Progress on Z genome biosynthetic pathway of bacteriophage.

There are abundant base modifications in bacteriophages' genomes, mainly for avoiding the digestion of host endonucleases. More than 40 years ago, researchers discovered that 2-amino-adenine (Z) completely replaced adenine (A) and forms a complementary pairing with three hydrogen bonds with thymine (T) in the DNA of cyanophage S-2L, forming a distinct "Z-genome". In recent years, researchers have discovered and validated the biosynthetic pathway of Z-genome in various bacteriophages, constituting a multi-enzyme system. This system includes the phage-encoded enzymes deoxy-2'-aminoadenylosuccinate synthetase (PurZ), deoxyadenosine triphosphate hydrolase (dATPase/DatZ), deoxyadenosine/deoxyguanosine triphosphate pyrophosphatase (DUF550/MazZ) and DNA polymerase (DpoZ). In this review, we provide a concise overview of the historical discovery on diversely modified nucleosides in bacteriophages, then we comprehensively summarize the research progress on multiple enzymes involved in the Z-genome biosynthetic pathway. Finally, the potential applications of the Z-genome and the enzymes in its biosynthetic pathway are discussed in order to provide reference for research in this field.

Senescence and oxidative stress toxicities induced by lamivudine and tenofovir in Drosophila melanogaster.

BACKGROUND: Lamivudine and tenofovir disoproxil fumarate act against the replication of hepatitis B and human immunodeficiency viruses via inhibition of the reverse transcriptase enzyme activity, thereby preventing the synthesis of viral DNA. Chronic administration of these drugs has been associated with toxicities, including senescence, oxidative stress and premature death. A study of these toxicities in Drosophila melanogaster, which share 75% genomic similarity with humans could help to develop a pharmacologic intervention. METHODS: Susceptibility of D. melanogaster for lamivudine and tenofovir-induced toxicities were investigated. First, flies (≤3 days old) were fed with drugs-supplemented diet at varying concentrations (1mg to 300mg/10-gram diet) or distilled water for seven days to determine LD50. Secondly, five groups of 60 flies were fed with four concentrations of test drugs: 2.9mg, 5.82mg, 11.64mg and 23.28mg each per 10-gram diet for 28 days survival and lifespan assays. Then 5-day treatment plan was utilized to determine drugs toxicities on climbing ability and some biomarkers of oxidative stress. Finally, molecular docking was carried out using the Auto-dock vina mode to predict the biological interactions between the test drugs and D. melanogaster acetylcholinesterase (AChE) or glutathione-S-transferase (GST). RESULTS: The LD50 of lamivudine or tenofovir was 47.07 or 43.95mg/10g diet, respectively. Each drug significantly (P<0.05) reduced the survival rate, longevity and climbing performance of the flies dose-dependently. These drugs also altered levels of biochemical parameters: AChE, GST, superoxide dismutase (SOD), catalase (CAT), total thiol (T-SH), and malondialdehyde (MDA) of the flies significantly (P<0.05). In silico molecular analysis showed that the test drugs interacted with significantly (P<0.05) higher binding affinities at the same catalytic sites of D. melanogaster GST and AChE compared with substrates (glutathione or acetylcholine). CONCLUSION: The significant lamivudine and tenofovir-induced toxicities observed as increased mortality, climbing deficits and compromised antioxidant defence in D. melanogaster demands further research for possible pharmacological intervention.

Limitations of bacterial culture, viral PCR, and tulathromycin susceptibility from upper respiratory tract samples in predicting clinical outcome of tulathromycin control or treatment of bovine respiratory disease in high-risk feeder heifers.

