromatin immunoprecipitation assay revealed that HDAC inhibition induced enriched histone acetylation in the promoter region of PTPRR and recovered PTPRR transcription. The combination of the HDAC inhibitor SAHA and SHP2 inhibitor SHP099 suppressed the progression of lung cancer markedly in vitro and in vivo. Therefore, we revealed the epigenetic silencing mechanism of PTPRR and demonstrated that combination therapy targeting HDAC and SHP2 might represent a novel strategy to treat RAS-mutant lung cancer.

Computational Exploration of Single-Nucleotide Polymorphisms in the Human hRAS Gene: Implications and Insights.

Background A group of genes called oncogenes includes the Harvey rat sarcoma virus (hRAS) gene. Along with hRAS, Kirsten rat sarcoma viral oncogene homolog (kRAS) and neuroblastoma RAS viral oncogene homolog(nRAS) genes belong to the Rat sarcoma (Ras) family of oncogenes. These three genes result in Rho guanosine triphosphate hydrolases (GTPases) as their protein product. Instructions for producing the protein hRAS, which is mainly involved in controlling cell division, are provided by the hRAS gene. The hRAS protein transfers signals from outside through a process called signal transduction.Because the hRAS protein is a GTPase, it changes the chemical guanosine-5'-triphosphate (GTP) into guanosine diphosphate (GDP). GTP and GDP molecules operate as switches to turn on and off the hRAS. This study aimed to anticipate the structure and stability of the protein resulting from missense single-nucleotide polymorphisms (SNPs) in the human hRAS genes. Methodology To investigate the possible negative effects associated with these SNPs, bioinformatic analysis is typically essential. The following tools were employed for forecasting harmful SNPs: Scale-Invariant Feature Transform (SIFT), Protein Analysis Through Evolutionary Relationships (PANTHER), non-synonymous SNP by Protein Variation Effect Analyzer (PROVEAN), and non-synonymous SNP by Single Nucleotide Polymorphism Annotation Platform (SNAP). Results The present study identified a total of 11 SNPs using the SIFT approach, which were shown to have functional significance. Only two of these 11 SNPs were determined to be tolerable, whereas nine were shown to be detrimental. Among the 11 SNPs analyzed, seven (Q61H, Q99H, K117R, A121D, A146V, R169W, R169Q) were classified as possibly damaging,and four (G13V, Q22K, Q61K, Q13V) were categorized as probably benign according to the predictions made by PANTHER tools. Therefore, the seven SNPs were identified as high-risk SNPs. Conclusions Given that SNPs have the potential to be candidates for cellular alterations brought on by mutations that are associated with cancer, this study provides vital information about how SNPs might be utilized as a diagnostic marker for cancer.

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.

Incongruence between confirmed and suspected clinical cases of Japanese encephalitis virus infection.

Japanese encephalitis (JE) is a notifiable infectious disease in China. Information on every case of JE is reported to the superior health administration department. However, reported cases include both laboratory-confirmed and clinically diagnosed cases. This study aimed to differentiate between clinical and laboratory-confirmed cases of Japanese encephalitis virus (JEV) infection, and improve the accuracy of reported JE cases by analyzing the acute-phase serum and cerebrospinal fluid of all reported JE cases in the Sichuan province from 2012 to 2022. All acute-phase serum and/or cerebrospinal fluid samples of the reported JE cases were screened for IgM(ImmunoglobulinM)to JEV using the enzyme-linked immunosorbent assay (ELISA), and the detection of the viral genes of JEV and 9 other pathogens including enterovirus (EV), using reverse transcription PCR was attempted. Epidemiological analyses of JE and non-JE cases based on sex, age, onset time, and geographical distribution were also performed. From 2012 to 2022, 1558 JE cases were reported in the Sichuan province. The results of serological (JEV-specific IgM) and genetic testing for JEV showed that 81% (1262/1558) of the reported cases were confirmed as JEV infection cases (laboratory-confirmed cases). Among the 296 cases of non-JEV infection, 6 viruses were detected in the cerebrospinal fluid in 62 cases, including EV and the Epstein-Barr virus (EBV), constituting 21% (62/296) of all non-JE cases. Among the 62 non-JEV infection cases with confirmed pathogens, infections with EV and EBV included 17 cases each, herpes simplex virus (HSV-1/2) included 14 cases, varicella- zoster virus included 6 cases, mumps virus included 2 cases, and human herpes viruses-6 included 1 case. Additionally, there were five cases involving mixed infections (two cases of EV/EBV, one case of HSV-1/HSV-2, one case of EBV/HSV-1, and one case of EV/herpes viruses-6). The remaining 234 cases were classified as unknown viral encephalitis cases. Our analysis indicated that those aged 0-15 y were the majority of the patients among the 1558 reported JE cases. However, the incidence of laboratory-confirmed JE cases in the >40 y age group has increased in recent years. The temporal distribution of laboratory-confirmed cases of JE revealed that the majority of cases occurred from May to September each year, with the highest incidence in August. The results of this study indicate that there is a certain discrepancy between clinically diagnosed and laboratory-confirmed cases of JE. Each reported case should be based on laboratory detection results, which is of great importance in improving the accuracy of case diagnosis and reducing misreporting. Our results are not only important for addressing JE endemic to the Sichuan province, but also provide a valuable reference for the laboratory detection of various notifiable infectious diseases in China and other regions outside China.

