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Real-time Genome Imaging of Host Interactions in Adeno-Associated Virus Genome Release
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Parvoviruses are small non‐enveloped single‐stranded DNA viruses, which depend on host cell nuclear transcriptional and replication machinery. After endosomal exposure of nuclear localization sequence and a phospholipase A2 domain on the capsid surface, and escape into the cytosol, parvovirus capsids enter the nucleus. Due to the small capsid diameter of 18–26 nm, intact capsids can potentially pass into the nucleus through nuclear pore complexes (NPCs). This might be facilitated by active nuclear import, but capsids may also follow an alternative entry pathway that includes activation of mitotic factors and local transient disruption of the nuclear envelope. The nuclear entry is followed by currently undefined events of viral genome uncoating. After genome release, viral replication compartments are initiated and infection proceeds. Parvoviral genomes replicate during cellular S phase followed by nuclear capsid assembly during virus‐induced S/G2 cell cycle arrest. Nuclear egress of capsids occurs upon nuclear envelope degradation during apoptosis and cell lysis. An alternative pathway for nuclear export has been described using active transport through the NPC mediated by the chromosome region maintenance 1 protein, CRM1, which is enhanced by phosphorylation of the N‐terminal domain of VP2. However, other alternative but not yet uncharacterized nuclear export pathways cannot be excluded. Parvovirus capsids enter the nucleus by active import or by an alternative entry pathway that includes local transient disruption of the nuclear envelope. Parvoviral genomes replicate during cellular S phase followed by nuclear capsid assembly. The egress of progeny capsids occurs upon nuclear envelope degradation during cell lysis or by using active transport through the nuclear pore complexes.
Nucleoli are membrane-less structures located within the nucleus and are known to be involved in many cellular functions, including stress response and cell cycle regulation. Besides, many viruses can employ the nucleolus or nucleolar proteins to promote different steps of their life cycle such as replication, transcription and assembly. While adeno-associated virus type 2 (AAV2) capsids have previously been reported to enter the host cell nucleus and accumulate in the nucleolus, both the role of the nucleolus in AAV2 infection, and the viral uncoating mechanism remain elusive. In all prior studies on AAV uncoating, viral capsids and viral genomes were not directly correlated on the single cell level, at least not in absence of a helper virus. To elucidate the properties of the nucleolus during AAV2 infection and to assess viral uncoating on a single cell level, we combined immunofluorescence analysis for detection of intact AAV2 capsids and capsid proteins with fluorescence in situ hybridization for detection of AAV2 genomes. The results of our experiments provide evidence that uncoating of AAV2 particles occurs in a stepwise process that is completed in the nucleolus and supported by alteration of the nucleolar structure.
Adenoviruses (AdV) are wide‐spread in vertebrates. They infect the respiratory and gastrointestinal tracts, the eyes, heart, liver and kidney, and are lethal to immunosuppressed people. Mastadenoviruses infecting mammals comprise several hundred different types, and many specifically infect humans. Human adenoviruses are the most widely used vectors in clinical applications, including cancer treatment and COVID‐19 vaccination. AdV vectors are physically and genetically stable, and generally safe in humans. The particles have an icosahedral coat and a nucleo‐protein core with a DNA genome. We describe a coherent concept of AdV entry, and highlight recent advances in cytoplasmic transport, uncoating and nuclear import of the viral DNA, including microtubule motors. We highlight a ‘linchpin’ function of the virion protein V ensuring cytoplasmic particle stability, which is relaxed at the nuclear pore complex by cues from the E3‐ubiquitin ligase Mind bomb 1 (MIB1) and the proteasome triggering disruption. Capsid disruption by kinesin motor proteins and microtubules then exposes the linchpin and renders protein V a target for MIB1 ubiquitination, which dissociates V from viral DNA and enhances DNA nuclear import. These advances uncover surprising mechanisms controlling capsid stability and premature uncoating, and provide insight into nuclear transport of nucleic acids. Viruses invade organisms and cells, cause disease, persist for a lifetime or are eliminated by host defense. Viral vectors are beneficial for clinical therapy or preventive vaccination. Adenoviruses are wide‐spread human pathogens and the most widely used vectors in gene therapy. Here we provide an updated coherent concept for nuclear entry of human adenoviruses, a key process in infection and immunity.
Adeno-associated viruses (AAVs) are the safest and most effective gene delivery vehicles to drive long-term transgene expression in gene therapy. While animal studies have shown promising results, the translatability of AAVs into clinical settings has been partly limited due to their restricted gene packaging capacities, off-target transduction, and immunogenicity. In this study, we analysed over two decades of AAV applications, in 136 clinical trials. This meta-analysis aims to provide an up-to-date overview of the use and successes of AAVs in clinical trials, while evaluating the approaches used to address the above challenges. First, this study reveals that the speed of novel AAV development has varied between therapeutic areas, with particular room for improvement in Central Nervous System disorders, where development has been slow. Second, the lack of dose-dependent toxicity and efficacy data indicates that optimal dosing regimes remain elusive. Third, more clinical data on the effectiveness of various immune-modulation strategies and gene editing approaches are required to direct future research and to accelerate the translation of AAV-mediated gene therapy into human applications.
The capsid structures of most Adeno-associated virus (AAV) serotypes, already assigned to an antigenic clade, have been previously determined. This study reports the remaining capsid structures of AAV7, AAV11, AAV12, and AAV13 determined by cryo-electron microscopy and three-dimensional image reconstruction to 2.96, 2.86, 2.54, and 2.76 Å resolution, respectively. These structures complete the structural atlas of the AAV serotype capsids. AAV7 represents the first clade D capsid structure; AAV11 and AAV12 are of a currently unassigned clade that would include AAV4; and AAV13 represents the first AAV2-AAV3 hybrid clade C capsid structure. These newly determined capsid structures all exhibit the AAV capsid features including 5-fold channels, 3-fold protrusions, 2-fold depressions, and a nucleotide binding pocket with an ordered nucleotide in genome-containing capsids. However, these structures have viral proteins that display clade-specific loop conformations. This structural characterization completes our three-dimensional library of the current AAV serotypes to provide an atlas of surface loop configurations compatible with capsid assembly and amenable for future vector engineering efforts. Derived vectors could improve gene delivery success with respect to specific tissue targeting, transduction efficiency, antigenicity or receptor retargeting.
