Cytotoxic-T-Lymphocyte-Mediated Elimination of Target Cells Transduced with Engineered Adeno-Associated Virus Type 2 Vector In Vivo

A recent clinical trial in patients with hemophilia B has suggested that adeno-associated virus (AAV) capsid-specific cytotoxic T lymphocytes (CTLs) eliminated AAV-transduced hepatocytes and resulted in therapeutic failure. AAV capsids elicit a CTL response in animal models; however, these capsid-specific CTLs fail to kill AAV-transduced target cells in mice. To better model the human clinical trial data in mice, we introduced an immunodominant epitope derived from ovalbumin (OVA; SIINFEKL) into the AAV capsid and tested CTL-mediated killing of AAV2-transduced target tissues in vivo. Initially, in vitro experiments demonstrated both classical class I and cross-presentation of the OVA antigen, following endogenous expression or AAV2-OVA vector transduction, respectively. Furthermore, an OVA-specific CTL response was elicited after muscular or systemic injection of the AAV2-OVA vector. Finally, CTL reactivity was enhanced in mice with established SIINFEKL-specific immunity after AAV2-OVA/α1 anti-trypsin (AAT) administration. Most importantly, these OVA-specific CTLs decreased AAT expression in mice treated with AAV2-OVA/AAT vector that followed a time course mimicking uncoating kinetics of AAV2 transduction in OVA-immunized mice. These results demonstrate that AAV capsid-derived antigens elicit CD8⁺ CTL reactivity, and these CTLs eliminated AAV-transduced target cells in mice. Notably, this model system can be exploited to study the kinetics of capsid presentation from different serotypes of AAV and permit the design of novel strategies to block CTL-mediated killing of AAV-transduced cells.Adeno-associated virus (AAV) is a single-stranded DNA parvovirus. Its replication relies on coinfection of a helper virus such as adenovirus or herpesvirus. In the absence of a helper virus, AAV establishes latency to integrate into the AAVS1 site of host chromosome 19 (11). The genome of AAV is ∼4.7 kb and contains two open reading frames encoding replication proteins and structural capsid proteins (21). The capsid proteins (VP) are composed of VP1, VP2, and VP3. The VP3 protein is the major structural component and constitutes nearly 80% of the virion shell with an overall ratio of 1:1:8 for VP1, VP2, VP3, respectively. While VP2 is thought to be nonessential for AAV transduction (30), the VP1 subunit contains a phospholipase A2 domain required for infectivity (9). Recombinant AAV (rAAV) vectors require only the 145-bp terminal repeats of the AAV genome in cis and all other viral factors supplied in trans for production (18). rAAV vectors have rapidly gained popularity in gene therapy applications and have proven effective in preclinical studies/clinical trials for a number of diseases (20, 31, 33).AAV vectors mount a potent humoral immune response against capsid in animals and human. However, AAV vectors only contain the therapeutic gene flanked by two 145-bp AAV terminal repeats devoid of any AAV genes(23). In addition, AAV initiates long-term stable therapeutic gene expression in animal models (3-5, 17, 31). Based on these observations AAV has been thought to be relatively nonimmunogenic regarding the induction of cytotoxic T lymphocytes (CTLs) specific for capsid proteins. In spite of all of these observations, the recent clinical trial for hemophilia B (F9) gene therapy has otherwise suggested that AAV2 capsid initiates cell-mediated immunity that eliminates the AAV2 encoding F9 (AAV2/F9) vector transduced liver cells (15). Against this backdrop, numerous attempts to replicate aforementioned observations in animal models have been made. Preliminary results from these studies support direct presentation and cross-presentation of the AAV2 capsid in animal models (6, 12, 13, 22, 29). However, capsid-specific CTLs did not eliminate AAV2-transduced target cells in mice (12, 13, 29), inconsistent with observations made in a clinical trial for hemophilia B with AAV2/F9 gene therapy. A potential explanation for this discrepancy is the weak immunogenicity of the AAV2 capsid in mice. Accordingly, we hypothesized that incorporation of a peptide epitope into the AAV2 capsid would increase immunogenicity of the rAAV and therefore could be exploited to mimic events ongoing in humans and study approaches to block capsid-specific CTL reactivity in mice.We chose to introduce the MHC-H2K^b-restricted SIINFEKL peptide derived from ovalbumin (OVA) into AAV2 capsid. Integration of the OVA epitope into AAV capsids elicited a specific CTL response. Most importantly, after administration of genetically engineered AAV2 vectors into OVA peptide-immunized mice, OVA-specific CTL reactivity was further enhanced, thereby limiting transgene expression in vivo. The modified vector described herein is a potentially valuable tool for future studies focused on developing strategies to evade capsid-specific CTL-mediated elimination of AAV-transduced target cells in animal models.     

Enhancement of Adeno-Associated Virus Infection by Mobilizing Capsids into and Out of the Nucleolus

