DNA Encapsidation Research Articles

Blood biodistribution and vector shedding of valoctocogene roxaparvovec in people with severe hemophilia A

AbstractFollowing systemically administered adeno-associated virus gene therapy, vector particles are widely distributed, raising concerns about horizontal or germline vector transmission. Characterization of biodistribution and kinetics of vector DNA in body fluids can address these concerns and provide insights into vector behavior in accessible samples. We investigated biodistribution and vector shedding profile of valoctocogene roxaparvovec in men with severe hemophilia A enrolled in the phase 3 GENEr8-1 trial. Participants (n = 134) received a single 6 × 1013 vector genome (vg)/kg infusion and were assessed over 3 years. Vector DNA was measured using 4 different assays. Total vector DNA was evaluated in blood, saliva, stool, semen, and urine by quantitative polymerase chain reaction (qPCR). Encapsidated vector DNA was measured in plasma and semen with immunocapture-based qPCR. Contiguity of vgs and assembly of inverted terminal repeat fusions were measured in whole blood and peripheral blood mononuclear cells (PBMCs) using multicolor digital PCR. Median peak vector DNA levels observed 1 to 8 days after dosing were highest in blood, followed by saliva, semen, stool, and urine. Concentrations declined steadily. Encapsidated vector DNA cleared faster than total vector DNA, achieving clearance by ≤12 weeks in plasma and semen. Predominant vector genome forms transitioned from noncontiguous to full-length over time in whole blood and PBMCs, indicating formation of stable circularized episomes within nucleated cells. The replication-incompetent nature of valoctocogene roxaparvovec, coupled with steady clearance of total and encapsidated vector DNA from shedding matrices, indicates transmission risk is low. This trial was registered at www.ClinicalTrials.gov as #NCT03370913.

Mutagenesis and functional analysis of the varicella-zoster virus portal protein.

Herpesviruses replicate by cleaving concatemeric dsDNA into single genomic units that are packaged through an oligomeric portal present in preformed procapsids. In contrast to what is known about phage portal proteins, details concerning herpesvirus portal structure and function are not as well understood. A panel of 65 Varicella-Zoster virus (VZV) recombinant portal proteins with five amino acid in-frame insertions were generated by random transposon mutagenesis of the VZV portal gene, ORF54. Subsequently, 65 VZVLUC recombinant viruses (TNs) were generated via recombineering. Insertions were mapped to predicted portal domains (clip, wing, stem, wall, crown, and β-hairpin tunnel-loop) and recombinant viruses were characterized for plaque morphology, replication kinetics, pORF54 expression, and classified based on replication in non-complementing (ARPE19) or complementing (ARPE54C50) cell lines. The N- and C-termini were tolerant to insertion mutagenesis, as were certain clip sub-domains. The majority of mutants mapping to the wing, wall, β-hairpin tunnel loop, and stem domains were lethal. Elimination of the predicted ORF54 start codon revealed that the first 40 amino acids of the N-terminus were not required for viral replication. Stop codon insertions in the C-terminus showed that the last 100 amino acids were not required for viral replication. Lastly, a putative protease cleavage site was identified in the C-terminus of pORF54. Cleavage was likely orchestrated by a viral protease; however, processing was not required for DNA encapsidation and viral replication. The panel of recombinants should prove valuable in future studies to dissect mammalian portal structure and function.IMPORTANCEThough nucleoside analogs and a live-attenuated vaccine are currently available to treat some human herpesvirus family members, alternate methods of combating herpesvirus infection could include blocking viral replication at the DNA encapsidation stage. The approval of Letermovir provided proof of concept regarding the use of encapsidation inhibitors to treat herpesvirus infections in the clinic. We propose that small-molecule compounds could be employed to interrupt portal oligomerization, assembly into the capsid vertex, or affect portal function/dynamics. Targeting portal at any of these steps would result in disruption of viral DNA packaging and a decrease or absence of mature infectious herpesvirus particles. The oligomeric portals of herpesviruses are structurally conserved, and therefore, it may be possible to find a single compound capable of targeting portals from one or more of the herpesvirus subfamilies. Drug candidates from such a series would be effective against viruses resistant to the currently available antivirals.