RAD52 is a member of the homologous recombination pathway that is important for survival of BRCA-deficient cells. Inhibition of RAD52 leads to lethality in BRCA-deficient cells. However, the exact mechanism of how RAD52 contributes to viability of BRCA-deficient cells remains unknown. Two major activities of RAD52 were previously identified: DNA or RNA pairing, which includes DNA/RNA annealing and strand exchange, and mediator, which is to assist RAD51 loading on RPA-covered ssDNA. Here, we report that the N-terminal domain (NTD) of RAD52 devoid of the potential mediator function is essential for maintaining viability of BRCA-deficient cells owing to its ability to promote DNA/RNA pairing. We show that RAD52 NTD forms nuclear foci upon DNA damage in BRCA-deficient human cells and promotes DNA double-strand break repair through two pathways: homology-directed repair (HDR) and single-strand annealing (SSA). Furthermore, we show that mutations in the RAD52 NTD that disrupt these activities fail to maintain viability of BRCA-deficient cells.
The autonomous parvovirus Minute Virus of Mice (MVM) localizes to cellular DNA damage sites to establish and sustain viral replication centers, which can be visualized by focal deposition of the essential MVM non-structural phosphoprotein NS1. How such foci are established remains unknown. Here, we show that NS1 localized to cellular sites of DNA damage independently of its ability to covalently bind the 5’ end of the viral genome, or its consensus DNA binding sequence. Many of these sites were identical to those occupied by virus during infection. However, localization of the MVM genome to DNA damage sites occurred only when wild-type NS1, but not its DNA-binding mutant was expressed. Additionally, wild-type NS1, but not its DNA binding mutant, could localize a heterologous DNA molecule containing the NS1 binding sequence to DNA damage sites. These findings suggest that NS1 may function as a bridging molecule, helping the MVM genome localize to cellular DNA damage sites to facilitate ongoing virus replication.
Background:
Hepatitis B virus (HBV) integration has implications for cancer development and surface antigen (HBsAg) production, but methods to quantify integrations are lacking. The aim of this study was to develop a digital PCR (ddPCR) assay discriminating between circular and integrated HBV DNA, and to relate the distribution between the two forms to other HBV markers.
Methods:
ddPCR comprising primers spanning the typical linearization breakpoint in the HBV genome allowed for quantification of the absolute copy numbers of total and circular HBV DNA, and calculation of linear HBV DNA.
Results:
Analysis of 70 liver biopsies from patients with chronic HBV infection revealed that the fraction of linear HBV DNA, which includes integrations, was higher in HBeAg-negative patients than HBeAg-positive. The ratio between HBsAg and HBV DNA levels in serum correlated with the intrahepatic proportion of linear HBV DNA. Furthermore, ddPCR experiments on serum samples and experiments with nuclease indicated the contribution of encapsidated double-stranded linear DNA and replication intermediates to be limited.
Conclusions:
The degree of integration of intrahepatic HBV DNA in the HBeAg-negative stage may be higher than previously anticipated, and integrated DNA may explain the persistence of high HBsAg serum levels in patients with low HBV DNA levels.
The adeno-associated virus (AAV) is a small, nonpathogenic parvovirus, which depends on helper factors to replicate. Those helper factors can be provided by coinfecting helper viruses such as adenoviruses, herpesviruses, or papillomaviruses. We review the basic biology of AAV and its most-studied helper viruses, adenovirus type 5 (AdV5) and herpes simplex virus type 1 (HSV-1). We further outline the direct and indirect interactions of AAV with those and additional helper viruses.
During virus entry, members of the Polyomaviridae transit the endolysosomal network en route to the endoplasmic reticulum (ER), from which degraded capsids escape into the cytoplasm and enter the nucleus. Emerging evidence suggests that viruses require both endosomal acidification and the correct ionic balance of K⁺ and Ca²⁺ ions in endosomes for correct virus trafficking and genome release. Here, using two polyomaviruses with different capsid architectures, namely Simian virus 40 (SV40) and Merkel cell polyomavirus (MCPyV), we describe methods to rapidly quantify virus infection using IncuCyte ZOOM imaging analysis, and use this system to investigate the role of both K⁺ and Ca²⁺ channels during the early stages of virus entry. Using broad spectrum blockers of both K⁺ and Ca²⁺ channels to specifically target host cell ion channel functionality, we show that MCPyV, but not SV40 can be inhibited by K⁺ channel modulators, whilst both viruses are restricted by the broad spectrum Ca²⁺ channel inhibitor verapamil. Using a panel of more specific Ca²⁺ blockers, we show that both MCPyV and SV40 are dependent on the activity of two-pore Ca²⁺ channels (TPCs), as the TPC-specific blocker tetrandrine could prevent the capsid disassembly and nuclear transport required for virus entry. We therefore reveal a novel target to restrict the entry of polyomaviruses, which given the known role of TPCs during endolysosomal-ER fusion, is likely to be applicable to other viruses that transit this pathway.
In this Special Issue, we would like to focus on the various functions of the RAD52 helicase-like protein and the current implications of such findings for cancer treatment. Over the last few years, various laboratories have discovered particular activities of mammalian RAD52—both in S and M phase—that are distinct from the auxiliary role of yeast RAD52 in homologous recombination. At DNA double-strand breaks, RAD52 was demonstrated to spur alternative pathways to compensate for the loss of homologous recombination functions. At collapsed replication forks, RAD52 activates break-induced replication. In the M phase, RAD52 promotes the finalization of DNA replication. Its compensatory role in the resolution of DNA double-strand breaks has put RAD52 in the focus of synthetic lethal strategies, which is particularly relevant for cancer treatment.
Parvoviruses are an important platform for gene and cancer therapy. Their cell entry and the following steps including nuclear import are inefficient limiting their use in therapeutic applications. Two models exist on parvoviral nuclear entry: classical import of the viral capsid using nuclear transport receptors of the importin (karyopherin) family, or direct attachment of the capsid to the nuclear pore complex leading to local disintegration of the nuclear envelope. Here, by laser scanning confocal microscopy and in situ proximity ligation analysis combined with co-immunoprecipitation we showed that infection requires importin β-mediated access into the nuclear pore complex and nucleoporin 153-mediated interactions on the nuclear side. Importin β-capsid interaction continued within the nucleoplasm, which suggests that a mixed model of nuclear entry in which the classical nuclear import across the nuclear pore complex is accompanied by transient ruptures of the nuclear envelope allowing also passive entry of importin β-capsid complexes into the nucleus.