Adeno-associated virus (AAV) serotypes are being tailored for numerous therapeutic applications, but the parameters governing the subcellular fate of even the most highly characterized serotype, AAV2, remain unclear. To understand how cellular conditions control capsid trafficking, we have tracked the subcellular fate of recombinant AAV2 (rAAV2) vectors using confocal immunofluorescence, three-dimensional infection analysis, and subcellular fractionation. Here we report that a population of rAAV2 virions enters the nucleus and accumulates in the nucleolus after infection, whereas empty capsids are excluded from nuclear entry. Remarkably, after subcellular fractionation, virions accumulating in nucleoli were found to retain infectivity in secondary infections. Proteasome inhibitors known to enhance transduction were found to potentiate nucleolar accumulation. In contrast, hydroxyurea, which also increases transduction, mobilized virions into the nucleoplasm, suggesting that two separate pathways influence vector delivery in the nucleus. Using a small interfering RNA (siRNA) approach, we then evaluated whether nucleolar proteins B23/nucleophosmin and nucleolin, previously shown to interact with AAV2 capsids, affect trafficking and transduction efficiency. Similar to effects observed with proteasome inhibition, siRNA-mediated knockdown of nucleophosmin potentiated nucleolar accumulation and increased transduction 5- to 15-fold. Parallel to effects from hydroxyurea, knockdown of nucleolin mobilized capsids to the nucleoplasm and increased transduction 10- to 30-fold. Moreover, affecting both pathways simultaneously using drug and siRNA combinations was synergistic and increased transduction over 50-fold. Taken together, these results support the hypothesis that rAAV2 virions enter the nucleus intact and can be sequestered in the nucleolus in stable form. Mobilization from the nucleolus to nucleoplasmic sites likely permits uncoating and subsequent gene expression or genome degradation. In summary, with these studies we have refined our understanding of AAV2 trafficking dynamics and have identified cellular parameters that mobilize virions in the nucleus and significantly influence AAV infection.Adeno-associated virus (AAV) is classified as a dependovirus because it requires the presence of a helper virus, such as adenovirus or herpesvirus, in order to enter into a productive lytic cycle (6). Because of its nonpathogenicity and ability to promote sustained, long-term transgene expression in a wide variety of tissues such as the brain, liver, muscle, retina, and vasculature (51), several recombinant AAV (rAAV) serotypes are emerging as attractive vectors for gene therapy. Despite many advances in AAV vector design, barriers such as a preexisting immune response and off-target binding have necessitated administration of high viral titers to achieve efficient transduction (24, 51).Beyond the barriers of the immune response (9, 42) and cell surface targeting (52), researchers are becoming increasingly aware that subcellular processing is a significant barrier to infection (16, 29, 52). Subcellular processing may include conformational changes within the endosome or similar compartments, endosomal escape, nuclear targeting, and uncoating, but the factors that control these events are not well defined. Understanding how cellular conditions affect subcellular processing of virions will lead to improved gene delivery through exploitation of these parameters and promote better vector design.Given that the virion is an icosahedral particle only 25 nm in diameter, rAAV must contain all of the molecular components required to navigate through the subcellular environment in a remarkably small structure. Wild-type AAV is a nonenveloped parvovirus that packages a single-stranded DNA genome of approximately 4.7 kb in length. The viral genome is flanked by two inverted terminal repeats and contains two open reading frames, one that codes for replication proteins and another that codes for capsid proteins. Three capsid proteins (VP1, VP2, and VP3) are encoded in the second overlapping reading frame, each beginning with a different start codon but sharing a common C terminus and stop codon. Capsids are comprised of 60 copies of V1, VP2, and VP3 in a ratio of approximately 1:1:10, respectively (11, 43). During production, AAV capsids are known to assemble at early time points in the nucleolus (64), a subdomain of the nucleus and one of the oldest known cellular structures. Intact capsids have been shown to interact with nucleolar proteins such as nucleolin (NCL) and B23/nucleophosmin (NPM1) in the context of assembly (8, 46), but how these proteins affect infection or vector delivery is currently unknown.Initial cell surface binding of AAV capsids is mediated by expression of glycoprotein receptors and specified by residues in VP3 (45, 58, 59). After binding receptors on the host cell plasma membrane, AAV serotype 2 (AAV2) is endocytosed from the cell surface in a clathrin- and dynamin-dependent process (3, 5, 19). Following endocytosis, many AAV particles accumulate in late endosomes, lysosomes, or other compartments and do not deliver their genome to the nucleus (17). This impediment to gene delivery is exacerbated when particles lack VP1 or contain specific mutations in the unique N terminus of VP1 (23). The N terminus of VP1 is normally folded inside the capsid, harboring a phospholipase domain and putative nuclear localization signals necessary for infection (13, 23, 74). These regions of VP1 are thought to translocate to the capsid exterior during subcellular processing of the virus (10, 35, 57). Even with proper capsid composition, the vast majority of internalized particles remain clearly outside the nuclear membrane, and although recent evidence suggests that successful infection occurs when the capsid uncoats inside the nucleus (57, 61), whether AAV can enter the nucleus as an intact capsid is still vehemently debated.In general, it has proven difficult to discern whether infectious particles truly cross the nuclear membrane, due to the limitations of fluorescence microscopy (5, 67). In an in vitro setting it has been demonstrated that unmodified AAV capsids are capable of entering purified nuclei (28), yet these conditions do not accurately represent what occurs physiologically, since virus directly microinjected into cytoplasm will not enter the nucleus or efficiently transduce the cell (17, 57). In one instance, single-particle tracking of AAV has been used to follow capsids in a live-cell imaging paradigm and has found that they can be quickly and directly transported to the nucleus (54). However, another recent study has parsed confocal images of green fluorescent protein-tagged AAV2 particles during infection and has reported that few if any particles enter the nucleus during infection (38).Although it is unclear whether capsids enter the nucleus intact, it has been well established that nuclear delivery of the genome is highly inefficient and significantly limits transduction. Several studies have identified agents that surmount subcellular barriers to transduction (20, 22, 69). Two of the most well-documented agents known to improve subcellular processing are proteasome inhibitors and hydroxyurea (HU); however, their mechanisms of action remain unknown. Therefore, we set out to determine what effect, if any, these agents had on subcellular trafficking of rAAV2 in the hope of identifying specific cellular parameters that promote efficient transduction.Here we report that rAAV2 capsids accumulate in the nucleolus during infection. Proteasome inhibitors were found to potentiate nucleolar accumulation, while HU reduced nucleolar accumulation and appeared to mobilize capsids to the nucleoplasm. Acting independently, both proteasome inhibitors and HU increased transduction, and together they were cooperative, which suggests that these treatments operate through separate pathways to improve gene delivery. In addition, we found that small interfering RNA (siRNA) knockdown of nucleolar proteins NCL and NPM1 had effects similar to those of proteasome inhibition or HU and increased transduction. Based on our results, we have proposed a model wherein AAV virions initially enter the nucleus intact and can be sequestered in the nucleolus in stable form. Disruption of the nucleolus subsequently mobilizes virions from the nucleolus to nucleoplasmic sites and likely permits uncoating.     

Adeno-Associated Virus Replication Induces a DNA Damage Response Coordinated by DNA-Dependent Protein Kinase

The parvovirus adeno-associated virus (AAV) contains a small single-stranded DNA genome with inverted terminal repeats that form hairpin structures. In order to propagate, AAV relies on the cellular replication machinery together with functions supplied by coinfecting helper viruses such as adenovirus (Ad). Here, we examined the host cell response to AAV replication in the context of Ad or Ad helper proteins. We show that AAV and Ad coinfection activates a DNA damage response (DDR) that is distinct from that seen during Ad or AAV infection alone. The DDR was also triggered when AAV replicated in the presence of minimal Ad helper proteins. We detected autophosphorylation of the kinases ataxia telangiectasia mutated (ATM) and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and signaling to downstream targets SMC1, Chk1, Chk2, H2AX, and XRCC4 and multiple sites on RPA32. The Mre11 complex was not required for activation of the DDR to AAV infection. Additionally, we found that DNA-PKcs was the primary mediator of damage signaling in response to AAV replication. Immunofluorescence revealed that some activated damage proteins were found in a pan-nuclear pattern (phosphorylated ATM, SMC1, and H2AX), while others such as DNA-PK components (DNA-PKcs, Ku70, and Ku86) and RPA32 accumulated at AAV replication centers. Although expression of the large viral Rep proteins contributed to some damage signaling, we observed that the full response required replication of the AAV genome. Our results demonstrate that AAV replication in the presence of Ad helper functions elicits a unique damage response controlled by DNA-PK.Replication of viral genomes produces a large amount of extrachromosomal DNA that may be recognized by the cellular DNA damage machinery. This is often accompanied by activation of DNA damage response (DDR) signaling pathways and recruitment of cellular repair proteins to sites of viral replication. Viruses therefore provide good model systems to study the recognition and response to DNA damage (reviewed in reference 48). The Mre11/Rad50/Nbs1 (MRN) complex functions as a sensor of chromosomal DNA double-strand breaks (DSBs) and is involved in activation of damage signaling (reviewed in reference 41). The MRN complex also localizes to DNA DSBs and is found at viral replication compartments during infection with a number of DNA viruses (6, 40, 47, 70, 75, 77, 87, 93). The phosphatidylinositol 3-kinase-like kinases (PIKKs) ataxia telangiectasia mutated (ATM), ATM and Rad3-related kinase (ATR), and the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) are involved in the signal transduction cascades activated by DNA damage (reviewed in references 43, 51, and 71). These kinases respond to distinct types of damage and regulate DSB repair during different phases of the cell cycle (5), either through nonhomologous end-joining (NHEJ) or homologous recombination pathways (reviewed in references 63, 81, and 86). The DNA-PK holoenzyme is composed of DNA-PKcs and two regulatory subunits, the Ku70 and Ku86 heterodimer. DNA-PK functions with XRCC4/DNA ligase IV to repair breaks during NHEJ, and works with Artemis to process DNA hairpin structures during VDJ recombination and during a subset of DNA DSB events (46, 50, 86). While the kinase activity of DNA-PKcs leads to phosphorylation of a large number of substrates in vitro as well as autophosphorylation of specific residues (reviewed in references 16 and 85), it is currently unclear how DNA-PKcs contributes to signaling in cells upon different types of damage.The adeno-associated virus (AAV) genome consists of a molecule of single-stranded DNA with inverted terminal repeats (ITRs) at both ends that form double-hairpin structures due to their palindromic sequences (reviewed in reference 52). The ITRs are important for replication and packaging of the viral genome and for integration into the host genome. Four viral Rep proteins (Rep78, Rep68, Rep52, and Rep40) are also required for replication and packaging of the AAV genome into virions assembled from the Cap proteins. Although the Rep and Cap genes are replaced in recombinant AAV vectors (rAAV) that retain only the ITRs flanking the gene of interest, these vectors can be replicated by providing Rep in trans (reviewed in reference 7). Productive AAV infection requires helper functions supplied by adenovirus (Ad) or other viruses such as herpes simplex virus (HSV) (reviewed in reference 27), together with components of the host cell DNA replication machinery (54, 55, 58). In the presence of helper viruses or minimal helper proteins from Ad or HSV, AAV replicates in the nucleus at centers where the viral DNA and Rep proteins accumulate (35, 76, 84, 89). Cellular and viral proteins involved in AAV replication, including replication protein A (RPA), Ad DNA-binding protein (DBP), and HSV ICP8, localize with Rep proteins at these viral centers (29, 33, 76).A number of published reports suggest associations between AAV and the cellular DNA damage machinery. For example, transduction by rAAV vectors is increased by genotoxic agents and DNA damaging treatments (1, 62, 91) although the mechanisms involved remain unclear. Additionally, the ATM kinase negatively regulates rAAV transduction (64, 92), and we have shown that the MRN complex poses a barrier to both rAAV transduction and wild-type AAV replication (11, 67). UV-inactivated AAV particles also appear to activate a DDR involving ATM and ATR kinases that perturbs cell cycle progression (39, 60, 88). It has been suggested that this response is provoked by the AAV ITRs (60) and that UV-treated particles mimic stalled replication forks in infected cells (39). In addition to AAV genome components, the viral Rep proteins have been observed to exhibit cytotoxicity and induce S-phase arrest (3, 65).The role of cellular repair proteins in AAV genome processing has also been explored by examining the molecular fate of rAAV vectors, which are converted into circular and concatemeric forms that persist episomally (18, 19, 66). Proteins shown to regulate circularization in cell culture include ATM and the MRN complex (14, 64), while in vivo experiments using mouse models have implicated ATM and DNA-PK in this process (14, 20, 72). Additionally, DNA-PKcs and Artemis have recently been shown to cleave the ITR hairpins of rAAV vectors in vivo in a tissue-dependent manner (36). Despite these studies, it is not clear how damage response factors function together and how they impact AAV transduction and replication in human cells.In this study we examined the cellular response to AAV replication in the context of Ad infection or helper proteins. We show that coinfection with AAV and Ad activates a DDR that is distinct from that seen during infection with Ad alone. The ATM and DNA-PKcs damage kinases are activated and signal to downstream substrates, but the response does not require the MRN complex and is primarily mediated by DNA-PKcs. Although expression of the large Rep proteins induced some DDR events, full signaling appeared to require AAV replication and was accompanied by accumulation of DNA-PK at viral replication compartments. Our results demonstrate that AAV replication induces a unique DNA damage signal transduction response and provides a model system for studying DNA-PK.     