Maribavir: Mechanism of action, clinical, and translational science.

Maribavir is an oral benzimidazole riboside for treatment of post-transplant cytomegalovirus (CMV) infection/disease that is refractory to prior antiviral treatment (with or without resistance). Through competitive inhibition of adenosine triphosphate, maribavir prevents the phosphorylation actions of UL97 to inhibit CMV DNA replication, encapsidation, and nuclear egress. Maribavir is active against CMV strains with viral DNA polymerase mutations that confer resistance to other CMV antivirals. After oral administration, maribavir is rapidly and highly absorbed (fraction absorbed >90%). The approved dose of 400 mg twice daily (b.i.d.) achieves a steady-state area under the curve per dosing interval of 128 h*μg/mL and trough concentration of 4.90 μg/mL (13.0 μM). Maribavir is highly bound to human plasma proteins (98%) with a small apparent volume of distribution of 27.3 L. Maribavir is primarily cleared by hepatic CYP3A4 metabolism; its major metabolite, VP44669 (pharmacologically inactive), is excreted in the urine and feces. There is no clinically relevant impact on maribavir pharmacokinetics by age, sex, race/ethnicity, body weight, transplant type, or hepatic/renal impairment status. In phase II dose-ranging studies, maribavir showed similar rates of CMV viral clearance across 400, 800, or 1200 mg b.i.d. groups, ranging from 62.5-70% in study 202 (NCT01611974) and 74-83% in study 203 (EudraCT 2010-024247-32). In the phase III SOLSTICE trial (NCT02931539), maribavir 400 mg b.i.d. demonstrated superior CMV viremia clearance at week 8 versus investigator-assigned treatments, with lower treatment discontinuation rates. Dysgeusia, nausea, vomiting, and diarrhea were commonly experienced adverse events among patients treated with maribavir in clinical trials.

Optimal size of DNA encapsidated by plant produced human papillomavirus pseudovirions

Human papillomaviruses (HPVs) are known to be the cause of anogenital and oropharyngeal cancers as well as genital and common warts. HPV pseudovirions (PsVs) are synthetic viral particles that are made up of the L1 major and L2 minor HPV capsid proteins and up to 8 Kb of encapsidated pseudogenome dsDNA. HPV PsVs are used to test novel neutralising antibodies elicited by vaccines, for studying the virus life cycle, and potentially for the delivery of therapeutic DNA vaccines. HPV PsVs are typically produced in mammalian cells, however, it has recently been shown that Papillomavirus PsVs can be produced in plants, a potentially safer, cheaper and more easily scalable means of production. We analysed the encapsidation frequencies of pseudogenomes expressing EGFP, ranging in size from 4.8 Kb to 7.8 Kb, by plant-made HPV-35 L1/L2 particles. The smaller pseudogenomes were found to be packaged more efficiently into PsVs as higher concentrations of encapsidated DNA and higher levels of EGFP expression were obtained with the 4.8 Kb pseudogenome, compared to the larger 5.8–7.8 Kb pseudogenomes. Thus, smaller pseudogenomes, of 4.8 Kb, should be used for efficient plant production of HPV-35 PsVs.

Helical organization of DNA-like liquid crystal filaments in cylindrical viral capsids

We study equilibrium configurations of double-stranded DNA in a cylindrical viral capsid. The state of the encapsidated DNA consists of a disordered inner core enclosed by an ordered outer region, next to the capsid wall. The DNA configuration is described by a unit helical vector field, tangent to an associated centre curve, passing through properly selected locations. We postulate an expression for the energy of the encapsulated DNA based on that of columnar chromonic liquid crystals. A thorough analysis of the Euler–Lagrange equations yields multiple solutions. We demonstrate that there is a trivial, non-helical solution, together with two solutions with non-zero helicity of opposite sign. Using bifurcation analysis, we derive the conditions for local stability and determine when the preferred coiling state is helical. The bifurcation parameters are the ratio of the twist versus the bend moduli of DNA and the ratio between the sizes of the ordered and the disordered regions.