IMPORTANCE Parvoviruses are small DNA viruses that deliver their DNA into the post-mitotic nuclei, which is an important step for parvoviral gene and cancer therapies. Limitations in virus-receptor interaction or endocytic entry do not fully explain the low transduction/infection efficiency, indicating a bottleneck after virus entry into the cytoplasm. We thus investigated the transfer of parvovirus capsids from the cytoplasm to the nucleus showing that the nuclear import of parvovirus capsid follows a unique strategy, which differs from classical nuclear import and that of other viruses.
Staphylococcal pathogens adhere to their human targets with exceptional resilience to mechanical stress, some propagating force to the bacterium via small, Ig-like folds called B domains. We examine the mechanical stability of these folds using atomic force microscopy-based single-molecule force spectroscopy. The force required to unfold a single B domain is larger than 2 nN – the highest mechanostability of a protein to date by a large margin. B domains coordinate three calcium ions, which we identify as crucial for their extreme mechanical strength. When calcium is removed through chelation, unfolding forces drop by a factor of four. Through systematic mutations in the calcium coordination sites we can tune the unfolding forces from over 2 nN to 0.15 nN, and dissect the contribution of each ion to B domain mechanostability. Their extraordinary strength, rapid refolding and calcium-tunable force response make B domains interesting protein design targets.
Adenoviruses are DNA viruses with a lytic infection cycle. Following the fate of incoming as well as recently replicated genomes during infections is a challenge. In this study, we used the ANCHOR3 technology based on a bacterial partitioning system to establish a versatile in vivo imaging system for adenoviral genomes. The system allows the visualization of both individual incoming and newly replicated genomes in real time in living cells. We demonstrate that incoming adenoviral genomes are attached to condensed cellular chromatin during mitosis, facilitating the equal distribution of viral genomes in daughter cells after cell division. We show that the formation of replication centers occurs in conjunction with in vivo genome replication and determine replication rates. Visualization of adenoviral DNA revealed that adenoviruses exhibit two kinetically distinct phases of genome replication. Low-level replication occurred during early replication, while high-level replication was associated with late replication phases. The transition between these phases occurred concomitantly with morphological changes of viral replication compartments and with the appearance of virus-induced postreplication (ViPR) bodies, identified by the nucleolar protein Mybbp1A. Taken together, our real-time genome imaging system revealed hitherto uncharacterized features of adenoviral genomes in vivo. The system is able to identify novel spatiotemporal aspects of the adenovirus life cycle and is potentially transferable to other viral systems with a doublestranded DNA phase.
Human cytomegalovirus (HCMV) induces latent lifelong infections in all human populations. Between 30% and nearly 100% of individuals are affected depending on the geographic area and socioeconomic conditions. The biology of the virus is difficult to explore due to its extreme sophistication and the lack of a pertinent animal model. Here, we present the first application of the ANCHOR DNA labeling system to a herpesvirus, enabling real-time imaging and direct monitoring of HCMV infection and replication in living human cells. The ANCHOR system is composed of a protein (OR) that specifically binds to a short, nonrepetitive DNA target sequence (ANCH) and spreads onto neighboring sequences by protein oligomerization. When the OR protein is fused to green fluorescent protein (GFP), its accumulation results in a site-specific fluorescent focus. We created a recombinant ANCHOR-HCMV harboring an ANCH target sequence and the gene encoding the cognate OR-GFP fusion protein. Infection of permissive cells with ANCHOR-HCMV enables visualization of nearly the complete viral cycle until cell fragmentation and death. Quantitative analysis of infection kinetics and of viral DNA replication revealed cell-type-specific HCMV behavior and sensitivity to inhibitors. Our results show that the ANCHOR technology provides an efficient tool for the study of complex DNA viruses and a new, highly promising system for the development of innovative biotechnology applications.
Virus families have evolved different strategies for genome uncoating, which are also followed by recombinant vectors. Vectors derived from adeno-associated viruses (AAV) are considered as leading delivery tools for in vivo gene transfer, and in particular gene therapy. Using a combination of atomic force microscopy (AFM), biochemical experiments, and physical modeling, we investigated here the physical properties and stability of AAV vector particles. We first compared the morphological properties of AAV vectors derived from two different serotypes (AAV8 and AAV9). Furthermore, we triggered ssDNA uncoating by incubating vector particles to increasing controlled temperatures. Our analyses, performed at the single-particle level, indicate that genome release can occur in vitro via two alternative pathways: either the capsid remains intact and ejects linearly the ssDNA molecule, or the capsid is ruptured, leaving ssDNA in a compact entangled conformation. The analysis of the length distributions of ejected genomes further revealed a two-step ejection behavior. We propose a kinetic model aimed at quantitatively describing the evolution of capsids and genomes along the different pathways, as a function of time and temperature. This model allows quantifying the relative stability of AAV8 and AAV9 particles.
Parvoviral genome translocation from the plasma membrane into the nucleus is a coordinated multistep process mediated by capsid proteins. We used fast confocal microscopy line scan imaging combined with image correlation methods including auto-, pair- and cross-correlation, and number and brightness analysis, to study the parvovirus entry pathway at the single-particle level in living cells. Our results show that the endosome-associated movement of virus particles fluctuates from fast to slow. Fast transit of single cytoplasmic capsids to the nuclear envelope is followed by slow movement of capsids and fast diffusion of capsid fragments in the nucleoplasm. The unique combination of image analyses allowed us to follow the fate of intracellular single virus particles and their interactions with importin β revealing previously unknown dynamics of the entry pathway.
Protoparvoviruses target the nucleus due to their dependence on the cellular reproduction machinery during the replication and expression of their single-stranded DNA genome. In recent years, our understanding of the multistep process of the capsid nuclear import has improved, and led to the discovery of unique viral nuclear entry strategies. Preceded by endosomal transport, endosomal escape and microtubule-mediated movement to the vicinity of the nuclear envelope, the protoparvoviruses interact with the nuclear pore complexes. The capsids are transported actively across the nuclear pore complexes using nuclear import receptors. The nuclear import is sometimes accompanied by structural changes in the nuclear envelope, and is completed by intranuclear disassembly of capsids and chromatinization of the viral genome. This review discusses the nuclear import strategies of protoparvoviruses and describes its dynamics comprising active and passive movement, and directed and diffusive motion of capsids in the molecularly crowded environment of the cell.