Conserved Motifs within Ebola and Marburg Virus VP40 Proteins Are Important for Stability,Localization, and Subsequent Budding of Virus-Like Particles

The filovirus VP40 protein is capable of budding from mammalian cells in the form of virus-like particles (VLPs) that are morphologically indistinguishable from infectious virions. Ebola virus VP40 (eVP40) contains well-characterized overlapping L domains, which play a key role in mediating efficient virus egress. L domains represent only one component required for efficient budding and, therefore, there is a need to identify and characterize additional domains important for VP40 function. We demonstrate here that the ₉₆LPLGVA₁₀₁ sequence of eVP40 and the corresponding ₈₄LPLGIM₈₉ sequence of Marburg virus VP40 (mVP40) are critical for efficient release of VP40 VLPs. Indeed, deletion of these motifs essentially abolished the ability of eVP40 and mVP40 to bud as VLPs. To address the mechanism by which the ₉₆LPLGVA₁₀₁ motif of eVP40 contributes to egress, a series of point mutations were introduced into this motif. These mutants were then compared to the eVP40 wild type in a VLP budding assay to assess budding competency. Confocal microscopy and gel filtration analyses were performed to assess their pattern of intracellular localization and ability to oligomerize, respectively. Our results show that mutations disrupting the ₉₆LPLGVA₁₀₁ motif resulted in both altered patterns of intracellular localization and self-assembly compared to wild-type controls. Interestingly, coexpression of either Ebola virus GP-WT or mVP40-WT with eVP40-ΔLPLGVA failed to rescue the budding defective eVP40-ΔLPLGVA mutant into VLPs; however, coexpression of eVP40-WT with mVP40-ΔLPLGIM successfully rescued budding of mVP40-ΔLPLGIM into VLPs at mVP40-WT levels. In sum, our findings implicate the LPLGVA and LPLGIM motifs of eVP40 and mVP40, respectively, as being important for VP40 structure/stability and budding.Ebola and Marburg viruses are members of the family Filoviridae. Filoviruses are filamentous, negative-sense, single-stranded RNA viruses that cause lethal hemorrhagic fevers in both humans and nonhuman primates (5). Filoviruses encode seven viral proteins including: NP (major nucleoprotein), VP35 (phosphoprotein), VP40 (matrix protein), GP (glycoprotein), VP30 (minor nucleoprotein), VP24 (secondary matrix protein), and L (RNA-dependent RNA polymerase) (2, 5, 10, 12, 45). Numerous studies have shown that expression of Ebola virus VP40 (eVP40) alone in mammalian cells leads to the production of virus-like particles (VLPs) with filamentous morphology which is indistinguishable from infectious Ebola virus particles (12, 17, 18, 25, 26, 27, 30, 31, 34, 49). Like many enveloped viruses such as rhabdovirus (11) and arenaviruses (44), Ebola virus encodes late-assembly or L domains, which are sequences required for the membrane fission event that separates viral and cellular membranes to release nascent virion particles (1, 5, 7, 10, 12, 18, 25, 27, 34). Thus far, four classes of L domains have been identified which were defined by their conserved amino acid core sequences: the Pro-Thr/Ser-Ala-Pro (PT/SAP) motif (25, 27), the Pro-Pro-x-Tyr (PPxY) motif (11, 12, 18, 19, 41, 53), the Tyr-x-x-Leu (YxxL) motif (3, 15, 27, 37), and the Phe-Pro-Ile-Val (FPIV) motif (39). Both PTAP and the PPxY motifs are essential for efficient particle release for eVP40 (25, 27, 48, 49), whereas mVP40 contains only a PPxY motif. L domains are believed to act as docking sites for the recruitment of cellular proteins involved in endocytic trafficking and multivesicular body biogenesis to facilitate virus-cell separation (8, 13, 14, 16, 28, 29, 33, 36, 43, 50, 51).In addition to L domains, oligomerization, and plasma-membrane localization of VP40 are two functions of the protein that are critical for efficient budding of VLPs and virions. Specific sequences involved in self-assembly and membrane localization have yet to be defined precisely. However, recent reports have attempted to identify regions of VP40 that are important for its overall function in assembly and budding. For example, the amino acid region ₂₁₂KLR₂₁₄ located at the C-terminal region was found to be important for efficient release of eVP40 VLPs, with Leu₂₁₃ being the most critical (30). Mutation of the ₂₁₂KLR₂₁₄ region resulted in altered patterns of cellular localization and oligomerization of eVP40 compared to those of the wild-type genotype (30). In addition, the proline at position 53 was also implicated as being essential for eVP40 VLP release and plasma-membrane localization (54).In a more recent study, a YPLGVG motif within the M protein of Nipah virus (NiV) was shown to be important for stability, membrane binding, and budding of NiV VLPs (35). Whether this NiV M motif represents a new class of L domain remains to be determined. However, it is clear that this YPLGVG motif of NiV M is important for budding, perhaps involving a novel mechanism (35). Our rationale for investigating the corresponding, conserved motifs present within the Ebola and Marburg virus VP40 proteins was based primarily on these findings with NiV. In addition, Ebola virus VP40 motif maps close to the hinge region separating the N- and C-terminal domains of VP40 (4). Thus, the ₉₆LPLGVA₁₀₁ motif of eVP40 is predicted to be important for the overall stability and function of VP40 during egress. Findings presented here indicate that disruption of these filovirus VP40 motifs results in a severe defect in VLP budding, due in part to impairment in overall VP40 structure, stability and/or intracellular localization.     