Epigenetic Restriction Factors (eRFs) in Virus Infection.

The ongoing arms race between viruses and their hosts is constantly evolving. One of the ways in which cells defend themselves against invading viruses is by using restriction factors (RFs), which are cell-intrinsic antiviral mechanisms that block viral replication and transcription. Recent research has identified a specific group of RFs that belong to the cellular epigenetic machinery and are able to restrict the gene expression of certain viruses. These RFs can be referred to as epigenetic restriction factors or eRFs. In this review, eRFs have been classified into two categories. The first category includes eRFs that target viral chromatin. So far, the identified eRFs in this category include the PML-NBs, the KRAB/KAP1 complex, IFI16, and the HUSH complex. The second category includes eRFs that target viral RNA or, more specifically, the viral epitranscriptome. These epitranscriptomic eRFs have been further classified into two types: those that edit RNA bases-adenosine deaminase acting on RNA (ADAR) and pseudouridine synthases (PUS), and those that covalently modify viral RNA-the N6-methyladenosine (m6A) writers, readers, and erasers. We delve into the molecular machinery of eRFs, their role in limiting various viruses, and the mechanisms by which viruses have evolved to counteract them. We also examine the crosstalk between different eRFs, including the common effectors that connect them. Finally, we explore the potential for new discoveries in the realm of epigenetic networks that restrict viral gene expression, as well as the future research directions in this area.

Interference of pseudorabies virus infection on functions of porcine granulosa cells via apoptosis modulated by MAPK signaling pathways

BackgroundPseudorabies virus (PRV) is one of the major viral pathogens leading to reproductive disorders in swine. However, little is known about the effects of PRV infection on porcine reproductive system. Ovarian granulosa cells are somatic cells surrounding oocytes in ovary and required for folliculogenesis. The present study aimed to investigate the interference of PRV on functions of porcine ovarian granulosa cells in vitro.MethodsPrimary granulosa cells were isolated from porcine ovaries. To investigate the PRV infectivity, transmission electron microscopy (TEM) was used to check the presence of viral particles, and the expression of viral gE gene was detected by quantitative real-time PCR (qPCR) in PRV-inoculated cells. After PRV infection, cell viability was detected by MTS assay, Ki67 for proliferative status was determined by immunofluorescence assay (IFA), cell cycle and apoptosis were detected by flow cytometry, and progesterone (P4) and estradiol (E2) were determined by radioimmunoassay. The checkpoint genes of cell cycle and apoptosis-related proteins were studied by qPCR and western blotting.ResultsVirus particles were observed in the nucleus and cytoplasm of PRV-infected granulosa cells by TEM imaging, and the expression of viral gE gene increased in a time-dependent manner post infection. PRV infection inhibited cell viability and blocked cell cycle at S phase in porcine granulosa cells, accompanied by decreases in expression of Ki67 protein and checkpoint genes related to S phase. Radioimmunoassay revealed decreased levels in P4 and E2, and the expressions of key steroidogenic enzymes were also down-regulated post PRV-infection. In addition, PRV induced apoptosis with an increase in Bax expression and activation of caspase 9, and the phosphorylation of JNK, ERK and p38 MAPKs were significantly up-regulated in porcine ovarian granulosa cells post PRV infection.ConclusionsThe data indicate that PRV causes infection on porcine ovarian granulosa cells and interferes the cell functions through apoptosis, and the MAPK signaling pathway is involved in the viral pathogenesis.

