Wild-type Capsid Research Articles

NEDD4 family ubiquitin ligase AIP4 interacts with Alix to enable HBV naked capsid egress in an Alix ubiquitination-independent manner.

Hepatitis B virus (HBV) exploits the endosomal sorting complexes required for transport (ESCRT)/multivesicular body (MVB) pathway for virion budding. In addition to enveloped virions, HBV-replicating cells nonlytically release non-enveloped (naked) capsids independent of the integral ESCRT machinery, but the exact secretory mechanism remains elusive. Here, we provide more detailed information about the existence and characteristics of naked capsid, as well as the viral and host regulations of naked capsid egress. HBV capsid/core protein has two highly conserved Lysine residues (K7/K96) that potentially undergo various types of posttranslational modifications for subsequent biological events. Mutagenesis study revealed that the K96 residue is critical for naked capsid egress, and the intracellular egress-competent capsids are associated with ubiquitinated host proteins. Consistent with a previous report, the ESCRT-III-binding protein Alix and its Bro1 domain are required for naked capsid secretion through binding to intracellular capsid, and we further found that the ubiquitinated Alix binds to wild type capsid but not K96R mutant. Moreover, screening of NEDD4 E3 ubiquitin ligase family members revealed that AIP4 stimulates the release of naked capsid, which relies on AIP4 protein integrity and E3 ligase activity. We further demonstrated that AIP4 interacts with Alix and promotes its ubiquitination, and AIP4 is essential for Alix-mediated naked capsid secretion. However, the Bro1 domain of Alix is non-ubiquitinated, indicating that Alix ubiquitination is not absolutely required for AIP4-induced naked capsid secretion. Taken together, our study sheds new light on the mechanism of HBV naked capsid egress in viral life cycle.

Production of recombinant adeno-associated virus 5 using a novel self-attenuating adenovirus production platform

Recombinant adeno-associated virus (rAAV) has become a prominent vector for clinical use. Despite an increase in successful clinical outcomes, the amount of high-quality rAAVs required for clinical trials and eventual commercial demand is difficult to produce, especially for genetic diseases that are prevalent or require high doses. Many groups are focused on establishing production processes that can produce sufficient rAAV while maintaining potency and quality. Our group used a novel production platform to increase our yield of rAAV5. This production platform uses tetracycline-enabled self-silencing adenovirus (TESSA) to deliver the wild-type AAV replication and capsid genes alongside the adenovirus helper genes necessary for production. Here, we describe our efforts to evaluate the TESSA platform in house. We conducted numerous experiments to determine the optimal conditions for producing rAAV5 from the TESSA production system. We then produced rAAV5 from the TESSA system to compare against rAAV5 produced from triple transfection. Ultimately, we generated data that showed that the vector genome yield of rAAV5 produced with TESSA was >20-fold higher than rAAV5 produced with triple transfection. Additionally, our data show that quality as well as potency in mice of rAAV5 produced with the TESSA system and by triple transfection are equivalent.

Virus specificity and nucleoporin requirements for MX2 activity are affected by GTPase function and capsid-CypA interactions.

Human myxovirus resistance 2 (MX2/MXB) is an interferon-induced GTPase that inhibits human immunodeficiency virus-1 (HIV-1) infection by preventing nuclear import of the viral preintegration complex. The HIV-1 capsid (CA) is the major viral determinant for sensitivity to MX2, and complex interactions between MX2, CA, nucleoporins (Nups), cyclophilin A (CypA), and other cellular proteins influence the outcome of viral infection. To explore the interactions between MX2, the viral CA, and CypA, we utilized a CRISPR-Cas9/AAV approach to generate CypA knock-out cell lines as well as cells that express CypA from its endogenous locus, but with specific point mutations that would abrogate CA binding but should not affect enzymatic activity or cellular function. We found that infection of CypA knock-out and point mutant cell lines with wild-type HIV-1 and CA mutants recapitulated the phenotypes observed upon cyclosporine A (CsA) addition, indicating that effects of CsA treatment are the direct result of blocking CA-CypA interactions and are therefore independent from potential interactions between CypA and MX2 or other cellular proteins. Notably, abrogation of GTP hydrolysis by MX2 conferred enhanced antiviral activity when CA-CypA interactions were abolished, and this effect was not mediated by the CA-binding residues in the GTPase domain, or by phosphorylation of MX2 at position T151. We additionally found that elimination of GTPase activity also altered the Nup requirements for MX2 activity. Our data demonstrate that the antiviral activity of MX2 is affected by CypA-CA interactions in a virus-specific and GTPase activity-dependent manner. These findings further highlight the importance of the GTPase domain of MX2 in regulation of substrate specificity and interaction with nucleocytoplasmic trafficking pathways.