In vitro and in vivo gene therapy vector evolution via multispecies interbreeding and retargeting of adeno-associated viruses

Adeno-associated virus (AAV) serotypes differ broadly in transduction efficacies and tissue tropisms and thus hold enormous potential as vectors for human gene therapy. In reality, however, their use in patients is restricted by prevalent anti-AAV immunity or by their inadequate performance in specific targets, exemplified by the AAV type 2 (AAV-2) prototype in the liver. Here, we attempted to merge desirable qualities of multiple natural AAV isolates by an adapted DNA family shuffling technology to create a complex library of hybrid capsids from eight different wild-type viruses. Selection on primary or transformed human hepatocytes yielded pools of hybrids from five of the starting serotypes: 2, 4, 5, 8, and 9. More stringent selection with pooled human antisera (intravenous immunoglobulin [IVIG]) then led to the selection of a single type 2/type 8/type 9 chimera, AAV-DJ, distinguished from its closest natural relative (AAV-2) by 60 capsid amino acids. Recombinant AAV-DJ vectors outperformed eight standard AAV serotypes in culture and greatly surpassed AAV-2 in livers of naïve and IVIG-immunized mice. A heparin binding domain in AAV-DJ was found to limit biodistribution to the liver (and a few other tissues) and to affect vector dose response and antibody neutralization. Moreover, we report the first successful in vivo biopanning of AAV capsids by using a new AAV-DJ-derived viral peptide display library. Two peptides enriched after serial passaging in mouse lungs mediated the retargeting of AAV-DJ vectors to distinct alveolar cells. Our study validates DNA family shuffling and viral peptide display as two powerful and compatible approaches to the molecular evolution of novel AAV vectors for human gene therapy applications.A large number of inherited or acquired diseases remain promising targets for human gene therapy. One vector that has shown outstanding potential thus far in numerous preclinical and clinical evaluations is based on nonpathogenic adeno-associated virus (AAV). A unique asset among various properties that make AAV especially attractive over its competitors, such as adenoviral or lentiviral vectors, is the availability of a vast number of natural isolates which differ significantly in their properties (24). We and others have shown previously that the function of an AAV vector particle is determined mainly by the capsid protein and that viral Rep proteins and genomic packaging elements are largely interchangeable (24, 27, 85). Paradoxically, the ever-increasing repertoire of naturally occurring and synthetically generated AAV capsid sequences (>300 to date) is currently creating a dilemma for the rational selection of the optimal serotype for a given application. The importance of finding the ideal capsid for efficient and safe gene transfer has been exemplified in many preclinical studies, as well as in a clinical trial using the AAV type 2 (AAV-2) prototype in human liver tissue (36, 47). In one previous study, the treatment of patients with severe hemophilia B with recombinant AAV-2 expressing human factor IX (hFIX) resulted in mildly elevated, yet therapeutic, levels of this blood coagulation factor. However, expression was short lived, and the hFIX decline was accompanied by a transient asymptomatic increase of liver transaminases, due to a T-cell immune response against the AAV-2 capsid (47). Also, preexisting neutralizing anti-AAV-2 antibodies (frequent in humans) in these individuals likely inhibited the linear vector dose response previously observed in animals.We and others have suggested previously that the use of novel AAV serotypes, in particular, nonhuman isolates, will help to overcome some of these problems (19, 24, 63). Important examples are AAV-8 and AAV-9, which can transduce mouse liver far better than AAV-2, albeit the difference in dogs or primates is less clear (17, 52, 54, 75). The potential for the complete transduction of liver tissue and perhaps other tissues makes these two non-AAV-2 serotypes also particularly interesting for therapeutic RNA interference (RNAi) (28). We recently demonstrated the feasibility of efficiently and persistently suppressing hepatitis B virus with RNAi from a double-stranded AAV-8 vector (28). On the other hand, a potential drawback of AAV-8 and AAV-9 is their lack of specific tissue tropism (34, 52). The resulting frequent vector dissemination into all organs, including the brain, even from low peripheral doses in mice or monkeys (52, 54) is a particular concern for RNAi therapies in which control over vector biodistribution and the limitation of off-target effects will be imperative for the success of the approach (28).In order to overcome the constraints of wild-type AAV serotypes, numerous groups have recently begun to develop novel strategies to engineer “designer” AAVs tailored for the therapeutic transduction of clinically relevant organs (reviewed in detail in references 9, 12, 35, 41, 51, and 85). Briefly, the variety of strategies can be grouped into indirect or chemical approaches and direct physical modification strategies. In the indirect approaches, specific molecules (e.g., bispecific antibodies [6] or avidin-coupled ligands [4]) are allowed to react with the viral surface (biotinylated in the case of avidin [4]), as well as a cellular receptor, forming a conjugate ideally able to retarget the capsid to a refractory cell type. Yet, numerous pharmacological problems, such as concerns about in vivo complex stability and difficulties in upscaling complex manufacturing, continue to prevent the broad adaptation of these approaches. Alternative, more powerful strategies rely on the direct physical modification of the AAV capsid protein and gene. Early examples include the generation of mosaic AAV capsids via the mixing of helper plasmids carrying capsid genes from distinct serotypes, such as AAV-1 and AAV-2 (30) and pairwise combinations of AAV-1 through AAV-5 (62). Similar mosaics were generated previously via a marker rescue approach, yielding AAV-2/AAV-3 recombinants with unique properties (8). A related strategy is the rational creation of chimeric virions via domain swapping among multiple parental serotypes, involving either entire capsid loops or parts thereof or individual residues. Notable examples include AAV-1/AAV-2 chimeras with improved tropism in muscle tissue (31), with one of these chimeras presently being studied in a phase I clinical trial for the treatment of Duchenne muscular dystrophy (85). Most recently, our own group described a battery of unique chimeras comprising elements from serotypes 2 and 8, which were exploited to identify capsid subdomains responsible for efficient AAV transduction in murine liver tissue in vivo (64).A special type of chimeric capsids are those containing foreign proteins or peptides inserted into various positions of the virion shell. The methods and strategies used are widely diverse, and again, we refer to comprehensive reviews (12, 35, 41). Noteworthy here are approaches to fuse targeting ligands to the N termini of AAV capsid proteins (ideally, VP2 [45, 83]), or more powerful, to insert short peptides (up to 14 amino acids [21], typically 7) into exposed regions of the assembled virion. This strategy is referred to as viral display, in analogy to phage display, and has already been used extensively to retarget AAV-2 virions to a multitude of refractory or hard-to-infect cell types, such as vascular endothelial, smooth muscle, and pancreatic islet cells (43, 55, 77, 81, 82) and various tumor lines (22, 58, 65, 66). It has particularly benefited from comprehensive mutational analyses by various groups (e.g., references 21, 33, 56, and 83) that have resulted in the identification of prominent locations within the AAV-2 capsid tolerating peptide insertion. Most notable is the heparin binding domain (HBD), consisting of a total of four arginine (R) residues and one lysine residue, with R585 and R588 representing the most crucial components (37, 56). Numerous groups have now consistently shown that the insertion of 7-mer peptides into this region not only is frequently well tolerated and efficiently mediates virus retargeting, but also provides the extra benefit that the endogenous AAV-2 tropism can be abolished, thus enhancing target specificity (e.g., reference 21).In addition to identifying sites for vector engineering, some of the mutational AAV studies directly yielded novel capsid variants with potential benefits for clinical use. A remarkable case was a recent study by Lochrie et al. (42) in which a set of 127 AAV-2 variants with point or insertion mutations were generated and screened for multiple properties. Several capsids were isolated which differed from the wild-type AAV-2 capsid in having better in vitro transduction efficiencies (albeit being equally efficient in vivo) or, clinically most relevant, higher-level resistance to individual or pooled human antisera. Nonetheless, the limitations of the approach also became clear, most notably, the extreme effort required to generate and manually screen a large number of mutants, which in fact prevented the interesting analyses of all possible combinations of beneficial point mutations in further capsids.Indeed, the factors of time and labor are the main reasons why an increasing number of groups have recently begun to develop novel means for AAV vector evolution that no longer rely on the rational modification of the AAV-2 capsid. Instead, the new combinatorial methodologies allow for the far more efficient creation and selection of interesting candidates in a library-based high-throughput format. Thus far, two different strategies have been reported, both principally expanding on previously developed techniques. One is the use of viral display libraries, in which random 7-mer peptides are inserted into the AAV-2 HBD (at amino acid 587 or 588), yielding between 4 × 10⁶ and 1.7 × 10⁸ capsids potentially exposing new ligands on their surfaces (50, 58, 76). Subsequent iterative selection on diverse cell types refractory to the wild type, e.g., coronary artery endothelial cells, cardiomyoblasts, and carcinoma, leukemia, and megakaryocytic cell lines, led to enrichment with peptide mutants with increased target specificities and efficacies (48, 50, 58, 76). The second library type, independently described by two groups in 2006, relies on error-prone PCR amplification of the AAV-2 capsid gene (46, 59). Similar to the methods in earlier mutational studies, this approach resulted in the identification of AAV-2 point mutations (usually up to two per capsid) which yielded mutants that differed from the wild type in having mildly enhanced efficacies in vitro and/or improved transduction efficiencies in the presence of neutralizing anti-AAV-2 antibodies either generated in rabbits or preexisting in individual human sera.Here, for the first time, we introduce the technology of DNA family shuffling into the realm of AAV vector evolution. The basic concept of this technology is the in vitro recombination of related parental genes with >50% homology, which are first fragmented and then reassembled based on partial homology, resulting in libraries of chimeric genes. Iterative amplification under pressure can then yield hybrids not only combining parental assets, but also ideally exhibiting novel and synergistic properties (70, 71). DNA family shuffling has been used extensively in recent years to evolve and improve all types of proteins, from markers and enzymes to vaccines (e.g., references 10, 13-15, and 39). Importantly, a set of reports also suggested its power to enhance viral gene therapy vectors by creating retro- or lentiviruses with improved stability or efficacy compared to that of the parental wild types (57, 61, 69). Here, we describe the novel use of DNA family shuffling for the highly efficient molecular interbreeding of eight multispecies AAVs to create chimeric capsids and, moreover, document its compatibility and synergism with existing AAV vector evolution technology.     