A cross-sectional prospective cohort study including 1026 heifers administered tulathromycin due to high risk of clinical signs of bovine respiratory disease (BRD), measured poor association between BRD clinical outcomes and results of bacterial culture and tulathromycin susceptibility from BRD isolates of deep nasopharyngeal swabs (DNS) and adequate association with viral polymerase chain reaction (PCR) results from nasal swabs. Isolation rates from DNS collected on day-0 and at 1st BRD-treatment respectively were: Mannheimia haemolytica (10.9% & 34.1%); Pasteurella multocida (10.4% & 7.4%); Mycoplasma bovis (1.0% & 36.6%); and Histophilus somni (0.7% & 6.3%). Prevalence of BRD viral nucleic acid on nasal swabs collected exclusively at 1st BRD-treatment were: bovine parainfluenza virus type-3 (bPIV-3) 34.1%; bovine viral diarrhea virus (BVDV) 26.3%; bovine herpes virus type-1 (BHV-1) 10.8%; and bovine respiratory syncytial virus (BRSV) 54.1%. Increased relative risk, at 95% confidence intervals, of 1st BRD-treatment failure was associated with positive viral PCR results: BVDV 1.39 (1.17-1.66), bPIV-3 1.26 (1.06-1.51), BHV-1 1.52 (1.25-1.83), and BRSV 1.35 (1.11-1.63) from nasal swabs collected at 1st BRD-treatment and culture of M. haemolytica 1.23 (1.00-1.51) from DNS collected at day-0. However, in this population of high-risk feeder heifers, the predictive values of susceptible and resistant isolates had inadequate association with BRD clinical outcome. These results indicate, that using tulathromycin susceptibility testing of isolates of M. haemolytica or P. multocida from DNS collected on arrival or at 1st BRD-treatment to evaluate tulathromycin clinical efficacy, is unreliable.

The phage defence island of a multidrug resistant plasmid uses both BREX and type IV restriction for complementary protection from viruses.

Bacteria have evolved a multitude of systems to prevent invasion by bacteriophages and other mobile genetic elements. Comparative genomics suggests that genes encoding bacterial defence mechanisms are often clustered in 'defence islands', providing a concerted level of protection against a wider range of attackers. However, there is a comparative paucity of information on functional interplay between multiple defence systems. Here, we have functionally characterised a defence island from a multidrug resistant plasmid of the emerging pathogen Escherichia fergusonii. Using a suite of thirty environmentally-isolated coliphages, we demonstrate multi-layered and robust phage protection provided by a plasmid-encoded defence island that expresses both a type I BREX system and the novel GmrSD-family type IV DNA modification-dependent restriction enzyme, BrxU. We present the structure of BrxU to 2.12 Å, the first structure of the GmrSD family of enzymes, and show that BrxU can utilise all common nucleotides and a wide selection of metals to cleave a range of modified DNAs. Additionally, BrxU undergoes a multi-step reaction cycle instigated by an unexpected ATP-dependent shift from an intertwined dimer to monomers. This direct evidence that bacterial defence islands can mediate complementary layers of phage protection enhances our understanding of the ever-expanding nature of phage-bacterial interactions.

Mechanism of Hepatitis B Virus cccDNA Formation.

Hepatitis B virus (HBV) remains a major medical problem affecting at least 257 million chronically infected patients who are at risk of developing serious, frequently fatal liver diseases. HBV is a small, partially double-stranded DNA virus that goes through an intricate replication cycle in its native cellular environment: human hepatocytes. A critical step in the viral life-cycle is the conversion of relaxed circular DNA (rcDNA) into covalently closed circular DNA (cccDNA), the latter being the major template for HBV gene transcription. For this conversion, HBV relies on multiple host factors, as enzymes capable of catalyzing the relevant reactions are not encoded in the viral genome. Combinations of genetic and biochemical approaches have produced findings that provide a more holistic picture of the complex mechanism of HBV cccDNA formation. Here, we review some of these studies that have helped to provide a comprehensive picture of rcDNA to cccDNA conversion. Mechanistic insights into this critical step for HBV persistence hold the key for devising new therapies that will lead not only to viral suppression but to a cure.

The Influence of Psychological Stress on HPV Infection Manifestations and Carcinogenesis.