Exploring modified chitosan-based gene delivery technologies for therapeutic advancements

One of the critical steps in gene therapy is the successful delivery of the genes. Immunogenicity and toxicity are major issues for viral gene delivery systems. Thus, non-viral vectors are explored. A cationic polysaccharide like chitosan could be used as a nonviral gene delivery vector owing to its significant interaction with negatively charged nucleic acid and biomembrane, providing effective cellular uptake. However, the native chitosan has issues of targetability, unpacking ability, and solubility along with poor buffer capability, hence requiring modifications for effective use in gene delivery. Modified chitosan has shown that the “proton sponge effect” involved in buffering the endosomal pH results in osmotic swelling owing to the accumulation of a greater amount of proton and chloride along with water. The major challenges include limited exploration of chitosan as a gene carrier, the availability of high-purity chitosan for toxicity reduction, and its immunogenicity. The genetic drugs are in their infancy phase and require further exploration for effective delivery of nucleic acid molecules as FDA-approved marketed formulations soon.

Human Pluripotent Stem Cell-Derived Retinal Organoids: A Viable Platform for Investigating the Efficacy of Adeno-Associated Virus Gene Therapy.

With recent advances in adeno-associated virus (AAV)-based gene therapy, efficacy and toxicity screening have become essential for developing gene therapeutic drugs for retinal diseases. Retinal organoids from human pluripotent stem cells (hPSCs) offer a more accessible and reproducible human test platform for evaluating AAV-based gene therapy. In this study, hPSCs were differentiated into retinal organoids composed of various types of retinal cells. The transduction efficiencies of AAV2 and AAV8, which are widely used in clinical trials of inherited retinal diseases, were analyzed using retinal organoids. These results suggest that retinal organoids derived from hPSCs serve as suitable screening platforms owing to their diverse retinal cell types and similarity to the human retina. In summary, we propose an optimal stepwise protocol that includes the generation of retinal organoids and analysis of AAV transduction efficacy, providing a comprehensive approach for evaluating AAV-based gene therapy for retinal diseases.

Characterization of a Very Short Meq Protein Isoform in a Marek's Disease Virus Strain in Japan.

Marek's disease virus (MDV) causes malignant lymphoma (Marek's disease; MD) in chickens. The Meq protein is essential for tumorigenesis since it regulates the expression of host and viral genes. Previously, we reported that the deletion of the short isoform of Meq (S-Meq) decreases the pathogenicity of MDV. Recently, we identified a further short isoform of Meq (very short isoform of Meq, VS-Meq) in chickens with MD in Japan. A 64-amino-acid deletion was confirmed at the C-terminus of VS-Meq. We measured the transcriptional regulation by VS-Meq in three gene promoters to investigate the effect of VS-Meq on protein function. Wild-type VS-Meq decreased the transrepression of the pp38 promoter but did not alter the transactivation activity of the Meq and Bcl-2 promoters. The deletion in VS-Meq did not affect the activity of the pp38 promoter but enhanced the transactivation activities of the Meq and Bcl-2 promoters. Collectively, the deletion of VS-Meq potentially enhanced the activity of the Meq promoter, while other amino acid sequences in wild-type VS-Meq seemed to affect the weak transrepression of the pp38 promoter. Further investigation is required to clarify the effects of these changes on pathogenicity.