Adeno-Associated Virus Small Rep Proteins Are Modified with at Least Two Types of Polyubiquitination

Adeno-associated virus (AAV) type 2 and 5 proteins Rep52 and Rep40 were polyubiquitinated during AAV-adenovirus type 5 (Ad5) coinfection and during transient transfection in either the presence or absence of Ad5 E4orf6 and E1b-55k. Polyubiquitination of small Rep proteins via lysine 48 (K48) linkages, normally associated with targeting of proteins for proteasomal degradation, was detected only in the presence of E4orf6. The small Rep proteins were ubiquitinated via lysine 63 (K63) following transfection in either the presence or absence of E4orf6 or following coinfection with Ad5. E4orf6/E1b-55k-dependent K48-specific polyubiquitination of small Rep proteins could be inhibited using small interfering RNA (siRNA) to cullin 5.Together, adenovirus type 5 (Ad5) early gene products E1a, E1b-55k, E2a, E4orf6, and virus-associated (VA) RNA can support efficient replication of adeno-associated virus (AAV) (4, 31). E4orf6 and E1b-55k are known to interact with cellular cullin 5 (cul5), elongins B and C, and the ring box protein Rbx1 to form an E3 ubiquitin ligase complex that specifically targets a small population of cellular proteins for degradation by the proteasome (1, 7, 21, 22, 24, 27). This property has been implicated in a number of functions presumed to be required for both Ad and AAV replication (3, 8-10, 17, 23, 24, 34, 35).Previously, only p53, Mre11, DNA ligase IV, and integrin α3 had been shown to be substrates of the Ad5 E3 ubiquitin ligase complex (1, 7, 21, 22, 24, 27); however, we have recently shown (16, 17) that the small Rep proteins and capsid proteins of AAV5 are also degraded in the presence of Ad E4orf6 and E1b-55k in a proteasome-dependent manner. These proteins were restored to levels required during infection by the action of VA RNA (17). The targeting for degradation of AAV5 protein by the E4orf6/E1b-55k E3 ubiquitin ligase complex required functional BC-box motifs in E4orf6 and could be inhibited by depletion of the scaffolding protein cullin 5 using directed small interfering RNA (siRNA) (16). In addition, the degradation of AAV5 protein was partially prevented by overexpression of pUBR7, a plasmid that generates a dominant-negative ubiquitin (16). The role this targeted degradation plays in the life cycle of AAV has not yet been clarified; however, E4orf6 mutants that cannot function in this regard do not support AAV replication as well as wild-type E4orf6 (R. Nayak and D. J. Pintel, unpublished data). Degradation of Mre11 by the Ad5 E3 ligase has also been implicated in allowing efficient Ad5 and AAV replication (24). Ubiquitination of AAV Rep proteins during viral infection, however, has not previously been reported.     

Mutations Abrogating VP35 Interaction with Double-Stranded RNA Render Ebola Virus Avirulent in Guinea Pigs