Seroprevalence of binding and neutralizing antibodies against 18 adeno-associated virus types in patients with neuromuscular disorders.

High levels of pre-existing antibodies are a major challenge for the application of viral vectors since they can severely limit their efficacy. To identify promising candidates among adeno-associated virus (AAV) based vectors for future gene therapies for the treatment of hereditary neuromuscular disorders (NMDs), we investigated the antibody levels in sera from patients with NMDs against 18 AAV types, including 11 AAVs with wild-type capsids, 5 AAVs with peptide-modified capsids and 2 AAVs with shuffled capsids. With regard to the wild-type capsid AAVs, the lowest binding antibody levels were detected against AAV6, AAV5, AAV12 and AAV9, whereas the highest binding antibody levels were detected against AAV10, AAV8, AAV1, and AAV2. The lowest neutralizing antibody levels against wild-type AAVs were detected against AAV12, AAV5, AAV9, AAV7, AAV8 and AAV10, and the highest neutralizing antibody levels were detected against AAV13, AAV2 and AAV3. Interestingly, the influence of peptide modifications or shuffling of AAV capsids on antibody binding and AAV neutralization seemed to depend on the parental AAV. While the sex of the serum donors had no significant impact on binding or neutralizing antibody levels, we observed a trend to higher binding antibodies in older serum donors against some AAV types and a clear positive correlation of neutralizing antibody titers with the age of the serum donors. The disease status on the other hand did not have a meaningful impact on antibody levels, with no changes in AAV neutralization. Our data indicate that several wild-type or peptide-modified AAV may be good candidates for therapeutic application due to low pre-existing antibody levels, and that the age of potential recipients rather than their health status with regard to NMDs has the biggest impact on vector applicability.

Chemical approaches to probe and engineer AAV vectors.

Adeno-associated virus (AAV) has emerged as the most promising vector for in vivo human gene therapy, with several therapeutic approvals in the last few years and countless more under development. Underlying this remarkable success are several attractive features that AAV offers, including lack of pathogenicity, low immunogenicity, long-term gene expression without genomic integration, the ability to infect both dividing and non-dividing cells, etc. However, the commonly used wild-type AAV capsids in therapeutic development present significant challenges, including inadequate tissue specificity and the need for large doses to attain therapeutic effectiveness, raising safety concerns. Additionally, significant preexisting adaptive immunity against most natural capsids, and the development of such anti-capsid immunity after the first treatment, represent major challenges. Strategies to engineer the AAV capsid are critically needed to address these challenges and unlock the full promise of AAV gene therapy. Chemical modification of the AAV capsid has recently emerged as a powerful new approach to engineer its properties. Unlike genetic strategies, which can be more disruptive to the delicate capsid assembly and packaging processes, "late-stage" chemical modification of the assembled capsid-whether at natural amino acid residues or site-specifically installed noncanonical amino acid residues-often enables a versatile approach to introducing new properties to the capsid. This review summarizes the significant recent progress in AAV capsid engineering strategies, with a particular focus on chemical modifications in advancing the next generation of AAV-based gene therapies.

Immunobiology of a rationally-designed AAV2 capsid following intravitreal delivery in mice.