Therapeutic potential of Lonicerae japonicae flos against emerging respiratory viral infections

BackgroundRespiratory infections continue to rank among the leading global causes of death, and the emergence of new infectious respiratory diseases remains a persistent concern. Throughout Chinese history, particularly in the context of combating the plague, Lonicerae japonicae flos (LJF), a vital component of traditional Chinese herbal medicine, has been extensively utilized for its therapeutic potential due to its antioxidant, anti-inflammatory, and antimicrobial properties. This review concentrated on summarizing the roles and mechanism of LJF on the treatment of respiratory infections, and analyzing the potential therapeutic values of emerging respiratory infectious disease. MethodsThe literature with desired information about LJF was gained from electronic databases, including PubMed, Science Direct, Web of Science, Embase, and China National Knowledge Infrastructure. The literature search was conducted using the keywords “Lonicerae japonicae flos”, “Honeysuckle”, “Jin Yin Hua” and “LJF”, from 1985 to 2023. ResultsThrough summarizing and analyzing extensive fundamental and clinical studies on LJF, we have discovered that the bioactive molecules derived from LJF and prescriptions containing LJF could play a crucial role in combating viral infections. The antiviral mechanism involves interacting with viral genes and proteins, enhancing innate and acquired immune responses, influencing the processes of apoptosis, autophagy, pyroptosis, and etc. ConclusionIn light of preparing for future outbreaks of emerging respiratory infectious disease, pharmacological research and drug development based on LJF hold great significance and offer a promising trajectory.

Kaposi's sarcoma-associated herpesvirus terminal repeat regulates inducible lytic gene promoters.

The Kaposi's sarcoma-associated herpesvirus (KSHV) genome consists of an approximately 140-kb unique coding region flanked by 30-40 copies of a 0.8-kb terminal repeat (TR) sequence. A gene enhancer recruits transcription-related enzymes by having arrays of transcription factor binding sites. Here, we show that KSHV TR possesses transcription regulatory function with latency-associated nuclear antigen (LANA). Cleavage under targets and release using nuclease demonstrated that TR fragments were occupied by LANA-interacting histone-modifying enzymes in naturally infected cells. The TR was enriched with histone H3K27 acetylation (H3K27Ac) and H3K4 tri-methylation (H3K4me3) modifications and also expressed nascent RNAs. The sites of H3K27Ac and H3K4me3 modifications were also conserved in the KSHV unique region among naturally infected primary effusion lymphoma cells. KSHV origin of lytic replication (Ori-Lyt) showed similar protein and histone modification occupancies with that of TR. In the Ori-Lyt region, the LANA and LANA-interacting proteins colocalized with an H3K27Ac-modified nucleosome along with paused RNA polymerase II. The KSHV transactivator KSHV replication and transcription activator (K-Rta) recruitment sites franked the LANA-bound nucleosome, and reactivation evicted the LANA-bound nucleosome. Including TR fragments in reporter plasmid enhanced inducible viral gene promoter activities independent of the orientations. In the presence of TR in reporter plasmids, K-Rta transactivation was drastically increased, while LANA acquired the promoter repression function. KSHV TR, therefore, functions as an enhancer for KSHV inducible genes. However, in contrast to cellular enhancers bound by multiple transcription factors, perhaps the KSHV enhancer is predominantly regulated by the LANA nuclear body.IMPORTANCEEnhancers are a crucial regulator of differential gene expression programs. Enhancers are the cis-regulatory sequences determining target genes' spatiotemporal and quantitative expression. Here, we show that Kaposi's sarcoma-associated herpesvirus (KSHV) terminal repeats fulfill the enhancer definition for KSHV inducible gene promoters. The KSHV enhancer is occupied by latency-associated nuclear antigen (LANA) and its interacting proteins, such as CHD4. Neighboring terminal repeat (TR) fragments to lytic gene promoters drastically enhanced KSHV replication and transcription activator and LANA transcription regulatory functions. This study, thus, proposes a new latency-lytic switch model in which TR accessibility to the KSHV gene promoters regulates viral inducible gene expression.

Kaposi's sarcoma-associated herpesvirus (KSHV) LANA prevents KSHV episomes from degradation.