Psychological stress is an important factor involved in disease manifestations of human papillomavirus (HPV) infection, and it can participate in HPV-associated carcinogenesis. The impact or effect which stress can have (exert) depends on a person's genetic pool, experiences and behaviors. Due to inconsistencies in some study results, this issue remains a subject of research. Concerning the course of HPV manifestations, it has been observed that a higher number of life stressors in at least the previous 6 months, the absence of social support and the types of personal coping mechanisms employed, all influence HPV progression. In women with cervical dysplasia, a connection between greater stress experiences and dysregulation of specific immune responses has been observed. Once HPV enters a cell via the α6 integrin there are three possible sequences: latent infection, subclinical infection, and clinically manifest disease. HPV proliferation in differentiated epithelial cells induces morphologically cytopathic changes (koilocytosis, epidermal thickening, hyperplasia, hyperkeratosis). Oncogenic transformation requires the integration of the virus genome into the host genome. In doing so, DNA in the E1 region of E2 breaks down, leading to transcription disorders of E6 and E7. For the formation of irreversible malignancy, the following sequence is necessary: initial expression of E6 and E7 genes followed by suppression of apoptosis and the stabile expression of E6 and E7 proteins that protect transformed cells from apoptosis. A successful immune response is characterized by a strong, local cell-mediated immune response. Several factors are important for the regression of HPV manifestation/infection, among which is psychological stress which can prolong the duration and severity of HPV disease. Stress hormones may reactivate latent tumor viruses, stimulate viral oncogene expression, and inhibit antiviral host responses. In the regression of HPV infection, increased activity of Th1 cells was observed. However, during psychosocial stress, a decrease in the Th1 type of immune response is seen, and there is a shift towards a Th2 response. Understanding perceived stress and biological changes in stress, as well as the evaluation of immune parameters, gives researchers a better picture of how stress influences HPV infections and how to improve disease management and outcomes.

Curcumin and Photobiomodulation in Chronic Viral Hepatitis and Hepatocellular Carcinoma.

Immune modulation is a very modern medical field for targeting viral infections. In the race to develop the best immune modulator against viruses, curcumin, as a natural product, is inexpensive, without side effects, and can stimulate very well certain areas of the human immune system. As a bright yellow component of turmeric spice, curcumin has been the subject of thousands of scientific and clinical studies in recent decades to prove its powerful antioxidant properties and anticancer effects. Curcumin has been shown to influence inter- and intracellular signaling pathways, with direct effects on gene expression of the antioxidant proteins and those that regulate the immunity. Experimental studies have shown that curcumin modulates several enzyme systems, reduces nitrosative stress, increases the antioxidant capacity, and decreases the lipid peroxidation, protecting against fatty liver pathogenesis and fibrotic changes. Hepatitis B virus (HBV) affects millions of people worldwide, having sometimes a dramatic evolution to chronic aggressive infection, cirrhosis, and hepatocellular carcinoma. All up-to-date treatments are limited, there is still a gap in the scientific knowledge, and a sterilization cure may not yet be possible with the removal of both covalently closed circular DNA (cccDNA) and the embedded HBV DNA. With a maximum light absorption at 420 nm, the cytotoxicity of curcumin as photosensitizer could be expanded by the intravenous blue laser blood irradiation (IVBLBI) or photobiomodulation in patients with chronic hepatitis B infection, Hepatitis B e-antigen (HBeAg)-positive, noncirrhotic, but nonresponsive to classical therapy. Photobiomodulation increases DNA repair by the biosynthesis of complex molecules with antioxidant properties, the outset of repairing enzyme systems and new phospholipids for regenerating the cell membranes. UltraBioavailable Curcumin and blue laser photobiomodulation could suppress the virus and control better the disease by reducing inflammation/fibrosis and stopping the progression of chronic hepatitis, reversing fibrosis, and diminishing the progression of cirrhosis, and decreasing the incidence of hepatocellular carcinoma. Photodynamic therapy with blue light and curcumin opens new avenues for the effective prevention and cure of chronic liver infections and hepatocellular carcinoma. Blue laser light and UltraBioavailable Curcumin could be a new valuable alternative for medical applications in chronic B viral hepatitis and hepatocarcinoma, saving millions of lives.