Oncolytic virotherapy may be a means of improving the dismal prognosis of malignant brain tumors. The rat H-1 parvovirus (H-1PV) suppresses tumors in preclinical glioma models, through both direct oncolysis and stimulation of anticancer immune responses. This was the basis of ParvOryx01, the first phase I/IIa clinical trial of an oncolytic parvovirus in recurrent glioblastoma patients. H-1PV (escalating dose) was administered via intratumoral or intravenous injection. Tumors were resected 9 days after treatment, and virus was re-administered around the resection cavity. Primary endpoints were safety and tolerability, virus distribution, and maximum tolerated dose (MTD). Progression-free and overall survival and levels of viral and immunological markers in the tumor and peripheral blood were also investigated. H-1PV treatment was safe and well tolerated, and no MTD was reached. The virus could cross the blood-brain/tumor barrier and spread widely through the tumor. It showed favorable pharmacokinetics, induced antibody formation in a dose-dependent manner, and triggered specific T cell responses. Markers of virus replication, microglia/macrophage activation, and cytotoxic T cell infiltration were detected in infected tumors, suggesting that H-1PV may trigger an immunogenic stimulus. Median survival was extended in comparison with recent meta-analyses. Altogether, ParvOryx01 results provide an impetus for further H-1PV clinical development.
Graphical Abstract Highlights d RAD52 binds ssDNA rapidly and tightly using wrapping and bridging modes d RAD52 binding to dsDNA is slower, weaker, and often diffusive d RAD52 changes dsDNA mechanics and intercalates into the double helix d RAD52 prevents DNA melting by clamping DNA strands (M.M.) In Brief Brouwer et al. show that human RAD52 swiftly and tightly wraps ssDNA around itself. With dsDNA, interactions are weaker and diffusive but drastically change DNA mechanics, suggesting double helix intercalation. DNA-bound RAD52 efficiently captures ssDNA in trans. These features seem favorable for strand annealing, clamping, and second-end capture. SUMMARY Human RAD52 promotes annealing of complementary single-stranded DNA (ssDNA). In-depth knowledge of RAD52-DNA interaction is required to understand how its activity is integrated in DNA repair processes. Here, we visualize individual fluorescent RAD52 complexes interacting with single DNA molecules. The interaction with ssDNA is rapid, static, and tight, where ssDNA appears to wrap around RAD52 complexes that promote intra-molecular bridging. With double-stranded DNA (dsDNA), interaction is slower, weaker, and often diffusive. Interestingly, force spectroscopy experiments show that RAD52 alters the mechanics dsDNA by enhancing DNA flexibility and increasing DNA contour length, suggesting intercalation. RAD52 binding changes the nature of the overstretching transition of dsDNA and prevents DNA melting, which is advantageous for strand clamping during or after annealing. DNA-bound RAD52 is efficient at capturing ssDNA in trans. Together, these effects may help key steps in DNA repair, such as second-end capture during homo-logous recombination or strand annealing during RAD51-independent recombination reactions.
Hepatitis B virus (HBV) is an enveloped pararetrovirus with a DNA genome, which is found in an up to 36 nm-measuring capsid. Replication of the genome occurs via an RNA intermediate, which is synthesized in the nucleus. The virus must have thus ways of transporting its DNA genome into this compartment. This review summarizes the data on hepatitis B virus genome transport and correlates the finding to those from other viruses.
Hepatitis B virus (HBV) replicates its genomic information in the nucleus via transcription and therefore has to deliver its partially double stranded DNA genome into the nucleus. Like other viruses with a nuclear replication phase, HBV genomes are transported inside the viral capsids first through the cytoplasm towards the nuclear envelope. Following the arrival at the nuclear pore, the capsids are transported through, using classical cellular nuclear import pathways. The arrest of nuclear import at the nucleoplasmic side of the nuclear pore is unique, however, and is where the capsids efficiently disassemble leading to genome release. In the latter phase of the infection, newly formed nucleocapsids in the cytosol have to move to budding sites at intracellular membranes carrying the three viral envelope proteins. Capsids containing single stranded nucleic acid are not enveloped, in contrast to empty and double stranded DNA containing capsids. A small linear domain in the large envelope protein and two areas on the capsid surface have been mapped, where point mutations strongly block nucleocapsid envelopment. It is possible that these domains are involved in the envelope - with capsid interactions driving the budding process. Like other enveloped viruses, HBV also uses the cellular endosomal sorting complexes required for transport (ESCRT) machinery for catalyzing budding through the membrane and away from the cytosol.
RAD52 is a member of the homologous recombination (HR) pathway that is important for maintenance of genome integrity. While
single RAD52 mutations show no significant phenotype in mammals, their combination with mutations in genes that cause hereditary
breast cancer and ovarian cancer like BRCA1, BRCA2, PALB2 and RAD51C are lethal. Consequently, RAD52 may represent an important
target for cancer therapy. In vitro, RAD52 has ssDNA annealing and DNA strand exchange activities. Here, to identify small molecule inhibitors of RAD52 we screened
a 372,903-compound library using a fluorescence-quenching assay for ssDNA annealing activity of RAD52. The obtained 70 putative
inhibitors were further characterized using biochemical and cell-based assays. As a result, we identified compounds that specifically
inhibit the biochemical activities of RAD52, suppress growth of BRCA1- and BRCA2-deficient cells and inhibit RAD52-dependent
single-strand annealing (SSA) in human cells. We will use these compounds for development of novel cancer therapy and as a
probe to study mechanisms of DNA repair.
DNA viruses undertake their replication within the cell nucleus, and therefore they must first deliver their genome into the nucleus of their host cells. Thus, trafficking across the nuclear envelope is at the basis of DNA virus infections. Nuclear transport of molecules with diameters up to 39 nm is a tightly regulated process that occurs through the nuclear pore complex (NPC). Due to the enormous diversity of virus size and structure, each virus has developed its own strategy for entering the nucleus of their host cells, with no two strategies alike. For example, baculoviruses target their DNA-containing capsid to the NPC and subsequently enter the nucleus intact, while the hepatitis B virus capsid crosses the NPC but disassembles at the nuclear side of the NPC. For other viruses such as herpes simplex virus and adenovirus, although both dock at the NPC, they have each developed a distinct mechanism for the subsequent delivery of their genome into the nucleus. Remarkably, other DNA viruses, such as parvoviruses and human papillomaviruses, access the nucleus through an NPC-independent mechanism. This review discusses our current understanding of the mechanisms used by DNA viruses to deliver their genome into the nucleus, and further presents the experimental evidence for such mechanisms.