Protein knockdown with an inducible degradation system is a powerful tool for studying proteins of interest in living cells. Here, we adopted the auxin-inducible degron (AID) approach to detail Kaposi's sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen (LANA) function in latency maintenance and inducible viral lytic gene expression. We fused the mini-auxin-inducible degron (mAID) tag at the LANA N-terminus with KSHV bacterial artificial chromosome 16 recombination, and iSLK cells were stably infected with the recombinant KSHV encoding mAID-LANA. Incubation with 5-phenyl-indole-3-acetic acid, a derivative of natural auxin, rapidly degraded LANA within 1.5 h. In contrast to our hypothesis, depletion of LANA alone did not trigger lytic reactivation but rather decreased inducible lytic gene expression when we stimulated reactivation with a combination of ORF50 protein expression and sodium butyrate. Decreased overall lytic gene induction seemed to be associated with a rapid loss of KSHV genomes in the absence of LANA. The rapid loss of viral genomic DNA was blocked by a lysosomal inhibitor, chloroquine. Furthermore, siRNA-mediated knockdown of cellular innate immune proteins, cyclic AMP-GMP synthase (cGAS) and simulator of interferon genes (STING), and other autophagy-related genes rescued the degradation of viral genomic DNA upon LANA depletion. Reduction of the viral genome was not observed in 293FT cells that lack the expression of cGAS. These results suggest that LANA actively prevents viral genomic DNA from sensing by cGAS-STING signaling axis, adding novel insights into the role of LANA in latent genome maintenance.IMPORTANCESensing of pathogens' components is a fundamental cellular immune response. Pathogens have therefore evolved strategies to evade such cellular immune responses. KSHV LANA is a multifunctional protein and plays an essential role in maintaining the latent infection by tethering viral genomic DNA to the host chromosome. We adopted the inducible protein knockdown approach and found that depletion of LANA induced rapid degradation of viral genomic DNA, which is mediated by innate immune DNA sensors and autophagy pathway. These observations suggest that LANA may play a role in hiding KSHV episome from innate immune DNA sensors. Our study thus provides new insights into the role of LANA in latency maintenance.

Enhanced NF-κB activation via HIV-1 Tat-TRAF6 cross-talk.

The Tat proteins of HIV-1 and simian immunodeficiency virus (SIV) are essential for activating viral transcription. In addition, Tat stimulates nuclear factor κB (NF-κB) signaling pathways to regulate viral gene expression although its molecular mechanism is unclear. Here, we report that Tat directly activates NF-κB through the interaction with TRAF6, which is an essential upstream signaling molecule of the canonical NF-κB pathway. This interaction increases TRAF6 oligomerization and auto-ubiquitination, as well as the synthesis of K63-linked polyubiquitin chains to further activate the NF-κB pathway and HIV-1 transcription. Moreover, ectopic expression of TRAF6 significantly activates HIV-1 transcription, whereas TRAF6 knockdown inhibits transcription. Furthermore, Tat-mediated activation of NF-κB through TRAF6 is conserved among HIV-1, HIV-2, and SIV isolates. Our study uncovers yet another mechanism by which HIV-1 subverts host transcriptional pathways to enhance its own transcription.

Inhibition of Bromodomain Proteins Enhances Oncolytic HAdVC5 Replication and Efficacy in Pancreatic Ductal Adenocarcinoma (PDAC) Models.

Pancreatic ductal adenocarcinoma (PDAC) is the most aggressive type of pancreatic cancer, which rapidly develops resistance to the current standard of care. Several oncolytic Human AdenoViruses (HAdVs) have been reported to re-sensitize drug-resistant cancer cells and in combination with chemotherapeutics attenuate solid tumour growth. Obstacles preventing greater clinical success are rapid hepatic elimination and limited viral replication and spread within the tumour microenvironment. We hypothesised that higher intratumoural levels of the virus could be achieved by altering cellular epigenetic regulation. Here we report on the screening of an enriched epigenetics small molecule library and validation of six compounds that increased viral gene expression and replication. The greatest effects were observed with three epigenetic inhibitors targeting bromodomain (BRD)-containing proteins. Specifically, BRD4 inhibitors enhanced the efficacy of Ad5 wild type, Ad∆∆, and Ad-3∆-A20T in 3-dimensional co-culture models of PDAC and in vivo xenografts. RNAseq analysis demonstrated that the inhibitors increased viral E1A expression, altered expression of cell cycle regulators and inflammatory factors, and attenuated expression levels of tumour cell oncogenes such as c-Myc and Myb. The data suggest that the tumour-selective Ad∆∆ and Ad-3∆-A20T combined with epigenetic inhibitors is a novel strategy for the treatment of PDAC by eliminating both cancer and associated stromal cells to pave the way for immune cell access even after systemic delivery of the virus.