Adeno-associated virus serotype 2 (AAV2) is a viral vector that can be used to deliver therapeutic genes to diseased cells in the retina. One strategy for altering AAV2 vectors involves the mutation of phosphodegron residues, which are thought to be phosphorylated/ubiquitinated in the cytosol, facilitating degradation of the vector and the inhibition of transduction. As such, mutation of phosphodegron residues have been correlated with increased transduction of target cells, however, an assessment of the immunobiology of wild-type and phosphodegron mutant AAV2 vectors following intravitreal (IVT) delivery to immunocompetent animals is lacking in the current literature. In this study, we show that IVT of a triple phosphodegron mutant AAV2 capsid is associated with higher levels of humoral immune activation, infiltration of CD4 and CD8 T-cells into the retina, generation of splenic germinal centre reactions, activation of conventional dendritic cell subsets, and elevated retinal gliosis compared to wild-type AAV2 capsids. However, we did not detect significant changes in electroretinography arising after vector administration. We also demonstrate that the triple AAV2 mutant capsid is less susceptible to neutralisation by soluble heparan sulphate and anti-AAV2 neutralising antibodies, highlighting a possible utility for the vector in terms of circumventing pre-existing humoral immunity. In summary, the present study highlights novel aspects of rationally-designed vector immunobiology, which may be relevant to their application in preclinical and clinical settings.

Progress in Bioengineering of Myotropic Adeno-Associated Viral Gene Therapy Vectors

The ability to specifically, safely, and efficiently transfer therapeutic payloads to the striated musculature via a minimally invasive delivery route remains one of the most important but also most ambitious aims in human gene therapy. Over the past two decades, a flurry of groups have harnessed recombinant adeno-associated viruses (AAVs) for this purpose, carrying cargoes that were packaged either in one of the various wild-type capsids or in a synthetic protein shell derived by molecular bioengineering. In this study, we provide an overview over the most commonly used techniques for the enrichment of muscle-specific (myotropic) AAV capsids, typically starting off with the genetic diversification of one or more extant wild-type sequences, followed by the stratification of the ensuing capsid libraries in different muscle types in small or large animals. These techniques include the shuffling of multiple parental capsid genes, peptide display in exposed capsid loops, mutagenesis of individual capsid residues, creation of chimeras between two viral parents, or combinations thereof. Moreover, we highlight alternative experimental or bioinformatic strategies such as ancestral reconstruction or rational design, all of which have already been employed successfully to derive synthetic AAV capsids or vectors with unprecedented in vivo efficiency and/or specificity in the musculature. Most recently, these efforts have culminated in the isolation of unique clades of myotropic vectors called AAVMYO or MyoAAV that have in common the display of the amino acid motif RGD (arginine-glycine-aspartate) on the capsid surface and that exhibit the highest transduction rate in striated muscles of mice or nonhuman primates reported to date. Finally, we note essential looming improvements that will facilitate and accelerate clinical translation of these latest generations of myotropic AAVs, including the identification and utilization of capsid selection or validation schemes that promise optimal translation in humans, and continued efforts to enhance patient safety by minimizing hepatic off-targeting.

Equilibrium Dynamics of a Biomolecular Complex Analyzed at Single-amino Acid Resolution by Cryo-electron Microscopy

The biological function of macromolecular complexes depends not only on large-scale transitions between conformations, but also on small-scale conformational fluctuations at equilibrium. Information on the equilibrium dynamics of biomolecular complexes could, in principle, be obtained from local resolution (LR) data in cryo-electron microscopy (cryo-EM) maps. However, this possibility had not been validated by comparing, for a same biomolecular complex, LR data with quantitative information on equilibrium dynamics obtained by an established solution technique. In this study we determined the cryo-EM structure of the minute virus of mice (MVM) capsid as a model biomolecular complex. The LR values obtained correlated with crystallographic B factors and with hydrogen/deuterium exchange (HDX) rates obtained by mass spectrometry (HDX-MS), a gold standard for determining equilibrium dynamics in solution. This result validated a LR-based cryo-EM approach to investigate, with high spatial resolution, the equilibrium dynamics of biomolecular complexes. As an application of this approach, we determined the cryo-EM structure of two mutant MVM capsids and compared their equilibrium dynamics with that of the wild-type MVM capsid. The results supported a previously suggested linkage between mechanical stiffening and impaired equilibrium dynamics of a virus particle. Cryo-EM is emerging as a powerful approach for simultaneously acquiring information on the atomic structure and local equilibrium dynamics of biomolecular complexes.