Yin-Yang 1 and HBx protein activate HBV transcription by mediating the spatial interaction of cccDNA minichromosome with cellular chromosome 19p13.11.

HBV cccDNA stably exists in the nuclei of infected cells as an episomal munichromosome which is responsible for viral persistence and failure of current antiviral treatments. However, the regulatory mechanism of cccDNA transcription by viral and host cellular factors is not well understood. In this study, we investigated whether cccDNA could be recruited into a specific region of the nucleus via specific interaction with a cellular chromatin to regulate its transcription activity. To investigate this hypothesis, we used chromosome conformation capture (3C) technology to search for the potential interaction of cccDNA and cellular chromatin through rcccDNA transfection in hepatoma cells and found that cccDNA is specifically associated with human chromosome 19p13.11 region, which contains a highly active enhancer element. We also confirmed that cellular transcription factor Yin-Yang 1 (YY1) and viral protein HBx mediated the spatial regulation of HBV cccDNA transcription by 19p13.11 enhancer. Thus, These findings indicate that YY1 and HBx mediate the recruitment of HBV cccDNA minichromosomes to 19p13.11 region for transcription activation, and YY1 may present as a novel therapeutic target against HBV infection.

3,4,5-Tri-O-caffeoylquinic acid methyl ester isolated from Lonicera japonica Thunb. Flower buds facilitates hepatitis B virus replication in HepG2.2.15 cells.

Caffeoylquinic acids are well known for their prominent antiviral activities. Beyond our expectations, we initially found 3,4,5-Tri-O-caffeoylquinic acid methyl ester (3,4,5-CQME) from L. japonica can facilitate HBV DNA and antigens secretion. This study aimed to investigate its underlying molecular mechanism. The results indicate that 3,4,5-CQME signally increased intracellular and secreted HBsAg levels by more than two times in HepG2.2.15 cells and HepAD38 cells. Furthermore, levels of HBeAg, HBV DNA and RNA were significantly enhanced by 3-day 3,4,5-CQME treatment; it didn't directly affect intracellular cccDNA amount, although it slightly increased cccDNA accumulation as a HBV DNA replication feedback. In addition, treatment with 3,4,5-CQME significantly induced HBx protein expression for viral replication. We utilized a phospho-antibody assay to profile the signal transduction change by 3,4,5-CQME to illuminate its molecular mechanism. The results indicate that treatment with 3,4,5-CQME activated AKT/mTOR, MAPK and NF-κB pathways verified by immunoblot. Moreover, 3,4,5-CQME upregulated the expression of nuclear transcriptional factors PGC1α and PPARα. In short, 3,4,5-CQME promotes HBV transcription and replication by upregulating HBx expression and activating HBV transcriptional regulation-related signals. As caffeoylquinic acids are widely present in traditional Chinese medicines, the risk of intaking caffeoylquinic acids-containing herbs for hepatitis B treatment requires more evaluation and further research.

Screening for inhibitor of episomal DNA identified dicumarol as a hepatitis B virus inhibitor.

Currently, there is no available therapy to eradicate hepatitis B virus (HBV) in chronically infected individuals. This is due to the difficulty in eliminating viral covalently closed circular (ccc) DNA, which is central to the gene expression and replication of HBV. We developed an assay system for nuclear circular DNA using an integration-deficient lentiviral vector. This vector produced non-integrated circular DNA in nuclei of infected cells. We engineered this vector to encode firefly luciferase to monitor the lentiviral episome DNA. We screened 3,840 chemicals by this assay for luciferase-reducing activity and identified dicumarol, which is known to have anticoagulation activity. We confirmed that dicumarol reduced lentiviral episome DNA. Furthermore, dicumarol inhibited HBV replication in cell culture using NTCP-expressing HepG2 and primary human hepatocytes. Dicumarol reduced intracellular HBV RNA, DNA, supernatant HBV antigens and DNA. We also found that dicumarol reduced the cccDNA level in HBV infected cells, but did not affect HBV adsorption/entry. This is a novel assay system for screening inhibitors targeting nuclear cccDNA and is useful for finding new antiviral substances for HBV.