Involvement of Terminase Complex in Herpes Simplex Virus Mature Virion Egress.

The life cycle of the Herpes simplex virus starts with attachment to the host cell, injection of the nucleocapsid into the cytoplasm, replication, transcription and viral protein production, and finally, the assembly of the mature virion nucleopcapsid. The assembled nucleocapsid exits the host nucleus and gains a tegument layer bound within a bilayer of membrane phospholipid. The packaged virion particle then exits the host cell. The interaction of the (Deoxyribonucleic acid) DNA packaging complex- terminase, present on the mature viral nucleocapsid, with other proteins involved in nuclear egress and cytoplasmic tegumentation has led to the proposal of the model by which the terminase complex may be involved in these two events. The role of terminase complex in Herpes Simplex Virus (HSV) genomic DNA encapsidation into the capsid is previously established, but the role of the terminase subunits post DNA packaging remains unclear. The current review provides a model by which the terminase complex may have a role to play in the events of nuclear egress and secondary envelopment.

A Comparative Study on Delivery of Externally Attached DNA by Papillomavirus VLPs and Pseudoviruses.

Human papillomavirus (HPV) 16 capsids have been chosen as a DNA delivery vehicle in many studies. Our preliminary studies suggest that HPV58 capsids could be better vehicles than HPV16 capsids to deliver encapsidated DNA in vitro and in vivo. In the current study, we compared HPV16, HPV58, and the cottontail rabbit papillomavirus (CRPV) capsids either as L1/L2 VLPs or pseudoviruses (PSVs) to deliver externally attached GFP-expressing DNA. Both rabbit and human cells were used to test whether there was a species-specific effect. DNA delivery efficiency was determined by quantifying either GFP-expressing cell populations or mean fluorescent intensities (MFI) by flow cytometry. Interestingly, CRPV and 58-VLPs and PSVs were significantly more efficient at delivering attached DNA when compared to 16-VLPs and PSVs. A capsid/DNA ratio of 2:1 showed the highest efficiency for delivering external DNA. The PSVs with papillomavirus DNA genomes also showed higher efficiency than those with irrelevant plasmid DNA. HPV16L1/58L2 hybrid VLPs displayed increased efficiency compared to HPV58L1/16L2 VLPs, suggesting that L2 may play a critical role in the delivery of attached DNA. Additionally, we demonstrated that VLPs increased in vivo infectivity of CRPV DNA in rabbits. We conclude that choosing CRPV or 58 capsids to deliver external DNA could improve DNA uptake in in vitro and in vivo models.

High-throughput identification of viral termini and packaging mechanisms in virome datasets using PhageTermVirome

Viruses that infect bacteria (phages) are increasingly recognized for their importance in diverse ecosystems but identifying and annotating them in large-scale sequence datasets is still challenging. Although efficient scalable virus identification tools are emerging, defining the exact ends (termini) of phage genomes is still particularly difficult. The proper identification of termini is crucial, as it helps in characterizing the packaging mechanism of bacteriophages and provides information on various aspects of phage biology. Here, we introduce PhageTermVirome (PTV) as a tool for the easy and rapid high-throughput determination of phage termini and packaging mechanisms using modern large-scale metagenomics datasets. We successfully tested the PTV algorithm on a mock virome dataset and then used it on two real virome datasets to achieve the rapid identification of more than 100 phage termini and packaging mechanisms, with just a few hours of computing time. Because PTV allows the identification of free fully formed viral particles (by recognition of termini present only in encapsidated DNA), it can also complement other virus identification softwares to predict the true viral origin of contigs in viral metagenomics datasets. PTV is a novel and unique tool for high-throughput characterization of phage genomes, including phage termini identification and characterization of genome packaging mechanisms. This software should help researchers better visualize, map and study the virosphere. PTV is freely available for downloading and installation at https://gitlab.pasteur.fr/vlegrand/ptv.