The ATR/Chk1 pathway is a critical surveillance network that maintains genomic integrity during DNA replication by stabilizing the replication forks during normal replication to avoid replication stress. One of the many differences between normal cells and cancer cells is the amount of replication stress that occurs during replication. Cancer cells with activated oncogenes generate increased levels of replication stress. This creates an increased dependency on the ATR/Chk1 pathway in cancer cells and opens up an opportunity to preferentially kill cancer cells by inhibiting this pathway. In support of this idea, we have identified a small molecule termed HAMNO ((1Z)-1-[(2-hydroxyanilino)methylidene]naphthalen-2-one), a novel protein interaction inhibitor of replication protein A (RPA), a protein involved in the ATR/Chk1 pathway. HAMNO selectively binds the N-terminal domain of RPA70, effectively inhibiting critical RPA protein interactions which rely on this domain. HAMNO inhibits both ATR autophosphorylation and phosphorylation of RPA32 Ser33 by ATR. By itself, HAMNO treatment creates DNA replication stress in cancer cells that are already experiencing replication stress, but not in normal cells, and it acts synergistically with etoposide to kill cancer cells in vitro and slow tumor growth in vivo. Thus, HAMNO illustrates how RPA inhibitors represent candidate therapeutics for cancer treatment, providing disease selectivity in cancer cells by targeting their differential response to replication stress.
Unlabelled:
Herpes simplex virus (HSV) entry requires multiple interactions at the cell surface and activation of a complex calcium signaling cascade. Previous studies demonstrated that integrins participate in this process, but their precise role has not been determined. These studies were designed to test the hypothesis that integrin αvβ3 signaling promotes the release of intracellular calcium (Ca2+) stores and contributes to viral entry and cell-to-cell spread. Transfection of cells with small interfering RNA (siRNA) targeting integrin αvβ3, but not other integrin subunits, or treatment with cilengitide, an Arg-Gly-Asp (RGD) mimetic, impaired HSV-induced Ca2+ release, viral entry, plaque formation, and cell-to-cell spread of HSV-1 and HSV-2 in human cervical and primary genital tract epithelial cells. Coimmunoprecipitation studies and proximity ligation assays indicated that integrin αvβ3 interacts with glycoprotein H (gH). An HSV-2 gH-null virus was engineered to further assess the role of gH in the virus-induced signaling cascade. The gH-2-null virus bound to cells and activated Akt to induce a small Ca2+ response at the plasma membrane, but it failed to trigger the release of cytoplasmic Ca2+ stores and was impaired for entry and cell-to-cell spread. Silencing of integrin αvβ3 and deletion of gH prevented phosphorylation of focal adhesion kinase (FAK) and the transport of viral capsids to the nuclear pore. Together, these findings demonstrate that integrin signaling is activated downstream of virus-induced Akt signaling and facilitates viral entry through interactions with gH by activating the release of intracellular Ca2+ and FAK phosphorylation. These findings suggest a new target for HSV treatment and suppression.
Importance:
Herpes simplex viruses are the leading cause of genital disease worldwide, the most common infection associated with neonatal encephalitis, and a major cofactor for HIV acquisition and transmission. There is no effective vaccine. These epidemiological findings underscore the urgency to develop novel HSV treatment or prevention strategies. This study addresses this gap by further defining the signaling pathways the virus usurps to enter human genital tract epithelial cells. Specifically, the study defines the role played by integrins and by the viral envelope glycoprotein H in entry and cell-to-cell spread. This knowledge will facilitate the identification of new targets for the development of treatment and prevention.
Unlabelled:
Recombinant adeno-associated viral (rAAV) vectors have garnered much promise in gene therapy applications. However, widespread clinical use has been limited by transduction efficiency. Previous studies suggested that the majority of rAAV accumulates in the perinuclear region of cells, presumably unable to traffic into the nucleus. rAAV nuclear translocation remains ill-defined; therefore, we performed microscopy, genetic, and biochemical analyses in vitro in order to understand this mechanism. Lectin blockade of the nuclear pore complex (NPC) resulted in inhibition of nuclear rAAV2. Visualization of fluorescently labeled particles revealed that rAAV2 localized to importin-β-dense regions of cells in late trafficking steps. Additionally, small interfering RNA (siRNA) knockdown of importin-β partially inhibited rAAV2 nuclear translocation and inhibited transduction by 50 to 70%. Furthermore, coimmunopreciptation (co-IP) analysis revealed that capsid proteins from rAAV2 could interact with importin-β and that this interaction was sensitive to the small GTPase Ran. More importantly, mutations to key basic regions in the rAAV2 capsid severely inhibited interactions with importin-β. We tested several other serotypes and found that the extent of importin-β interaction varied, suggesting that different serotypes may utilize alternative import proteins for nuclear translocation. Co-IP and siRNA analyses were used to investigate the role of other karyopherins, and the results suggested that rAAV2 may utilize multiple import proteins for nuclear entry. Taken together, our results suggest that rAAV2 interacts with importin-β alone or in complex with other karyopherins and enters the nucleus via the NPC. These results may lend insight into the design of novel AAV vectors that have an enhanced nuclear entry capability and transduction potential.
Importance:
Use of recombinant adeno-associated viral (rAAV) vectors for gene therapy applications is limited by relatively low transduction efficiency, in part due to cellular barriers that hinder successful subcellular trafficking to the nucleus, where uncoating and subsequent gene expression occur. Nuclear translocation of rAAV has been regarded as a limiting step for successful transduction but it remains ill-defined. We explored potential nuclear entry mechanisms for rAAV2 and found that rAAV2 can utilize the classical nuclear import pathway, involving the nuclear pore complex, the small GTPase Ran, and cellular karyopherins. These results could lend insight into the rational design of novel rAAV vectors that can more efficiently translocate to the nucleus, which may lead to more efficient transduction.