Preclinical Characterization of NVR 3-778, a First-in-Class Capsid Assembly Modulator against Hepatitis B Virus.

NVR 3-778 is the first capsid assembly modulator (CAM) that has demonstrated antiviral activity in hepatitis B virus (HBV)-infected patients. NVR 3-778 inhibited the generation of infectious HBV DNA-containing virus particles with a mean antiviral 50% effective concentration (EC50) of 0.40 µM in HepG2.2.15 cells. The antiviral profile of NVR 3-778 indicates pan-genotypic antiviral activity and a lack of cross-resistance with nucleos(t)ide inhibitors of HBV replication. The combination of NVR 3-778 with nucleos(t)ide analogs in vitro resulted in additive or synergistic antiviral activity. Mutations within the hydrophobic pocket at the dimer-dimer interface of the core protein could confer resistance to NVR 3-778, which is consistent with the ability of the compound to bind to core and to induce capsid assembly. By targeting core, NVR 3-778 inhibits pregenomic RNA encapsidation, viral replication, and the production of HBV DNA- and HBV RNA-containing particles. NVR 3-778 also inhibited de novo infection and viral replication in primary human hepatocytes with EC50 values of 0.81 µM against HBV DNA and between 3.7 and 4.8 µM against the production of HBV antigens and intracellular HBV RNA. NVR 3-778 showed favorable pharmacokinetics and safety in animal species, allowing serum levels in excess of 100 µM to be achieved in mice and, thus, enabling efficacy studies in vivo The overall preclinical profile of NVR 3-778 predicts antiviral activity in vivo and supports its further evaluation for safety, pharmacokinetics, and antiviral activity in HBV-infected patients.

Beneficial Effects of Bushen Formula Combined with Enticavir on Chronic Hepatitis B Patients with Suboptimal Response to Enticavir by Regulating B-Cell Differentiation.

BACKGROUND/AIMS: To investigate the clinical effects of the combination therapy with Bushen Formula (BSF) plus enticavir (ETV) on chronic hepatitis B (CHB) patients with suboptimal response to ETV and explore the regulatory mechanisms of BSF on B cells-mediated humoral immunity. METHODS: Sixty-four HBeAg-positive CHB patients with suboptimal response to ETV were enrolled, and were randomly assigned into control group (C-Group, placebo combined with ETV for 12 months) or treatment group (T-Group, BSF combined with ETV for 12 months). Serum samples from 57 treatment-naïve CHB patients and 15 healthy controls were collected. Serum HBV DNA levels were evaluated by real-time PCR. Characteristics of peripheral blood B-cell subtypes were analyzed by flow cytometry. Serum HBV markers and B cell-activating factor (BAFF) levels were detected by ELISA. Chinese medicine symptom complex score was evaluated and recorded. RESULTS: After treatment, the rates of patients with a reduction of HBsAg > 0.5 log10 IU/ml or 1.0 log10 IU/ml and the rates of HBeAg clearance in T-Group were all higher than those in C-group, with no significant intergroup difference. Only in T-Group, Chinese medicine symptom complex score and the frequency of total B cells were significantly decreased, and the frequencies of Bm1, CD24+CD27-switched B cells and plasma cells were markedly increased after treatment compared with those before treatment. Compared with healthy controls, serum BAFF levels in treatment-naïve CHB patients were increased, and there was a significant positive correlation between serum BAFF and HBsAg levels. However, serum BAFF levels did not differ after treatment in T-Group and C-Group. CONCLUSIONS: The combination therapy with BSF plus ETV promotes the reduction of HBsAg level and the clearance of HBeAg in CHB patients with partial response to ETV through regulating the differentiation of B-cell subsets.