Ultra-sensitive AAV capsid detection by immunocapture-based qPCR following factor VIII gene transfer

Adeno-associated virus (AAV)-based gene therapy vectors are replication-incompetent and thus pose minimal risk for horizontal transmission or release into the environment. In studies with AAV5-FVIII-SQ (valoctocogene roxaparvovec), an investigational gene therapy for hemophilia A, residual vector DNA was detectable in blood, secreta, and excreta, but it remained unclear how long structurally intact AAV5 vector capsids were present. Since a comprehensive assessment of vector shedding is required by regulatory agencies, we developed a new method (termed iqPCR) that utilizes capsid-directed immunocapture followed by qPCR amplification of encapsidated DNA. The limit of detection for AAV5 vector capsids was 1.17E+04 and 2.33E+04 vg/mL in plasma and semen, respectively. Acceptable precision, accuracy, selectivity, and specificity were verified; up to 1.00E+09 vg/mL non-encapsidated vector DNA showed no interference. Anti-AAV5 antibody plasma concentrations above 141 ng/mL decreased AAV5 capsid quantification, suggesting that iqPCR mainly detects free capsids and not those complexed with antibodies. In a clinical study, AAV5-FVIII-SQ capsids were found in plasma and semen but became undetectable within nine weeks after dose administration. Hence, iqPCR monitors the presence and shedding kinetics of intact vector capsids following AAV gene therapy and informs the potential risk for horizontal transmission.

CRISPR-Guided Programmable Self-Assembly of Artificial Virus-Like Nucleocapsids.

Designer virus-inspired proteins drive the manufacturing of more effective, safer gene-delivery systems and simpler models to study viral assembly. However, self-assembly of engineered viromimetic proteins on specific nucleic acid templates, a distinctive viral property, has proved difficult. Inspired by viral packaging signals, we harness the programmability of CRISPR-Cas12a to direct the nucleation and growth of a self-assembling synthetic polypeptide into virus-like particles (VLP) on specific DNA molecules. Positioning up to ten nuclease-dead Cas12a (dCas12a) proteins along a 48.5 kbp DNA template triggers particle growth and full DNA encapsidation at limiting polypeptide concentrations. Particle growth rate is further increased when dCas12a is dimerized with a polymerization silk-like domain. Such improved self-assembly efficiency allows for discrimination between cognate versus noncognate DNA templates by the synthetic polypeptide. CRISPR-guided VLPs will help to develop programmable bioinspired nanomaterials with applications in biotechnology as well as viromimetic scaffolds to improve our understanding of viral self-assembly.

Multiple-parameter profiling of density gradient ultracentrifugation for characterization of empty and full capsid distribution in AAV preparations

Ultracentrifugation (UC) is a well-known technique for fractionating adeno-associated virus (AAV) capsids according to their density, which is mainly a function of their encapsidated DNA mass. Empty capsids represent the lowest density subpopulation. Full capsids represent the highest density subpopulation, sometimes accompanied by partially full capsids of intermediate density. Fractions can be collected after sedimentation for analysis but the practice is laborious and discourages application of multiple monitoring techniques that might provide deeper insights into sample composition. Anion exchange chromatography (AEC) also achieves fractionation of empty and full capsids for many AAV serotypes. The degree of separation varies among serotypes and does not correlate strictly with UC. This is not surprising since separation by AEC is highly influenced by capsid surface charge, which is independent of the amount of DNA packaged within the capsids. Chromatography methods however present a significant analytical advantage in the ease of monitoring the column effluent, including with multiple detectors. UV absorbance at 260 nm and 280 nm permits estimation of empty and full capsid proportions in any given peak. Intrinsic fluorescence enables estimation of relative areas of empty capsid peaks and full capsid peaks. Light scattering does the same and permits the further determination of capsid size and mass. In this report, we merge UC with an HPLC monitoring array to simultaneously analyze dual wavelength UV, intrinsic fluorescence, and light scattering through cesium chloride density gradient strata. Limitations of each monitoring method are discussed. UC results are compared with chromatography profiles to highlight distinction between separation methods. Practical application of results for final product characterization is considered, along with potential to support development of better purification processes.