Disassembly of the nuclear lamina is essential in mitosis and apoptosis requiring multiple coordinated enzymatic activities in nucleus and cytoplasm. Activation and coordination of the different activities is poorly understood and moreover complicated as some factors translocate between cytoplasm and nucleus in preparatory phases. Here we used the ability of parvoviruses to induce nuclear membrane breakdown to understand the triggers of key mitotic enzymes. Nuclear envelope disintegration was shown upon infection, microinjection but also upon their application to permeabilized cells. The latter technique also showed that nuclear envelope disintegration was independent upon soluble cytoplasmic factors. Using time-lapse microscopy, we observed that nuclear disassembly exhibited mitosis-like kinetics and occurred suddenly, implying a catastrophic event irrespective of cell- or type of parvovirus used. Analyzing the order of the processes allowed us to propose a model starting with direct binding of parvoviruses to distinct proteins of the nuclear pore causing structural rearrangement of the parvoviruses. The resulting exposure of domains comprising amphipathic helices was required for nuclear envelope disintegration, which comprised disruption of inner and outer nuclear membrane as shown by electron microscopy. Consistent with Ca(++) efflux from the lumen between inner and outer nuclear membrane we found that Ca(++) was essential for nuclear disassembly by activating PKC. PKC activation then triggered activation of cdk-2, which became further activated by caspase-3. Collectively our study shows a unique interaction of a virus with the nuclear envelope, provides evidence that a nuclear pool of executing enzymes is sufficient for nuclear disassembly in quiescent cells, and demonstrates that nuclear disassembly can be uncoupled from initial phases of mitosis.
We describe biophysical and ultrastructural differences in genome release from adeno-associated viral (AAV) capsids packaging wild type (wt), recombinant single-stranded (ss) or dimeric, self-complementary (sc) DNA genomes. Atomic force microscopy and electron micrographs (EM) revealed that AAV particles release packaged genomes and undergo marked changes in capsid morphology upon heating in physiological buffer (pH 7.2). When different AAV capsids packaging ss/scDNA varying in length from 72-123% of wtDNA (3.4-5.8kb) were incrementally heated, the proportion of uncoated AAV capsids decreased with genome length as observed by EM. Genome release was further characterized by a fluorimetric assay, which demonstrated that acidic pH and high osmotic pressure suppress genome release from AAV particles. In addition, fluorimetric analysis corroborated an inverse correlation between packaged genome length and the temperature needed to induce uncoating. Surprisingly, scAAV vectors required significantly higher temperatures to uncoat than their ssDNA packaging counterparts. However, externalization of VP1 N-termini appears to be unaffected by packaged genome length or self-complementarity. Further analysis by tungsten-shadowing EM revealed striking differences in the morphology of ssDNA and scDNA genomes upon release from intact capsids. Computational modeling and molecular dynamics simulations suggest that the unusual thermal stability of scAAV vectors might arise from partial base pairing and optimal organization of packaged scDNA. Our work further defines the biophysical mechanisms underlying adeno-associated viral uncoating and genome release.
Viruses that replicate in the nucleus need to pass the nuclear envelope barrier during infection. Research in recent years indicates that the nuclear envelope is a major hurdle for many viruses. This review describes strategies to overcome this obstacle developed by seven virus families: herpesviridae, adenoviridae, orthomyxoviridae, lentiviruses (which are part of retroviridae), hepdnaviridae, parvoviridae and polyomaviridae. Most viruses use the canonical nuclear pore complex (NPC) in order to get their genome into the nucleus. Viral capsids that are larger than the nuclear pore disassemble before or during passing through the NPC, thus allowing genome nuclear entry. Surprisingly, increasing evidence suggest that parvoviruses and polyomaviruses may bypass the nuclear pore by trafficking directly through the nuclear membrane. Additional studies are required for better understanding these processes. Since nuclear entry emerges as the limiting step in infection for many viruses, it may serve as an ideal target for antiviral drug development.
Adeno-associated virus type 2 (AAV2) has gained much interest as a gene delivery vector. A hallmark of AAV2-mediated gene
transfer is an intracellular conformational change of the virus capsid, leading to the exposure of infection-relevant protein
domains. These protein domains, which are located on the N-terminal portion of the structural proteins VP1 and VP2, include
a catalytic phospholipase A2 domain and three clusters of basic amino acids. We have identified additional protein sequence motifs located on the VP1/2
N terminus that also proved to be obligatory for virus infectivity. These motifs include signals that are known to be involved
in protein interaction, endosomal sorting and signal transduction in eukaryotic cells. Among different AAV serotypes they
are highly conserved and mutation of critical amino acids of the respective motifs led to a severe infection-deficient phenotype.
In particular, mutation of a YXXQ-sequence motif significantly reduced accumulation of virus capsids around the nucleus in
comparison to wild-type AAV2. Interestingly, intracellular trafficking of AAV2 was shown to be independent of PLA2 activity. Moreover, mutation of three PDZ-binding motifs, which are located consecutively at the very tip of the VP1 N terminus,
revealed a nuclear transport-defective phenotype, suggesting a role in nuclear uptake of the virus through an as-yet-unknown
mechanism.
The majority of viruses infecting animals and plants today are RNA viruses [1]. There are double-stranded (ds) RNA viruses with dsRNA genomes, as well as (+) and (-) RNA viruses whose genomes are single-stranded (ss) RNA of either positive or negative polarity. RNA viruses have small genomes that rarely exceed 30 kb in size, and a large portion of their genomes is used to encode proteins involved in viral RNA replication. Viral RNA synthesis is catalyzed by the virally encoded RNA-dependent RNA polymerase (RdRp), which lacks any proofreading activity. Although the details of RNA replication greatly vary among viruses, common principles clearly exist in the organization of the replication machinery of different RNA viruses.
Rad52 promotes the annealing of complementary strands of DNA bound by replication protein A (RPA) during discrete repair pathways. Here, we used a fluorescence resonance energy transfer (FRET) between two fluorescent dyes incorporated into DNA substrates to probe the mechanism by which human Rad52 (hRad52) interacts with and mediates annealing of ssDNA-hRPA complexes. Human Rad52 bound ssDNA or ssDNA-hRPA complex in two, concentration-dependent modes. At low hRad52 concentrations, ssDNA was wrapped around the circumference of the protein ring, while at higher protein concentrations, ssDNA was stretched between multiple hRad52 rings. Annealing by hRad52 occurred most efficiently when each complementary DNA strand or each ssDNA-hRPA complex was bound by hRad52 in a wrapped configuration, suggesting homology search and annealing occur via two hRad52-ssDNA complexes. In contrast to the wild type protein, hRad52(RQK/AAA) and hRad52(1-212) mutants with impaired ability to bind hRPA protein competed with hRPA for binding to ssDNA and failed to counteract hRPA-mediated duplex destabilization highlighting the importance of hRad52-hRPA interactions in promoting efficient DNA annealing.
Benzonase nuclease degrades all forms of DNA and RNA while having no photolytic activity. In this study we check possibility of use this reagent to prepare proteins to microcalorymetric experiments.