The use of melittin to enhance transgene expression mediated by recombinant adeno-associated virus serotype 2 vectors both in vitro and in vivo

ObjectiveMelittin, a cell-penetrating peptide, improves the efficiency of many non-viral gene delivery vectors, yet its application in viral vectors has not been well studied. The non-pathogenic recombinant adeno-associated virus (rAAV) vector is an ideal in vivo gene delivery vector. However, its full potential will only be achieved after improvement of its transduction efficiency. To improve the transduction efficiency of rAAV2 vectors, we attempted to develop a melittin-based rAAV2 vector delivery strategy. MethodsThe melittin peptide was inserted into the rAAV2 capsid either in the loop VIII of all viral proteins (VPs) or at the N terminus of VP2. Various rAAV2-gfp or -fluc vectors were subjected to quantitative real-time polymerase chain reaction and Western blot assays to determine their titers and integrity of capsid proteins, respectively. Alternatively, the vectors based on wild-type capsid were pre-incubated with melittin, followed by transduction of cultured cells or tail vein administration of the mixture to C57BL/6 and BALB/c nude mice. In vivo bioluminescence imaging was performed to evaluate the transgene expression. ResultsrAAV2 vectors with melittin peptide inserted in the loop VIII of VPs had low transduction efficiency, probably due to dramatically reduced ability to bind to the target cells. Fusing the melittin peptide at the N-terminus of VP2 produced vectors without the VP2 subunit. Interestingly, among the commonly used rAAV vectors, pre-incubation of rAAV2 and rAAV6 vectors with melittin significantly enhanced their transduction efficiency in HEK293 and Huh7 cells in vitro. Melittin also had the ability to increase the rAAV2-mediated transgene expression in mouse liver in vivo. Mechanistically, melittin did not change the vector-receptor interaction. Moreover, cell counting kit-8 assays of cultured cells and serum transaminase levels indicated melittin had little cytotoxicity. ConclusionPre-incubation with melittin, but not insertion of melittin into the rAAV2 capsid, significantly enhanced rAAV2-mediated transgene expression. Although further in vivo evaluations are required, this research not only expands the pharmacological potential of melittin, but also provides a new strategy to improve gene therapy mediated by rAAV vectors.

Selection of rAAV vectors that cross the human blood-brain barrier and target the central nervous system using a transwell model

A limitation for recombinant adeno-associated virus (rAAV)-mediated gene transfer into the central nervous system (CNS) is the low penetration of vectors across the human blood-brain barrier (BBB). High doses of intravenously delivered vector are required to reach the CNS, which has resulted in varying adverse effects. Moreover, selective transduction of various cell types might be important depending on the disorder being treated. To enhance BBB penetration and improve CNS cell selectivity, we screened an AAV capsid-shuffled library using an in vitro transwell BBB system with separate layers of human endothelial cells, primary astrocytes and/or human induced pluripotent stem cell-derived cortical neurons. After multiple passages through the transwell, we identified chimeric AAV capsids with enhanced penetration and improved transduction of astrocytes and/or neurons compared with wild-type capsids. We identified the amino acids (aa) from regions 451–470 of AAV2 associated with the capsids selected for neurons, and a combination of aa from regions 413–496 of AAV-rh10 and 538–598 of AAV3B/LK03 associated with capsids selected for astrocytes. A small interfering RNA screen identified several genes that affect transcytosis of AAV across the BBB. Our work supports the use of a human transwell system for selecting enhanced AAV capsids targeting the CNS and may allow for unraveling the underlying molecular mechanisms of BBB penetration.

The significance of triple-capsid-mutant AAV8 for treatment of Sanfilippo Syndrome Type B.