A method for characterizing dissolved DNA and its application to the North Pacific Subtropical Gyre

AbstractDissolved DNA (D‐DNA) is a ubiquitous component of dissolved organic matter in aquatic systems. It is operationally defined as the DNA that passes a membrane filter and thus includes pools of truly dissolved “free” DNA (F‐DNA), virion encapsidated DNA, DNA within membrane vesicles, and possibly other bound forms, each with different sources and lability. We investigated whether filtration (< 0.1 μm), concentration by tangential flow ultrafiltration (> 30 kDa), and fractionation in an equilibrium buoyant density gradient could be used to discriminate the mass contributions of the different pools of filterable DNA in seawater. Spike‐in experiments with a known range of DNA standards (75–20,000 bp) indicated that this method results in high recoveries of F‐DNA (68–86%) with minimal degradation. Application of the fractionation method to seawater samples collected from the oligotrophic North Pacific Ocean followed by analysis of fractions (epifluorescence and electron microscopy, DNase digestion) suggested that the low‐density fractions (1.30–1.35 g mL−1) were dominated by vesicle‐like particles, mid‐density fractions (1.45–1.55 g mL−1) by virus‐like particles, and high‐density fractions (1.60–1.70 g mL−1) by F‐DNA. The estimated concentration of DNA that is either F‐DNA, in viruses, or in vesicles was 0.13, 0.14, and 0.08 μg L−1, respectively in the euphotic zone and 0.09, 0.04, and 0.03 μg L−1, respectively in the mesopelagic zone. The approach described should be useful for more detailed investigations of the abundance, dynamics, and sources of DNA in the distinct pools that comprise filterable DNA in aquatic environments.

Multiple-Monitor HPLC Assays for Rapid Process Development, In-Process Monitoring, and Validation of AAV Production and Purification.

HPLC is established as a fast convenient analytical technology for characterizing the content of empty and full capsids in purified samples containing adeno-associated virus (AAV). UV-based monitoring unfortunately over-estimates the proportion of full capsids and offers little value for characterizing unpurified samples. The present study combines dual-wavelength UV monitoring with intrinsic fluorescence, extrinsic fluorescence, and light-scattering to extend the utility of HPLC for supporting development of therapeutic AAV-based drugs. Applications with anion exchange (AEC), cation exchange (CEC), and size exclusion chromatography (SEC) are presented. Intrinsic fluorescence increases sensitivity of AAV detection over UV and enables more objective estimation of empty and full capsid ratios by comparison of their respective peak areas. Light scattering enables identification of AAV capsids in complex samples, plus semiquantitative estimation of empty and full capsid ratios from relative peak areas of empty and full capsids. Extrinsic Picogreen fluorescence enables semiquantitative tracking of DNA with all HPLC methods at all stages of purification. It does not detect encapsidated DNA but reveals DNA associated principally with the exteriors of empty capsids. It also enables monitoring of host DNA contamination across chromatograms. These enhancements support many opportunities to improve characterization of raw materials and process intermediates, to accelerate process development, provide rapid in-process monitoring, and support process validation.

Characterization of Viral Genome Encapsidated in Adeno-associated Recombinant Vectors Produced in Yeast Saccharomyces cerevisiae.

Adeno-associated virus (AAV) is a small, non-enveloped virus used as vector in gene therapy, mainly produced in human cells and in baculovirus systems. Intense studies on these platforms led to the production of vectors with titers between 103 and 105 viral genomes (vg) per cells. In spite of this, vector yields need to be improved to satisfy the high product demands of clinical trials and future commercialization. Our studies and those of other groups have explored the possibility to exploit the yeast Saccharomyces cerevisiae to produce rAAV. We previously demonstrated that yeast supports AAV genome replication and capsid assembly. The purpose of this study was to evaluate the quality of the encapsidated AAV DNA. Here, we report the construction of a yeast strain expressing Rep68/40 from an integrated copy of the Rep gene under the control of the yeast constitutive ADH promoter and Capsid proteins from the Cap gene under the control of an inducible GAL promoter. Our results indicate that a portion of AAV particles generated by this system contains encapsidated AAV DNA. However, the majority of encapsidated DNA consists of fragmented regions of the transgene cassette, with ITRs being the most represented sequences. Altogether, these data indicate that, in yeast, encapsidation occurs with low efficiency and that rAAVs resemble pseudo-vectors that are present in clinical-grade rAAV preparations.