Ebola virus (EBOV) protein VP35 is a double-stranded RNA (dsRNA) binding inhibitor of host interferon (IFN)-α/β responses that also functions as a viral polymerase cofactor. Recent structural studies identified key features, including a central basic patch, required for VP35 dsRNA binding activity. To address the functional significance of these VP35 structural features for EBOV replication and pathogenesis, two point mutations, K319A/R322A, that abrogate VP35 dsRNA binding activity and severely impair its suppression of IFN-α/β production were identified. Solution nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography reveal minimal structural perturbations in the K319A/R322A VP35 double mutant and suggest that loss of basic charge leads to altered function. Recombinant EBOVs encoding the mutant VP35 exhibit, relative to wild-type VP35 viruses, minimal growth attenuation in IFN-defective Vero cells but severe impairment in IFN-competent cells. In guinea pigs, the VP35 mutant virus revealed a complete loss of virulence. Strikingly, the VP35 mutant virus effectively immunized animals against subsequent wild-type EBOV challenge. These in vivo studies, using recombinant EBOV viruses, combined with the accompanying biochemical and structural analyses directly correlate VP35 dsRNA binding and IFN inhibition functions with viral pathogenesis. Moreover, these studies provide a framework for the development of antivirals targeting this critical EBOV virulence factor.Ebola viruses (EBOVs) are zoonotic, enveloped negative-strand RNA viruses belonging to the family Filoviridae which cause lethal viral hemorrhagic fever in humans and nonhuman primates (47). Currently, information regarding EBOV-encoded virulence determinants remains limited. This, coupled with our lack of understanding of biochemical and structural properties of virulence factors, limits efforts to develop novel prophylactic or therapeutic approaches toward these infections.It has been proposed that EBOV-encoded mechanisms to counter innate immune responses, particularly interferon (IFN) responses, are critical to EBOV pathogenesis (7). However, a role for viral immune evasion functions in the pathogenesis of lethal EBOV infection has yet to be demonstrated. Of the eight major EBOV gene products, two viral proteins have been demonstrated to counter host IFN responses. The VP35 protein is a viral polymerase cofactor and structural protein that also inhibits IFN-α/β production by preventing the activation of interferon regulatory factor (IRF)-3 and -7 (3, 4, 8, 24, 27, 34, 41). VP35 also inhibits the activation of PKR, an IFN-induced, double-stranded RNA (dsRNA)-activated kinase with antiviral activity, and inhibits RNA silencing (17, 20, 48). The VP24 protein is a minor structural protein implicated in virus assembly and regulation of viral RNA synthesis, and changes in VP24 coding sequences are also associated with adaptation of EBOVs to mice and guinea pigs (2, 13, 14, 27, 32, 37, 50, 52). Further, VP24 inhibits cellular responses to both IFN-α/β and IFN-γ by preventing the nuclear accumulation of tyrosine-phosphorylated STAT1 (44, 45). The functions of VP35 and VP24 proteins are manifested in EBOV-infected cells by the absence of IRF-3 activation, impaired production of IFN-α/β, and severely reduced expression of IFN-induced genes, even after treatment of infected cells with IFN-α (3, 19, 21, 22, 24, 25, 28).Previous studies proposed that VP35 basic residues 305, 309, and 312 are required for VP35 dsRNA binding activity (26). VP35 residues K309 and R312 were subsequently identified as critical for binding to dsRNA, and mutation of these residues impaired VP35 suppression of IFN-α/β production (8). In vivo, an EBOV engineered to carry a VP35 R312A point mutation exhibited reduced replication in mice (23). However, because the parental recombinant EBOV into which the mutation was built did not cause disease in these animals, the impact of the mutation on viral pathogenesis could not be fully evaluated. Further, the lack of available structural and biochemical data to explain how the R312A mutation affects VP35 function limited avenues for the therapeutic targeting of critical VP35 functions. Recent structural analyses of the VP35 carboxy-terminal interferon inhibitory domain (IID) suggested that additional residues from the central basic patch may contribute to VP35 dsRNA binding activity and IFN-antagonist function (30). However, a direct correlation between dsRNA and IFN inhibitory functions of VP35 with viral pathogenesis is currently lacking.In order to further define the molecular basis for VP35 dsRNA binding and IFN-antagonist function and to define the contribution of these functions to EBOV pathogenesis, an integrated molecular, structural, and virological approach was taken. The data presented below identify two VP35 carboxy-terminal basic amino acids, K319 and R322, as required for its dsRNA binding and IFN-antagonist functions. Interestingly, these residues are outside the region originally identified as being important for dsRNA binding and IFN inhibition (26). However, they lie within the central basic patch identified by prior structural studies (26, 30). Introduction of these mutations (VP35 with these mutations is designated KRA) into recombinant EBOV renders this otherwise fully lethal virus avirulent in guinea pigs. KRA-infected animals also develop EBOV-specific antibodies and become fully resistant to subsequent challenge with wild-type (WT) virus. Our data further reveal that the KRA EBOV is immunogenic and likely replicates to low levels early after infection in vivo. However, the mutant virus is subsequently cleared by host immune responses. These data demonstrate that the VP35 central basic patch is important not only for IFN-antagonist function but also for EBOV immune evasion and pathogenesis in vivo. High-resolution structural analysis, coupled with our in vitro and in vivo analyses of the recombinant Ebola viruses, provides the molecular basis for loss of function by the VP35 mutant and highlights the therapeutic potential of targeting the central basic patch with small-molecule inhibitors and for future vaccine development efforts.     

Tryptophan Residues in the Portal Protein of Herpes Simplex Virus 1 Critical to the Interaction with Scaffold Proteins and Incorporation of the Portal into Capsids

Incorporation of the herpes simplex virus 1 (HSV-1) portal vertex into the capsid requires interaction with a 12-amino-acid hydrophobic domain within capsid scaffold proteins. The goal of this work was to identify domains and residues in the U_L6-encoded portal protein pU_L6 critical to the interaction with scaffold proteins. We show that whereas the wild-type portal and scaffold proteins readily coimmunoprecipitated with one another in the absence of other viral proteins, truncation beyond the first 18 or last 36 amino acids of the portal protein precluded this coimmunoprecipitation. The coimmunoprecipitation was also precluded by mutation of conserved tryptophan (W) residues to alanine (A) at positions 27, 90, 127, 163, 241, 262, 532, and 596 of U_L6. All of these W-to-A mutations precluded the rescue of a viral deletion mutant lacking U_L6, except W163A, which supported replication poorly, and W596A, which fully rescued replication. A recombinant virus bearing the W596A mutation replicated and packaged DNA normally, and scaffold proteins readily coimmunoprecipitated with portal protein from lysates of infected cells. Thus, viral functions compensated for the W596A mutation''s detrimental effects on the portal-scaffold interaction seen during transient expression of portal and scaffold proteins. In contrast, the W27A mutation precluded portal-scaffold interactions in infected cell lysates, reduced the solubility of pU_L6, decreased incorporation of the portal into capsids, and abrogated viral-DNA cleavage and packaging.Immature herpesvirus capsids or procapsids consist of two shells: an inner shell, or scaffold, and an outer shell that is roughly spherical and largely composed of the major capsid protein VP5 (24, 38).The capsid scaffold consists of a mixture of the U_L26.5 and U_L26 gene products, with the U_L26.5 gene product (pU_L26.5, ICP35, or VP22a) being the most abundant (1, 12, 20, 21, 32, 38). The U_L26.5 open reading frame shares its coding frame and C terminus with the U_L26 gene but initiates at codon 307 of U_L26 (17). The extreme C termini of both VP22a and the UL26-encoded protein (pU_L26) interact with the N terminus of VP5 (7, 14, 26, 40, 41). Capsid assembly likely initiates when the portal binds VP5/VP22a and/or VP5/pU_L26 complexes (22, 25). The addition of more of these complexes to growing capsid shells eventually produces a closed sphere bearing a single portal. pU_L26 within the scaffold contains a protease that cleaves itself between amino acids 247 and 248, separating pU_L26 into an N-terminal protease domain called VP24 and a C-terminal domain termed VP21 (4, 5, 8, 9, 28, 42). The protease also cleaves 25 amino acids from pU_L26 and VP22a to release VP5 (5, 8, 9). VP21 and VP22a are replaced with DNA when the DNA is packaged (12, 29).When capsids undergo maturation, the outer protein shell angularizes to become icosahedral (13). One fivefold-symmetrical vertex in the angularized outer capsid shell is biochemically distinct from the other 11 and is called the portal vertex because it serves as the channel through which DNA is inserted as it is packaged (23). In herpes simplex virus (HSV), the portal vertex is composed of 12 copies of the portal protein encoded by U_L6 (2, 23, 39). We and others have shown that interactions between scaffold and portal proteins are critical for incorporation of the portal into the capsid (15, 33, 44, 45). Twelve amino acids of scaffold proteins are sufficient to interact with the portal protein, and tyrosine and proline resides within this domain are critical for the interaction with scaffold proteins and incorporation of the portal into capsids (45).One goal of the current study was to map domains and residues within the U_L6-encoded portal protein that mediate interaction with scaffold proteins. We show that the portal-scaffold interaction requires all but the first 18 and last 36 amino acids of pU_L6, as well as several tryptophan residues positioned throughout the portal protein.     