Sanfilippo Syndrome Type-B remains an untreatable childhood neurodegenerative disease with great burden for both patient and caregiver. Very few clinical trials have been undertaken to treat the disease, and none of these have yet yielded clinically obtainable products for patients. Caused by a simple enzyme function deficiency, Sanfilippo Syndrome Type-B has been considered a great prospect for gene-therapy interventions. Adeno-associated virus (AAV) remains a major choice for therapeutic gene delivery due to its relatively low-immunogenicity, versatility and tissue tropism. However, many clinical trials with AAV continue to use wild-type capsids, which in many cases are not able to reach stable transgene expression for long enough to be clinically effective in most cases. Previous research in AAV gene-therapy has created a litany of novel AAV capsids that can improve overall transduction efficiency far above that of wild-type AAV capsids. One such example is the triple-capsid mutant AAV8 (TCM8), which has been shown to exhibit transgene expression far superior to other capsids in Sanfilippo mouse models, specifically. Originally designed to bypass capsid ubiquitination intracellularly, mouse studies suggest this TCM8 vector outperforms both AAV5 and AAV9 when delivered to the central nervous system. This implies it as an ideal contender for an effective gene-therapy clinical trial candidate and has the potential to advance the progress of Sanfilippo Syndrome treatment. Here we provide commentary on the TCM8 vector and its context in the field of Sanfilippo Syndrome Type-B research.

TRIM5α Restriction of HIV-1-N74D Viruses in Lymphocytes Is Caused by a Loss of Cyclophilin A Protection.

The core of HIV-1 viruses bearing the capsid change N74D (HIV-1-N74D) do not bind the human protein CPSF6. In primary human CD4+ T cells, HIV-1-N74D viruses exhibit an infectivity defect when compared to wild-type. We first investigated whether loss of CPSF6 binding accounts for the loss of infectivity. Depletion of CPSF6 in human CD4+ T cells did not affect the early stages of wild-type HIV-1 replication, suggesting that defective infectivity in the case of HIV-1-N74D viruses is not due to the loss of CPSF6 binding. Based on our previous result that cyclophilin A (Cyp A) protected HIV-1 from human tripartite motif-containing protein 5α (TRIM5αhu) restriction in CD4+ T cells, we found that depletion of TRIM5αhu in CD4+ T cells rescued the infectivity of HIV-1-N74D, suggesting that HIV-1-N74D cores interacted with TRIM5αhu. Accordingly, TRIM5αhu binding to HIV-1-N74D cores was increased compared with that of wild-type cores, and consistently, HIV-1-N74D cores lost their ability to bind Cyp A. In agreement with the notion that N74D capsids are defective in their ability to bind Cyp A, we found that HIV-1-N74D viruses were 20-fold less sensitive to TRIMCyp restriction when compared to wild-type viruses in OMK cells. Structural analysis revealed that N74D hexameric capsid protein in complex with PF74 is different from wild-type hexameric capsid protein in complex with PF74, which explains the defect of N74D capsids to interact with Cyp A. In conclusion, we showed that the decreased infectivity of HIV-1-N74D in CD4+ T cells is due to a loss of Cyp A protection from TRIM5αhu restriction activity.

HIV-1 requires capsid remodelling at the nuclear pore for nuclear entry and integration.

The capsid (CA) lattice of the HIV-1 core plays a key role during infection. From the moment the core is released into the cytoplasm, it interacts with a range of cellular factors that, ultimately, direct the pre-integration complex to the integration site. For integration to occur, the CA lattice must disassemble. Early uncoating or a failure to do so has detrimental effects on virus infectivity, indicating that an optimal stability of the viral core is crucial for infection. Here, we introduced cysteine residues into HIV-1 CA in order to induce disulphide bond formation and engineer hyper-stable mutants that are slower or unable to uncoat, and then followed their replication. From a panel of mutants, we identified three with increased capsid stability in cells and found that, whilst the M68C/E212C mutant had a 5-fold reduction in reverse transcription, two mutants, A14C/E45C and E180C, were able to reverse transcribe to approximately WT levels in cycling cells. Moreover, these mutants only had a 5-fold reduction in 2-LTR circle production, suggesting that not only could reverse transcription complete in hyper-stable cores, but that the nascent viral cDNA could enter the nuclear compartment. Furthermore, we observed A14C/E45C mutant capsid in nuclear and chromatin-associated fractions implying that the hyper-stable cores themselves entered the nucleus. Immunofluorescence studies revealed that although the A14C/E45C mutant capsid reached the nuclear pore with the same kinetics as wild type capsid, it was then retained at the pore in association with Nup153. Crucially, infection with the hyper-stable mutants did not promote CPSF6 re-localisation to nuclear speckles, despite the mutant capsids being competent for CPSF6 binding. These observations suggest that hyper-stable cores are not able to uncoat, or remodel, enough to pass through or dissociate from the nuclear pore and integrate successfully. This, is turn, highlights the importance of capsid lattice flexibility for nuclear entry. In conclusion, we hypothesise that during a productive infection, a capsid remodelling step takes place at the nuclear pore that releases the core complex from Nup153, and relays it to CPSF6, which then localises it to chromatin ready for integration.