Proteome profiling and vector yield optimization in a recombinant adeno-associated virus-producing yeast model.

ABSTRACTRecent studies on recombinant adeno‐associated viral (rAAV) vector production demonstrated the generation of infectious viral particles in Saccharomyces cerevisiae. Proof‐of‐concept results showed low vector yields that correlated with low AAV DNA encapsidation rates. In an attempt to understand the host cell response to rAAV production, we profiled proteomic changes throughout the fermentation process by mass spectrometry. By comparing an rAAV‐producing yeast strain with a respective non‐producer control, we identified a subset of yeast host proteins with significantly different expression patterns during the rAAV induction period. Gene ontology enrichment and network interaction analyses identified changes in expression patterns associated mainly with protein folding, as well as amino acid metabolism, gluconeogenesis, and stress response. Specific fold change patterns of heat shock proteins and other stress protein markers suggested the occurrence of a cytosolic unfolded protein response during rAAV protein expression. Also, a correlative increase in proteins involved in response to oxidative stress suggested cellular activities to ameliorate the effects of reactive oxygen species or other oxidants. We tested the functional relevance of the identified host proteins by overexpressing selected protein leads using low‐ and high‐copy number plasmids. Increased vector yields up to threefold were observed in clones where proteins SSA1, SSE1, SSE2, CCP1, GTT1, and RVB2 were overexpressed. Recombinant expression of SSA1 and YDJ insect homologues (HSP40 and HSC70, respectively) in Sf9 cells led to a volumetric vector yield increase of 50% relative to control, which validated the importance of chaperone proteins in rAAV‐producing systems. Overall, these results highlight the utility of proteomic‐based tools for the understanding and optimization of rAAV‐producing recombinant strains.

Requirements for the Packaging of Geminivirus Circular Single-Stranded DNA: Effect of DNA Length and Coat Protein Sequence.

Geminivirus particles, consisting of a pair of twinned isometric structures, have one of the most distinctive capsids in the virological world. Until recently, there was little information as to how these structures are generated. To address this, we developed a system to produce capsid structures following the delivery of geminivirus coat protein and replicating circular single-stranded DNA (cssDNA) by the infiltration of gene constructs into plant leaves. The transencapsidation of cssDNA of the Begomovirus genus by coat protein of different geminivirus genera was shown to occur with full-length but not half-length molecules. Double capsid structures, distinct from geminate capsid structures, were also generated in this expression system. By increasing the length of the encapsidated cssDNA, triple geminate capsid structures, consisting of straight, bent and condensed forms were generated. The straight geminate triple structures generated were similar in morphology to those recorded for a potato-infecting virus from Peru. These finding demonstrate that the length of encapsidated DNA controls both the size and stability of geminivirus particles.

DNA Encapsidation and Capsid Assembly Are Underexploited Antiviral Targets for the Treatment of Herpesviruses.

Although there are effective nucleoside analogs to treat HSV, VZV, and HCMV disease, herpesvirus infections continue to contribute to significant morbidity and mortality. Acyclovir is the drug of choice for HSV encephalopathy, yet there is an estimated 6–19% mortality rate with half of the survivors experiencing moderate to severe chronic neurological deficits. For VZV, current treatments are inadequate to prevent acute and persistent pain due to zoster. Treatment of HCMV with GCV requires close monitoring particularly in patients with impaired renal function and there are no approved treatments for congenital HCMV infections. New therapeutic options to control cytomegalovirus reactivation in bone marrow and stem cell transplant patients are needed to improve patient outcome. No successful chemotherapeutic options are available for EBV, HHV-6, 7, and 8. Drug resistance is a concern for HCMV, HSV, and VZV since approved drugs share common mechanisms of action. Targeting DNA encapsidation or capsid assembly provide additional options for the development of non-nucleoside, small molecule anti-herpesviral drugs.