A Single Coxsackievirus B2 Capsid Residue Controls Cytolysis and Apoptosis in Rhabdomyosarcoma Cells

Coxsackievirus B2 (CVB2), one of six human pathogens of the group B coxsackieviruses within the enterovirus genus of Picornaviridae, causes a wide spectrum of human diseases ranging from mild upper respiratory illnesses to myocarditis and meningitis. The CVB2 prototype strain Ohio-1 (CVB2O) was originally isolated from a patient with summer grippe in the 1950s. Later on, CVB2O was adapted to cytolytic replication in rhabdomyosarcoma (RD) cells. Here, we present analyses of the correlation between the adaptive mutations of this RD variant and the cytolytic infection in RD cells. Using reverse genetics, we identified a single amino acid change within the exposed region of the VP1 protein (glutamine to lysine at position 164) as the determinant for the acquired cytolytic trait. Moreover, this cytolytic virus induced apoptosis, including caspase activation and DNA degradation, in RD cells. These findings contribute to our understanding of the host cell adaptation process of CVB2O and provide a valuable tool for further studies of virus-host interactions.Virus infections depend on complex interactions between viral and cellular proteins. Consequently, the nature of these interactions has important implications for viral cell type specificity, tissue tropism, and pathogenesis. Group B coxsackieviruses (CVB1 to CVB6), members of the genus Enterovirus within the family of Picornaviridae, are human pathogens that cause a broad spectrum of diseases, ranging from mild upper respiratory illnesses to more severe infections of the central nervous system, heart, and pancreas (61). These viruses have also been associated with certain chronic muscle diseases and myocardial infarction (2, 3, 12, 13, 22).The positive single-stranded RNA genome (approximately 7,500 nucleotides in length) of CVBs is encapsidated within a small T=1, icosahedral shell (30 nm in diameter) comprised of repeating identical subunits made up of four structural proteins (VP1 to VP4). Parts of VP1, VP2, and VP3 are exposed on the outer surface of the capsid, whereas VP4 is positioned on the interior. The virion morphology is characterized by a star-shaped mesa at each 5-fold icosahedral symmetry axis, surrounded by a narrow depression referred to as the “canyon” (69). All six serotypes of CVB can use the coxsackie and adenovirus receptor (CAR) for cell attachment and entry (9, 55, 82). Some strains of CVB1, -3, and -5 also use decay accelerating factor ([DAF] CD55) for initial attachment to the host cell; however, binding to DAF alone is insufficient to permit entry into the cell (10, 54, 76).Picornaviruses are generally characterized by their cytolytic nature in cell culture. However, several in vivo and in vitro studies have shown that some picornaviruses, e.g., poliovirus, Theiler''s murine encephalomyelitis virus, foot-and-mouth disease virus, CVB3, CVB4, and CVB5, may also establish persistent, noncytolytic infections (4, 29, 35, 39, 62, 74). Recently, it has been shown that the diverse outcomes of picornaviral infections may depend on interactions between the virus and the apoptotic machinery of the infected cell (14, 30, 71). Several picornaviral proteins have been identified as inducers of an apoptotic response, including viral capsid proteins VP1, VP2, and VP3, as well as nonstructural proteins 2A and 3C (7, 20, 32, 33, 42, 50, 63). In addition, antiapoptotic activity has been assigned to the nonstructural proteins 2B and 3A (16, 59).Picornaviruses have the potential to adapt rapidly to new host environments. Virus features affecting adaptability include high mutation rates, short replication times, large populations, and frequent incidences of recombination (25-27, 53). Consequently, picornaviruses exist as genetically heterogenous populations, referred to as viral quasispecies (25, 26).Previously, the CVB2 prototype strain Ohio-1 (CVB2O) was adapted to cytolytic replication in rhabdomyosarcoma (RD) cells (66). Two amino acid changes were identified in the capsid-coding region, and one was identified in the 2C-coding region of the adapted virus. Further characterization of the virus-host interaction showed that the infection was not affected by anti-DAF antibodies, indicating the use of an alternative receptor.In this study, the amino acid substitutions associated with the adaptation of CVB2O to cytolytic infection of RD cells were evaluated. Site-directed mutagenesis studies showed that a single amino acid change in the VP1 capsid protein was responsible for the cytolytic RD phenotype. In addition, as indicated by caspase activation and DNA degradation, the apoptotic pathway was activated in RD cells infected by the cytolytic virus.     

Characterization of a Putative Ancestor of Coxsackievirus B5

Like other RNA viruses, coxsackievirus B5 (CVB5) exists as circulating heterogeneous populations of genetic variants. In this study, we present the reconstruction and characterization of a probable ancestral virion of CVB5. Phylogenetic analyses based on capsid protein-encoding regions (the VP1 gene of 41 clinical isolates and the entire P1 region of eight clinical isolates) of CVB5 revealed two major cocirculating lineages. Ancestral capsid sequences were inferred from sequences of these contemporary CVB5 isolates by using maximum likelihood methods. By using Bayesian phylodynamic analysis, the inferred VP1 ancestral sequence dated back to 1854 (1807 to 1898). In order to study the properties of the putative ancestral capsid, the entire ancestral P1 sequence was synthesized de novo and inserted into the replicative backbone of an infectious CVB5 cDNA clone. Characterization of the recombinant virus in cell culture showed that fully functional infectious virus particles were assembled and that these viruses displayed properties similar to those of modern isolates in terms of receptor preferences, plaque phenotypes, growth characteristics, and cell tropism. This is the first report describing the resurrection and characterization of a picornavirus with a putative ancestral capsid. Our approach, including a phylogenetics-based reconstruction of viral predecessors, could serve as a starting point for experimental studies of viral evolution and might also provide an alternative strategy for the development of vaccines.The group B coxsackieviruses (CVBs) (serotypes 1 to 6) were discovered in the 1950s in a search for new poliovirus-like viruses (33, 61). Infections caused by CVBs are often asymptomatic but may occasionally result in severe diseases of the heart, pancreas, and central nervous system (99). CVBs are small icosahedral RNA viruses belonging to the Human enterovirus B (HEV-B) species within the family Picornaviridae (89). In the positive single-stranded RNA genome, the capsid proteins VP1 to VP4 are encoded within the P1 region, whereas the nonstructural proteins required for virus replication are encoded within the P2 and P3 regions (4). The 30-nm capsid has an icosahedral symmetry and consists of 60 copies of each of the four structural proteins. The VP1, VP2, and VP3 proteins are surface exposed, whereas the VP4 protein lines the interior of the virus capsid (82). The coxsackievirus and adenovirus receptor (CAR), a cell adhesion molecule of the immunoglobulin superfamily, serves as the major cell surface attachment molecule for all six serotypes of CVB (5, 6, 39, 60, 98). Some strains of CVB1, CVB3 and CVB5 also interact with the decay-accelerating factor (DAF) (CD55), a member of the family of proteins that regulate the complement cascade. However, the attachment of CVBs to DAF alone does not permit the infection of cells (6, 7, 59, 85).Picornaviruses exist as genetically highly diverse populations within their hosts, referred to as quasispecies (20, 57). This genetic plasticity enables these viruses to adapt rapidly to new environments, but at the same time, it may compromise the structural integrity and enzymatic functionality of the virus. The selective constraints imposed on the picornavirus genome are reflected in the different regions used for different types of evolutionary studies. The highly conserved RNA-dependent RNA polymerase (3D^pol) gene is used to establish phylogenetic relationships between more-distantly related viruses (e.g., viruses belonging to different genera) (38), whereas the variable genomic sequence encoding the VP1 protein is used for the classification of serotypes (13, 14, 69, 71, 72).In 1963, Pauling and Zuckerkandl proposed that comparative analyses of contemporary protein sequences can be used to predict the sequences of their ancient predecessors (73). Experimental reconstruction of ancestral character states has been applied to evolutionary studies of several different proteins, e.g., galectins (49), G protein-coupled receptors (52), alcohol dehydrogenases (95), rhodopsins (15), ribonucleases (46, 88, 110), elongation factors (32), steroid receptors (10, 96, 97), and transposons (1, 45, 87). In the field of virology, reconstructed ancestral or consensus protein sequences have been used in attempts to develop vaccine candidates for human immunodeficiency virus type 1 (21, 51, 66, 81) but rarely to examine general phenotypic properties.In this study, a CVB5 virus with a probable ancestral virion (CVB5-P1anc) was constructed and characterized. We first analyzed in detail the evolutionary relationships between structural genes of modern CVB5 isolates and inferred a time scale for their evolutionary history. An ancestral virion sequence was subsequently inferred by using a maximum likelihood (ML) method. This sequence was then synthesized de novo, cloned into a replicative backbone of an infectious CVB5 cDNA clone, and transfected into HeLa cells. The hypothetical CVB5-P1anc assembled into functional virus particles that displayed phenotypic properties similar to those of contemporary clinical isolates. This is the first report describing the reconstruction and characterization of a fully functional picornavirus with a putative ancestral capsid.     