Molecular Dynamics Free Energy Simulations Reveal the Mechanism for the Antiviral Resistance of the M66I HIV-1 Capsid Mutation.

While drug resistance mutations can often be attributed to the loss of direct or solvent-mediated protein−ligand interactions in the drug-mutant complex, in this study we show that a resistance mutation for the picomolar HIV-1 capsid (CA)-targeting antiviral (GS-6207) is mainly due to the free energy cost of the drug-induced protein side chain reorganization in the mutant protein. Among several mutations, M66I causes the most suppression of the GS-6207 antiviral activity (up to ~84,000-fold), and only 83- and 68-fold reductions for PF74 and ZW-1261, respectively. To understand the molecular basis of this drug resistance, we conducted molecular dynamics free energy simulations to study the structures, energetics, and conformational free energy landscapes involved in the inhibitors binding at the interface of two CA monomers. To minimize the protein−ligand steric clash, the I66 side chain in the M66I−GS-6207 complex switches to a higher free energy conformation from the one adopted in the apo M66I. In contrast, the binding of GS-6207 to the wild-type CA does not lead to any significant M66 conformational change. Based on an analysis that decomposes the absolute binding free energy into contributions from two receptor conformational states, it appears that it is the free energy cost of side chain reorganization rather than the reduced protein−ligand interaction that is largely responsible for the drug resistance against GS-6207.

The Portal Vertex of KSHV Promotes Docking of Capsids at the Nuclear Pores.

Kaposi’s sarcoma-associated herpesvirus (KSHV) is a cancer-related herpesvirus. Like other herpesviruses, the KSHV icosahedral capsid includes a portal vertex, composed of 12 protein subunits encoded by open reading frame (ORF) 43, which enables packaging and release of the viral genome into the nucleus through the nuclear pore complex (NPC). Capsid vertex-specific component (CVSC) tegument proteins, which directly mediate docking at the NPCs, are organized on the capsid vertices and are enriched on the portal vertex. Whether and how the portal vertex is selected for docking at the NPC is unknown. Here, we investigated the docking of incoming ORF43-null KSHV capsids at the NPCs, and describe a significantly lower fraction of capsids attached to the nuclear envelope compared to wild-type (WT) capsids. Like WT capsids, nuclear envelope-associated ORF43-null capsids co-localized with different nucleoporins (Nups) and did not detach upon salt treatment. Inhibition of nuclear export did not alter WT capsid docking. As ORF43-null capsids exhibit lower extent of association with the NPCs, we conclude that although not essential, the portal has a role in mediating the interaction of the CVSC proteins with Nups, and suggest a model whereby WT capsids can dock at the nuclear envelope through a non-portal penton vertex, resulting in an infection ‘dead end’.

Nuclear pore blockade reveals that HIV-1 completes reverse transcription and uncoating in the nucleus.

Retroviral infection involves the reverse transcription of the viral RNA genome into DNA, which is subsequently integrated into the host cell genome. HIV-1 and other lentiviruses are able to mediate the infection of non-dividing cells through the ability of the capsid (CA) protein 1 to engage the cellular nuclear import pathways of the target cell and mediate their nuclear translocation through components of the nuclear pore complex (NPC) 2–4. Although recent studies have observed the presence of capsid in the nucleus during infection5–8, reverse transcription and disassembly of the viral core have conventionally been considered to be cytoplasmic events. Here, we use an inducible nuclear pore blockade to monitor the kinetics of HIV-1 nuclear import and define the biochemical staging of these steps of infection. Surprisingly, we observe that nuclear import occurs with relatively rapid kinetics (<5 hours) and precedes the completion of reverse transcription in target cells, demonstrating that reverse transcription completes in the nucleus. We also observe that HIV-1 remains susceptible to a capsid destabilizing compound PF74 following nuclear import, revealing that uncoating completes in the nucleus. We also observe that certain CA mutants are insensitive to a Nup62 mediated nuclear pore blockade in cells which potently block infection by wild type CA, demonstrating that HIV-1 can utilize distinct nuclear import pathways during infection. These studies collectively define the spatiotemporal staging of critical steps of HIV-1 infection and provide an experimental system to separate and thereby define the cytoplasmic and nuclear stages of infection by other viruses.