In Vitro Characteristics of Phages to Guide 'Real Life' Phage Therapy Suitability.

The increasing problem of antibiotic-resistant pathogens has put enormous pressure on healthcare providers to reduce the application of antibiotics and to identify alternative therapies. Phages represent such an alternative with significant application potential, either on their own or in combination with antibiotics to enhance the effectiveness of traditional therapies. However, while phage therapy may offer exciting therapeutic opportunities, its evaluation for safe and appropriate use in humans needs to be guided initially by reliable and appropriate assessment techniques at the laboratory level. Here, we review the process of phage isolation and the application of individual pathogens or reference collections for the development of specific or "off-the-shelf" preparations. Furthermore, we evaluate current characterization approaches to assess the in vitro therapeutic potential of a phage including its spectrum of activity, genome characteristics, storage and administration requirements and effectiveness against biofilms. Lytic characteristics and the ability to overcome anti-phage systems are also covered. These attributes direct phage selection for their ultimate application as antimicrobial agents. We also discuss current pitfalls in this research area and propose that priority should be given to unify current phage characterization approaches.

Establishment of Cre-mediated HBV recombinant cccDNA (rcccDNA) cell line for cccDNA biology and antiviral screening assays.

Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA), existing in hepatocyte nuclei as a stable minichromosome, plays a central role in the life cycle of the virus and permits the persistence of infection. Despite being essential for HBV infection, little is known about the molecular mechanisms of cccDNA formation, regulation and degradation, and there is no therapeutic agents directly targeting cccDNA, fore mostly due to the lack of robust, reliable and quantifiable HBV cccDNA models. In this study, combined the Cre/loxP and sleeping beauty transposons system, we established HepG2-derived cell lines integrated with 2-60 copies of monomeric HBV genome flanked by loxP sites (HepG2-HBV/loxP). After Cre expression via adenoviral transduction, 3.3-kb recombinant cccDNA (rcccDNA) bearing a chimeric intron can be produced in the nuclei of these HepG2-HBV/loxP cells. The rcccDNA could be accurately quantified by quantitative PCR using specific primers and cccDNA pool generated in this model could be easily detected by Southern blotting using the digoxigenin probe system. We demonstrated that the rcccDNA was epigenetically organized as the natural minichromosome and served as the template supporting pgRNA transcription and viral replication. As the expression of HBV S antigen (HBsAg) is dependent on the newly generated cccDNA, HBsAg is the surrogate marker of cccDNA. Additionally, the efficacies of 3 classes of anti-HBV agents were evaluated in HepG2-HBV/loxP cells and antiviral activities with different mechanisms were confirmed. These data collectively suggested that HepG2-HBV/loxP cell system will be powerful platform for studying cccDNA related biological mechanisms and developing novel cccDNA targeting drugs.

Novel stable HBV producing cell line systems for expression and screening antiviral inhibitor of hepatitis B virus in human hepatoma cell line.

Chronic hepatitis B virus (HBV) infection is currently a major public health burden. Therefore, there is an urgent need for the development of novel antiviral inhibitors. The stable HBV-producing cell lines of genotype D are widely used to investigate the HBV life cycle and to evaluate antiviral agents. However, stable HBV-producing cell lines of different genotypes do not exist. To construct more convenient and efficient novel cell systems, stable cell lines of genotypes A, B, and C were established using a full-length HBV genome sequence isolated from chronic HBV patients in human hepatoma HepG2 cells. Novel HBV clones were identified and stable HBV-producing cell lines derived from these clones were constructed. HBV replication activities demonstrated time-dependent expression, and the novel cell lines were susceptible to several antiviral inhibitors with no cytotoxicity. Furthermore, infectious viruses were produced from these cell lines. In conclusion, we have established novel stable HBV-producing cell line systems of genotypes A, B, and C. These systems can provide valuable tools for screening antiviral agents and analyzing viral phenotypes in vitro.