Fine structure of viral dsDNA encapsidation.

Unraveling the mechanisms of packing of DNA inside viral capsids is of fundamental importance to understanding the spread of viruses. It could also help develop new applications to targeted drug delivery devices for a large range of therapies. In this article, we present a robust, predictive mathematical model and its numerical implementation to aid the study and design of bacteriophage viruses for application purposes. Exploiting the analogies between the columnar hexagonal chromonic phases of encapsidated viral DNA and chromonic aggregates formed by plank-shaped molecular compounds, we develop a first-principles effective mechanical model of DNA packing in a viral capsid. The proposed expression of the packing energy, which combines relevant aspects of the liquid crystal theory, is developed from the model of hexagonal columnar phases, together with that describing configurations of polymeric liquid crystals. The method also outlines a parameter selection strategy that uses available data for a collection of viruses, aimed at applications to viral design. The outcome of the work is a mathematical model and its numerical algorithm, based on the method of finite elements, and computer simulations to identify and label the ordered and disordered regions of the capsid and calculate the inner pressure. It also presents the tools for the local reconstruction of the DNA "scaffolding" and the center curve of the filament within the capsid.

Characterization and genome analysis of B1 sub-cluster mycobacteriophage PDRPxv

Mycobacteriophages are viruses specific to mycobacteria that have gained attention as alternative therapeutic strategies for treating antibiotic-resistant infections. Mycobacteriophages are highly diverse and have been grouped into 29 clusters, 71 sub-clusters and 10 singletons based on the genome sequence. Here, we annotate the genome of PDRPxv, a lytic mycobacteriophage isolated from New Delhi; it belongs to the Siphoviridae family as determined by transmission electron microscopy. This phage survives at higher temperatures (up to 55 °C) and in alkaline conditions (up to pH11). PDRPxv phage genome is 69,171 bp in length with 66.35 % GC content and encodes 107 putative open reading frames and belongs to the B1 sub-cluster. Genome annotation indicated that genes for DNA encapsidation, structural proteins, replication/transcription and lysis of the host are present in functional clusters. Structural proteins encoded by Gp10-Gp12, Gp18, Gp25 and Gp28-Gp33 were identified by mass spectrometry. Interestingly, no gene encoding a holin function was found. Single-step growth curve revealed that PDRPxv has an adsorption time of 45 min, a latency time of 135 min and an average burst size of 99 phage particles per infected cell. The short latency period and the large burst size mark the lytic nature of the PDRPxv phage, which could therefore be a promising therapeutic candidate against pathogenic Mycobacterium species.

HBV X protein mutations affect HBV transcription and association of histone-modifying enzymes with covalently closed circular DNA

The hepatitis B X protein (HBx) plays a role in the epigenetic regulation of hepatitis B virus (HBV) replication. This study investigated the effects of HBx mutations on HBV transcription and the recruitment of HBx, histone acetyl-transferase P300 and histone deacetylase 1 (HDAC1) to circularized HBV DNA (which resembles covalently closed circular DNA [cccDNA]). Compared with wild type, majority of mutants had lower levels of intracellular HBV RNA (44–77% reduction) and secretory HBsAg (25–81% reduction), and 12 mutants had a reduction in intracellular encapsidated HBV DNA (33–64% reduction). Eight mutants with >70% reduction in HBV RNA and/or HBsAg were selected for chromatin immunoprecipitation analysis. Four HBx mutants with mutations in amino acid residues 55–60 and 121–126 had a lower degree of HBx-cccDNA association than wild type HBx (mean % input: 0.02–0.64% vs. 3.08% in wild type). A reduced association between cccDNA and P300 (mean % input: 0.69–1.81% vs. 3.48% in wild type) and an augmented association with HDAC1 (mean % input: 4.01–14.0% vs. 1.53% in wild type) were detected. HBx amino acid residues 55–60 and 121–126 may play an important role in HBV transcription regulation, via their impeded interaction with cccDNA and altered recruitment of histone modifying enzymes to cccDNA.