Utilizing minimally purified secreted rAAV for rapid and cost-effective manipulation of gene expression in the CNS

BackgroundRecombinant adeno-associated virus (rAAV) is widely used in the neuroscience field to manipulate gene expression in the nervous system. However, a limitation to the use of rAAV vectors is the time and expense needed to produce them. To overcome this limitation, we evaluated whether unpurified rAAV vectors secreted into the media following scalable PEI transfection of HEK293T cells can be used in lieu of purified rAAV.MethodsWe packaged rAAV2-EGFP vectors in 30 different wild-type and mutant capsids and subsequently collected the media containing secreted rAAV. Genomic titers of each rAAV vector were assessed and the ability of each unpurified virus to transduce primary mixed neuroglial cultures (PNGCs), organotypic brain slice cultures (BSCs) and the mouse brain was evaluated.ResultsThere was ~ 40-fold wide variance in the average genomic titers of the rAAV2-EGFP vector packaged in the 30 different capsids, ranging from a low ~ 4.7 × 1010 vector genomes (vg)/mL for rAAV2/5-EGFP to a high of ~ 2.0 × 1012 vg/mL for a capsid mutant of rAAV2/8-EGFP. In PNGC studies, we observed a wide range of transduction efficiency among the 30 capsids evaluated, with the rAAV2/6-EGFP vector demonstrating the highest overall transduction efficiency. In BSC studies, we observed robust transduction by wild-type capsid vectors rAAV2/6, 2/8 and 2/9, and by capsid mutants of rAAV2/1, 2/6, and 2/8. In the in vivo somatic brain transgenesis (SBT) studies, we found that intra-cerebroventricular injection of media containing unpurified rAAV2-EGFP vectors packaged with select mutant capsids resulted in abundant EGFP positive neurons and astrocytes in the hippocampus and forebrain of non-transgenic mice. We demonstrate that unpurified rAAV can express transgenes at equivalent levels to lysate-purified rAAV both in vitro and in vivo. We also show that unpurified rAAV is sufficient to drive tau pathology in BSC and neuroinflammation in vivo, recapitulating previous studies using purified rAAV.ConclusionsUnpurified rAAV vectors secreted into the media can efficiently transduce brain cells in vitro and in vivo, providing a cost-effective way to manipulate gene expression. The use of unpurified virus will greatly reduce costs of exploratory studies and further increase the utility of rAAV vectors for standard laboratory use.

Amino-acid inserts of HIV-1 capsid (CA) induce CA degradation and abrogate viral infectivity: Insights for the dynamics and mechanisms of HIV-1 CA decomposition

Accumulation of amino acid (AA) insertions/substitutions are observed in the Gag-protein of HIV-1 variants resistant to HIV-1 protease inhibitors. Here, we found that HIV-1 carrying AA insertions in capsid protein (CA) undergoes aberrant CA degradation. When we generated recombinant HIV-1s (rHIV-1s) containing 19-AAs in Gag, such insertions caused significant CA degradation, which initiated in CA’s C-terminal. Such rHIV-1s had remarkable morphological abnormality, decreased infectivity, and no replicative ability, which correlated with levels of CA degradation. The CA degradation observed was energy-independent and had no association with cellular/viral proteolytic mechanisms, suggesting that the CA degradation occurs due to conformational/structural incompatibility caused by the 19-AA insertions. The incorporation of degradation-prone CA into the wild-type CA resulted in significant disruption of replication competence in “chimeric” virions. The data should allow better understanding of the dynamics and mechanisms of CA decomposition/degradation and retroviral uncoating, which may lead to new approach for antiretroviral modalities.