Role of Litopenaeus vannamei Yin Yang 1 in the Regulation of the White Spot Syndrome Virus Immediate Early Gene ie1.

Yin Yang 1 (YY1) is a multifunctional zinc finger transcription factor that regulates many key cellular processes. In this study, we report the cloning of YY1 from Litopenaeus vannamei shrimp (LvYY1). This study shows that LvYY1 is ubiquitously expressed in shrimp tissues, and knockdown of LvYY1 expression by double-stranded RNA (dsRNA) injection in white spot syndrome virus (WSSV)-infected shrimp reduced both mRNA levels of the WSSV immediate early gene ie1 as well as overall copy numbers of the WSSV genome. The cumulative mortality rate of infected shrimp also declined with LvYY1 dsRNA injection. Using an insect cell model, we observed that LvYY1 activates ie1 expression, and a mutation introduced into the ie1 promoter subsequently repressed this capability. Moreover, reporter assay results suggested that LvYY1 is involved in basal transcriptional regulation via an interaction with L. vannamei TATA-binding protein (LvTBP). Electrophoretic mobility shift assay (EMSA) results further indicated that LvYY1 binds to a YY1-binding site in the region between positions -119 and -126 in the ie1 promoter. Chromatin immunoprecipitation analysis also confirmed that LvYY1 binds to the ie1 promoter in WSSV-infected shrimp. Taken together, these results indicate that WSSV uses host LvYY1 to enhance ie1 expression via a YY1-binding site and the TATA box in the ie1 promoter, thereby facilitating lytic activation and viral replication.IMPORTANCE WSSV has long been a scourge of the shrimp industry and remains a serious global threat. Thus, there is a pressing need to understand how the interactions between WSSV and its host drive infection, lytic development, pathogenesis, and mortality. Our successful cloning of L. vannamei YY1 (LvYY1) led to the elucidation of a critical virus-host interaction between LvYY1 and the WSSV immediate early gene ie1 We observed that LvYY1 regulates ie1 expression via a consensus YY1-binding site and TATA box. LvYY1 was also found to interact with L. vannamei TATA-binding protein (LvTBP), which may have an effect on basal transcription. Knockdown of LvYY1 expression inhibited ie1 transcription and subsequently reduced viral DNA replication and decreased cumulative mortality rates of WSSV-infected shrimp. These findings are expected to contribute to future studies involving WSSV-host interactions.

Cell cycle S phase markers are expressed in cerebral neuron nuclei of cats infected by the Feline Panleukopenia Virus.

The cell cycle-associated neuronal death hypothesis, which has been proposed as a common mechanism for most neurodegenerative diseases, is notably supported by evidencing cell cycle effectors in neurons. However, in naturally occurring nervous system diseases, these markers are not expressed in neuron nuclei but in cytoplasmic compartments. In other respects, the Feline Panleukopenia Virus (FPV) is able to complete its cycle in mature brain neurons in the feline species. As a parvovirus, the FPV is strictly dependent on its host cell reaching the cell cycle S phase to start its multiplication. In this retrospective study on the whole brain of 12 cats with naturally-occurring, FPV-associated cerebellar atrophy, VP2 capsid protein expression was detected by immunostaining not only in some brain neuronal nuclei but also in neuronal cytoplasm in 2 cats, suggesting that viral mRNA translation was still occurring. In these cats, double immunostainings demonstrated the expression of cell cycle S phase markers cyclin A, cdk2 and PCNA in neuronal nuclei. Parvoviruses are able to maintain their host cells in S phase by triggering the DNA damage response. S139 phospho H2A1, a key player in the cell cycle arrest, was detected in some neuronal nuclei, supporting that infected neurons were also blocked into the S phase. PCR studies did not support a co-infection with an adeno or herpes virus. ERK1/2 nuclear accumulation was observed in some neurons suggesting that the ERK signaling pathway might be involved as a mechanism driving these neurons far into the cell cycle.