Microtubule transport and nuclear import are functionally connected, and the nuclear pore complex (NPC) can interact with microtubule motors. For several alphaherpesvirus proteins, nuclear localization signals (NLSs) and their interactions with specific importin-α proteins have been characterized. Here, we review recent insights on the roles of microtubule motors, capsid-associated NLSs, and importin-α proteins for capsid transport, capsid docking to NPCs, and genome release into the nucleoplasm, as well as the role of importins for nuclear viral transcription, replication, capsid assembly, genome packaging, and nuclear capsid egress. Moreover, importin-α proteins exert antiviral effects by promoting the nuclear import of transcription factors inducing the expression of interferons (IFN), cytokines, and IFN-stimulated genes, and the IFN-inducible MxB restricts capsid docking to NPCs.
Ongoing development of recombinant vectors based on adeno-associated virus (rAAV) is providing an increasingly powerful and widely used toolkit for gene transfer and genome editing applications. While conceptually simple, the system harbours considerable complexity that presents many potential pitfalls for the inexperienced user. The short inverted terminal repeats (ITR) can prove to be particularly problematic during vector engineering due to inherent instability necessitating diligent quality control measures during vector manufacture. This is especially important from a clinical standpoint when consistent purity and potency are paramount, and all components of the system are rigorously scrutinized by regulatory agencies. Despite the discovery over thirty years ago that the AAV ITRs are the only cis-acting elements of the virus required for vector production, there is a scarcity of reviews specifically focused on these complex elements. This review provides an overview of the ITR with the dual purpose of acting as a user's guide in the application of AAV vector technology and as a roadmap for ongoing vector development and optimization.
The Mre11 nuclease has been the subject of intensive investigation for the past 20 years because of the central role that Mre11/Rad50 complexes play in genome maintenance. The last two decades of work on this complex has led to a much deeper understanding of the structure, biochemical activities, and regulation of Mre11/Rad50 complexes from archaea, bacteria, and eukaryotic cells. This review will discuss some of the important findings over recent years that have illuminated roles for the Mre11 nuclease in these different contexts as well as the insights from structural biology that have helped us to understand its mechanisms of action.
Adeno-associated virus vectors are a powerful tool for gene transfer approaches. We have established a simple and fast plasmid-based production system for achieving high adeno-associated virus titers within 6 working days. The same procedure can be used for all serotypes and thus allows direct comparability of different serotypes. In this protocol we describe a step-by-step procedure that results in well-characterized vectors suitable for both in vitro approaches and preclinical studies.
Parvoviruses are small, rugged, nonenveloped protein particles containing a linear, nonpermuted, single-stranded DNA genome of similar to 5 kb. Their limited coding potential requires optimal adaptation to the environment of particular host cells, where entry is mediated by a variable program of capsid dynamics, ultimately leading to genome ejection from intact particles within the host nucleus. Genomes are amplified by a continuous unidirectional strand-displacement mechanism, a linear adaptation of rolling circle replication that relies on the repeated folding and unfolding of small hairpin telomeres to reorient the advancing fork. Progeny genomes are propelled by the viral helicase into the preformed capsid via a pore at one of its icosahedral fivefold axes. Here we explore how the fine-tuning of this unique replication system and the mechanics that regulate opening and closing of the capsid fivefold portals have evolved in different viral lineages to create a remarkably complex spectrum of phenotypes.
The nuclear import of viral genomes is an important step of the infectious cycle for viruses that replicate in the nucleus of their host cells. Although most viruses use the cellular nuclear import machinery or some components of this machinery, others have developed sophisticated ways to reach the nucleus. Some of these have been known for some time; however, recent studies have changed our understanding of how some non-enveloped DNA viruses access the nucleus. For example, parvoviruses enter the nucleus through small disruptions of the nuclear membranes and nuclear lamina, and adenovirus tugs at the nuclear pore complex, using kinesin-1, to disassemble their capsids and deliver viral proteins and genomes into the nucleus. Here we review recent findings of the nuclear import strategies of three small non-enveloped DNA viruses, including adenovirus, parvovirus, and the polyomavirus simian virus 40.
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We have developed an in vitro system involving digitonin-permeabilized vertebrate cells to study biochemical events in the transport of macromolecules across the nuclear envelope. While treatment of cultured cells with digitonin permeabilizes the plasma membranes to macromolecules, the nuclear envelopes remain structurally intact and nuclei retain the ability to transport and accumulate proteins containing the SV40 large T antigen nuclear location sequence. Transport requires addition of exogenous cytosol to permeabilized cells, indicating the soluble cytoplasmic factor(s) required for nuclear import are released during digitonin treatment. In this reconstituted import system, a protein containing a nuclear location signal is rapidly accumulated in nuclei, where it reaches a 30-fold concentration compared to the surrounding medium within 30 min. Nuclear import is specific for a functional nuclear location sequence, requires ATP and cytosol, and is temperature dependent. Furthermore, accumulation of the transport substrate within nuclei is completely inhibited by wheat germ agglutinin, which binds to nuclear pore complexes and inhibits transport in vivo. Together, these results indicate that the permeabilized cell system reproduces authentic nuclear protein import. In a preliminary biochemical dissection of the system, we observe that the sulfhydryl alkylating reagent N-ethylmaleimide inactivates both cytosolic factor(s) and also component(s) in the insoluble permeabilized cell fraction required for nuclear protein import. Because this permeabilized cell model is simple, efficient, and works effectively with cells and cytosol fractions prepared from a variety of different vertebrate sources, it will prove powerful for investigating the biochemical pathway of nuclear transport.
The Replication Protein A (RPA) complex is an essential regulator of eukaryotic DNA metabolism. RPA avidly binds to single-stranded DNA (ssDNA) through multiple oligonucleotide/oligosaccharide-binding folds and coordinates the recruitment and exchange of genome maintenance factors to regulate DNA replication, recombination and repair. The RPA-ssDNA platform also constitutes a key physiological signal which activates the master ATR kinase to protect and repair stalled or collapsed replication forks during replication stress. In recent years, the RPA complex has emerged as a key target and an important regulator of post-translational modifications in response to DNA damage, which is critical for its genome guardian functions. Phosphorylation and SUMOylation of the RPA complex, and more recently RPA-regulated ubiquitination, have all been shown to control specific aspects of DNA damage signaling and repair by modulating the interactions between RPA and its partners. Here, we review our current understanding of the critical functions of the RPA-ssDNA platform in the maintenance of genome stability and its regulation through an elaborate network of covalent modifications.Cell Research advance online publication 18 November 2014; doi:10.1038/cr.2014.147.