Nuclear envelope impairment is facilitated by the herpes simplex virus 1 Us3 kinase

Background: Capsids of herpes simplex virus 1 (HSV-1) are assembled in the nucleus, translocated either to the perinuclear space by budding at the inner nuclear membrane acquiring tegument and envelope, or released to the cytosol in a “naked” state via impaired nuclear pores that finally results in impairment of the nuclear envelope. The Us3 gene encodes a protein acting as a kinase, which is responsible for phosphorylation of numerous viral and cellular substrates. The Us3 kinase plays a crucial role in nucleus to cytoplasm capsid translocation. We thus investigate the nuclear surface in order to evaluate the significance of Us3 in maintenance of the nuclear envelope during HSV-1 infection. Methods: To address alterations of the nuclear envelope and capsid nucleus to cytoplasm translocation related to the function of the Us3 kinase we investigated cells infected with wild type HSV-1 or the Us3 deletion mutant R7041(∆Us3) by transmission electron microscopy, focused ion-beam electron scanning microscopy, cryo-field emission scanning electron microscopy, confocal super resolution light microscopy, and polyacrylamide gel electrophoresis. Results: Confocal super resolution microscopy and cryo-field emission scanning electron microscopy revealed decrement in pore numbers in infected cells. Number and degree of pore impairment was significantly reduced after infection with R7041(∆Us3) compared to infection with wild type HSV-1. The nuclear surface was significantly enlarged in cells infected with any of the viruses. Morphometric analysis revealed that additional nuclear membranes were produced forming multiple folds and caveolae, in which virions accumulated as documented by three-dimensional reconstruction after ion-beam scanning electron microscopy. Finally, significantly more R7041(∆Us3) capsids were retained in the nucleus than wild-type capsids whereas the number of R7041(∆Us3) capsids in the cytosol was significantly lower. Conclusions: The data indicate that Us3 kinase is involved in facilitation of nuclear pore impairment and, concomitantly, in capsid release through impaired nuclear envelope.

Nuclear envelope impairment is facilitated by the herpes simplex virus 1 Us3 kinase.

Background: Capsids of herpes simplex virus 1 (HSV-1) are assembled in the nucleus, translocated either to the perinuclear space by budding at the inner nuclear membrane acquiring tegument and envelope, or released to the cytosol in a "naked" state via impaired nuclear pores that finally results in impairment of the nuclear envelope. The Us3 gene encodes a protein acting as a kinase, which is responsible for phosphorylation of numerous viral and cellular substrates. The Us3 kinase plays a crucial role in nucleus to cytoplasm capsid translocation. We thus investigate the nuclear surface in order to evaluate the significance of Us3 in maintenance of the nuclear envelope during HSV-1 infection. Methods: To address alterations of the nuclear envelope and capsid nucleus to cytoplasm translocation related to the function of the Us3 kinase we investigated cells infected with wild type HSV-1 or the Us3 deletion mutant R7041(∆Us3) by transmission electron microscopy, focused ion-beam electron scanning microscopy, cryo-field emission scanning electron microscopy, confocal super resolution light microscopy, and polyacrylamide gel electrophoresis. Results: Confocal super resolution microscopy and cryo-field emission scanning electron microscopy revealed decrement in pore numbers in infected cells. Number and degree of pore impairment was significantly reduced after infection with R7041(∆Us3) compared to infection with wild type HSV-1. The nuclear surface was significantly enlarged in cells infected with any of the viruses. Morphometric analysis revealed that additional nuclear membranes were produced forming multiple folds and caveolae, in which virions accumulated as documented by three-dimensional reconstruction after ion-beam scanning electron microscopy. Finally, significantly more R7041(∆Us3) capsids were retained in the nucleus than wild-type capsids whereas the number of R7041(∆Us3) capsids in the cytosol was significantly lower. Conclusions: The data indicate that Us3 kinase is involved in facilitation of nuclear pore impairment and, concomitantly, in capsid release through impaired nuclear envelope.