Structural Insights into the HIV-1 Minus-strand Strong-stop DNA.

An essential step of human immunodeficiency virus type 1 (HIV-1) reverse transcription is the first strand transfer that requires base pairing of the R region at the 3'-end of the genomic RNA with the complementary r region at the 3'-end of minus-strand strong-stop DNA (ssDNA). HIV-1 nucleocapsid protein (NC) facilitates this annealing process. Determination of the ssDNA structure is needed to understand the molecular basis of NC-mediated genomic RNA-ssDNA annealing. For this purpose, we investigated ssDNA using structural probes (nucleases and potassium permanganate). This study is the first to determine the secondary structure of the full-length HIV-1 ssDNA in the absence or presence of NC. The probing data and phylogenetic analysis support the folding of ssDNA into three stem-loop structures and the presence of four high-affinity binding sites for NC. Our results support a model for the NC-mediated annealing process in which the preferential binding of NC to four sites triggers unfolding of the three-dimensional structure of ssDNA, thus facilitating interaction of the r sequence of ssDNA with the R sequence of the genomic RNA. In addition, using gel retardation assays and ssDNA mutants, we show that the NC-mediated annealing process does not rely on a single pathway (zipper intermediate or kissing complex).

SJP-L-5, a novel small-molecule compound, inhibits HIV-1 infection by blocking viral DNA nuclear entry.

BACKGROUND: Small-molecule compounds that inhibit human immunodeficiency virus type 1 (HIV-1) infection can be used not only as drug candidates, but also as reagents to dissect the life cycle of the virus. Thus, it is desirable to have an arsenal of such compounds that inhibit HIV-1 infection by various mechanisms. Until now, only a few small-molecule compounds that inhibit nuclear entry of viral DNA have been documented. RESULTS: We identified a novel, small-molecule compound, SJP-L-5, that inhibits HIV-1 infection. SJP-L-5 is a nitrogen-containing, biphenyl compound whose synthesis was based on the dibenzocyclooctadiene lignan gomisin M2, an anti-HIV bioactive compound isolated from Schisandra micrantha A. C. Smith. SJP-L-5 displayed relatively low cytotoxicity (50% cytoxicity concentrations were greater than 200 µg/ml) and high antiviral activity against a variety of HIV strains (50% effective concentrations (EC50)) of HIV-1 laboratory-adapted strains ranged from 0.16-0.97 µg/ml; EC50s of primary isolates ranged from 1.96-5.33 µg/ml). Analyses of the viral DNA synthesis indicated that SJP-L-5 specifically blocks the entry of the HIV-1 pre-integration complex (PIC) into the nucleus. Further results implicated that SJP-L-5 inhibits the disassembly of HIV-1 particulate capsid in the cytoplasm of the infected cells. CONCLUSIONS: SJP-L-5 is a novel small-molecule compound that inhibits HIV-1 nuclear entry by blocking the disassembly of the viral core.

Digestion of Nucleic Acids Starts in the Stomach.

The ingestion of nucleic acids (NAs) as a nutritional supplement or in genetically modified food has attracted the attention of researchers in recent years. Discussions over the fate of NAs led us to study their digestion in the stomach. Interestingly, we found that NAs are digested efficiently by human gastric juice. By performing digests with commercial, recombinant and mutant pepsin, a protein-specific enzyme, we learned that the digestion of NAs could be attributed to pepsin rather than to the acidity of the stomach. Further study showed that pepsin cleaved NAs in a moderately site-specific manner to yield 3'-phosphorylated fragments and the active site to digest NAs is probably the same as that used to digest protein. Our results rectify the misunderstandings that the digestion of NAs in the gastric tract begins in the intestine and that pepsin can only digest protein, shedding new light on NA metabolism and pepsin enzymology.