With advances in therapeutic science, apart from drugs, newer bioactive moieties like oligonucleotides, proteins, peptides, enzymes and antibodies are constantly being introduced for the betterment of therapeutic efficacy. These moieties have intracellular components of the cells like
cytoplasm and nucleus as one of their pharmacological sites for exhibiting therapeutic activity. Despite their promising efficacy, their intracellular bioavailability has been critically hampered leading to failure in the treatment of numerous diseases and disorders. The endosomal uptake pathway
is known to be a rate-limiting barrier for such systems. Bioactive molecules get trapped in the endosomal vesicles and degraded in the lysosomal compartment, necessitating the need for effective strategies that facilitate the endosomal escape and enhance the cytosolic bioavailability of bioactives.
Microbes like viruses and bacteria have developed their innate mechanistic tactics to translocate their genome and toxins by efficiently penetrating the host cell membrane. Understanding this mechanism and exploring it further for intracellular delivery has opened new avenues to surmount the
endosomal barrier. These strategies include membrane fusion, pore formation and proton sponge effects. On the other hand, progress in designing a novel smart polymeric carrier system that triggers endosomal escape by undergoing modulations in the intracellular milieu has further led to an
improvement in intracellular delivery. These comprise pH, enzyme and temperature-induced modulators, synthetic cationic lipids and photo-induced physical disruption. Each of the aforementioned strategies has its own unique mechanism to escape the endosome. This review recapitulates the numerous
strategies designed to surmount the bottleneck of endosomal escape and thereby achieve successful intracellular uptake of bioactives.
Using laser scanning confocal microscopy, we have found that the in cells loaded with fluo-3/ AM, highest intracellular Ca2+ in the perinuclear region is associated with the Golgi apparatus. The spatiotemporal subcellular distribution of Ca2+ in living human fibroblasts exposing to calcium-free medium in response to agonists has been investigated. PDGF, which releases Ca2+ from intra-cellular stores by inositol(1, 4, 5)-trisphosphate pathway, produced a biphasic transient rise in intracellular calcium. The initial rise was resulted from a direct release of calcium from the Golgi apparatus. Calcium could be also released from and reaccumulated into the Golgi apparatus by the stimulation of thapsigargin, an inhibitor of the Ca2+ transport ATPase of intracellular calcium store. Permeablizing the plasma membrane by 10 μM digitonin resulted in the calcium release from the Golgi apparatus and depletion of the internal calcium store. These results suggest that the Golgi apparatus plays a role in Ca2+ regulation in signal transduction.
Poxviruses are large, enveloped viruses that replicate in the cytoplasm and encode proteins for DNA replication and gene expression. Hairpin ends link the two strands of the linear, double-stranded DNA genome. Viral proteins involved in DNA synthesis include a 117-kDa polymerase, a helicase-primase, a uracil DNA glycosylase, a processivity factor, a single-stranded DNA-binding protein, a protein kinase, and a DNA ligase. A viral FEN1 family protein participates in double-strand break repair. The DNA is replicated as long concatemers that are resolved by a viral Holliday junction endonuclease.
Both ends of the linear single-stranded parvoviral DNA genome contain short palindromic sequences which form duplex hairpins containingcis-acting information required for replication and encapsidation. DNA synthesis is primed directly by the 3′ end, and genomes are replicated through multimeric duplex intermediates by unidirectional, leading-strand synthesis. Unit-length genomes are excised from these concatemers, and their telomeres replicated, by the viral NS1 protein, which introduces a single-strand nick into specific origin sequences, becoming covalently attached to the 5′ end at the nick and providing a 3′ hydroxyl which primes synthesis of a new copy of the telomere. Progeny DNA synthesis requires ongoing replication and is dependent upon packaging.
The volume available in icosahedral virus capsids limits the size of viral genomes. To overcome this limitation, viruses have evolved strategies to increase their coding capacity by using more than one ORF while keeping the genome length constant. The assembly of virus capsids requires the coordinated interaction of a large number of subunits to generate a highly ordered structure in which the viral genome can be enclosed. To understand this process, it is essential to know which viral and nonviral components are involved in the assembly reaction. Here, we show that the adeno-associated virus (AAV) encodes a protein required for capsid formation by means of a nested, alternative ORF of the cap gene. Translation is initiated at a nonconventional translation start site, resulting in the expression of a protein with a calculated molecular weight of 23 kDa. This protein, designated assembly-activating protein (AAP), is localized in the host cell nucleolus, where AAV capsid morphogenesis occurs. AAP targets newly synthesized capsid proteins to this organelle and in addition fulfils a function in the assembly reaction itself. Sequence analysis suggests that also all other species of the genus Dependovirus encode a homologous protein in their cap gene. The arrangement of different ORFs that encode capsid proteins and an assembly factor within the same mRNA facilitates a timely coordinated expression of the components involved in the assembly process.
To infect a cell, the parvovirus or adeno-associated virus (AAV) genome must be delivered from outside the plasma membrane to the nucleus, and in the process, the capsid must follow a series of binding and trafficking steps and also undergo necessary changes that result in exposure or release the ssDNA genome at the appropriate time and place within the cell. The 25 nm parvovirus capsid is comprised of two or three forms of a single protein, and although it is robust and stable, it is still sufficiently flexible to allow the exposure of several internal components at appropriate times during cell infection. The capsid can also accommodate insertion of peptides into surface loops, and capsid proteins from different viral serotypes can be shuffled to create novel functional variants. The capsids of the different viruses bind to one or more cell receptors, and for at least some viruses, the insertion of additional or alternative receptor binding sequences or structures into the capsid can expand or redirect its tropism. The infection process after cell binding involves receptor-mediated endocytosis followed by viral trafficking through the endosomal systems. That endosomal trafficking may be complex and prolonged for hours or be relatively brief. Generally only a small proportion of the particles taken up enter the cytoplasm after altering the endosomal membrane through the activity of a VP1-encoded phospholipase A2 domain that becomes released to the outside of the viral particle. Modifications to the capsid that can occur within the endosome or cytoplasm include structural changes to expose internal components, ubiquination and proteosomal processing, and possible trafficking of particles on molecular motors. It is still not clear how the genomes enter the nucleus, but nuclear pore-dependent entry of particles or permeabilization of nuclear membranes have been proposed. Those processes control the infection, pathogenesis, and host ranges of the autonomous viruses and determine the effectiveness of gene therapy using AAV capsids.