A New Genetic Vaccine Platform Based on an Adeno-Associated Virus Isolated from a Rhesus Macaque

We created a hybrid adeno-associated virus (AAV) from two related rhesus macaque isolates, called AAVrh32.33, and evaluated it as a vaccine carrier for human immunodeficiency virus type 1 (HIV-1) and type A influenza virus antigens. The goal was to overcome the limitations of vaccines based on other AAVs, which generate dysfunctional T-cell responses and are inhibited by antibodies found in human sera. Injection of a Gag-expressing AAVrh32.33 vector into mice resulted in a high-quality CD8⁺ T-cell response. The resulting Gag-specific T cells express multiple cytokines at high levels, including interleukin-2, with many having memory phenotypes; a subsequent boost with an adenovirus vector yielded a brisk expansion of Gag-specific T cells. A priming dose of AAVrh32.33 led to high levels of Gag antibodies, which exceed levels found after injection of adenovirus vectors. Importantly, passive transfer of pooled human immunoglobulin into mice does not interfere with the efficacy of AAVrh32.33 expressing nucleoproteins from influenza virus, as measured by protection to a lethal dose of influenza virus, which is consistent with the very low seroprevalence to this virus in humans. Studies of macaques with vectors expressing gp140 from HIV-1 (i.e., with AAVrh32.33 as the prime and simian adenovirus type 24 as the boost) demonstrated results similar to those for mice with high-level and high-quality CD8⁺ T-cell responses to gp140 and high-titered neutralizing antibodies to homologous HIV-1. The biology of this novel AAV hybrid suggests that it should be a preferred genetic vaccine carrier, capable of generating robust T- and B-cell responses.The initial interest in vectors based on adeno-associated viruses (AAV) was for applications in gene therapy. Most of the initial work was with vectors derived from AAV serotype 2 (AAV2), which is one of the six initial isolates. In the first in vivo studies, several groups showed stable expression of the transgene Escherichia coli β-galactosidase following intramuscular (i.m.) injection of AAV2-LacZ without immune responses to the transgene (23, 44). The apparent tolerance of the host to AAV-encoded antigens to a variety of transgene products has been demonstrated in mice and some large animals (1, 35, 39). Several mechanisms have been proposed to explain the lack of T-cell responses following in vivo gene transfer with AAV, including ignorance (inadequate presentation of antigen), anergy, and suppression (1, 5, 18, 37).As applications of AAV vectors for in vivo gene transfer expanded, it became clear that the apparent immune privilege of AAV transgene products was not absolute. A number of examples emerged in which the host mounted vibrant T-cell responses to AAV-encoded transgene products. Several key parameters appeared to influence immunogenicity of the transgene. For example, Sarukhan et al. suggest that the subcellular localization of the protein influences the magnitude of the ensuing T-cell response after AAV gene transfer (37). The dose and route of administration of the AAV vector also contribute significantly to B- and T-cell responses to the transgene (3, 13). Wang et al. showed that inflammation at the site of AAV administration promotes antigen-specific immune responses to the transgene (47). A consistent observation has been that B-cell responses to AAV-encoded transgenes are much more intense and more consistently generated than CD8⁺ T-cell responses (8, 46, 51). A number of investigators have begun to explore AAV vectors as genetic vaccines against a variety of infectious and noninfectious diseases, based on the notion that it can be developed to stimulate transgene immune responses (14, 22, 26, 28, 48-50).The discovery of an expanded family of AAV capsids from human and nonhuman primates has provided an opportunity to evaluate the effects of capsid structure on vector performance. Most of this work has focused on the use of novel AAV serotypes for achieving higher levels of transgene expression for applications in gene therapy (7, 12, 36). Xin et al. recently evaluated, in mice, vectors as vaccines for human immunodeficiency virus type 1 (HIV-1) based on the original AAV isolates AAV1 to AAV6 and two novel AAVs we recently discovered, AAV7 and AAV8 (48). They showed significant capsid-dependent effects on T- and B-cell responses to HIV-1 gp160. We recently confirmed these observations and more thoroughly evaluated the quality of the CD8⁺ T-cell responses (26). AAV vectors of multiple serotypes encoding HIV-1 Gag were injected i.m. into mice, which all showed some level of CD8⁺ T-cell responses based on tetramer staining and peptide-induced gamma interferon (IFN-γ) expression. However, the quality of AAV-induced, Gag-specific T cells was substantially lower than that obtained with adenoviral vectors, based on several criteria. A majority of the tetramer-positive (Tet⁺) T cells were nonresponsive to antigen, and those that did respond to antigen produced low levels of IFN-γ and no interleukin-2 (IL-2). Very few memory T cells were generated, and animals primed with AAV vectors were not responsive to a boost with an adenoviral vector. However, all AAV serotypes studied did generate very high levels of antibodies to the Gag transgene product.A final issue to consider in the use of AAV as a genetic vaccine for HIV-1 is the presence of neutralizing antibodies (NAbs) to the vector due to prior AAV infections. We recently conducted an extensive screening of human populations from several continents and found high prevalence and high titers of NAbs to AAV1 and AAV2 and moderate levels of NAbs to AAV7 and -8 (4). In vivo gene transfer experiments indicate that AAV NAbs will likely impinge on vector efficacy (9, 33, 38).This study describes the creation of a novel AAV from rhesus macaque isolates, called AAVrh32.33, and its characterization as a genetic vaccine for HIV-1. AAVrh32.33 has properties unlike those of any others we have studied. We showed that vectors based on this novel capsid elicit strong CD8⁺ T-cell responses to reporter transgene products that are dependent on CD4⁺ T-cell help and dependent on signaling through CD40L and CD28 (L. E. Mays and J. M. Wilson, submitted for publication). Important to the use of this vector in the clinic is a very low incidence of NAbs to it in human populations. This study describes the development of vectors based on AAVrh32.33 as genetic vaccines.     

Effects of Major Capsid Proteins,Capsid Assembly,and DNA Cleavage/Packaging on the pUL17/pUL25 Complex of Herpes Simplex Virus 1

The U_L17 and U_L25 proteins (pU_L17 and pU_L25, respectively) of herpes simplex virus 1 are located at the external surface of capsids and are essential for DNA packaging and DNA retention in the capsid, respectively. The current studies were undertaken to determine whether DNA packaging or capsid assembly affected the pU_L17/pU_L25 interaction. We found that pU_L17 and pU_L25 coimmunoprecipitated from cells infected with wild-type virus, whereas the major capsid protein VP5 (encoded by the U_L19 gene) did not coimmunoprecipitate with these proteins under stringent conditions. In addition, pU_L17 (i) coimmunoprecipitated with pU_L25 in the absence of other viral proteins, (ii) coimmunoprecipitated with pU_L25 from lysates of infected cells in the presence or absence of VP5, (iii) did not coimmunoprecipitate efficiently with pU_L25 in the absence of the triplex protein VP23 (encoded by the U_L18 gene), (iv) required pU_L25 for proper solubilization and localization within the viral replication compartment, (v) was essential for the sole nuclear localization of pU_L25, and (vi) required capsid proteins VP5 and VP23 for nuclear localization and normal levels of immunoreactivity in an indirect immunofluorescence assay. Proper localization of pU_L25 in infected cell nuclei required pU_L17, pU_L32, and the major capsid proteins VP5 and VP23, but not the DNA packaging protein pU_L15. The data suggest that VP23 or triplexes augment the pU_L17/pU_L25 interaction and that VP23 and VP5 induce conformational changes in pU_L17 and pU_L25, exposing epitopes that are otherwise partially masked in infected cells. These conformational changes can occur in the absence of DNA packaging. The data indicate that the pU_L17/pU_L25 complex requires multiple viral proteins and functions for proper localization and biochemical behavior in the infected cell.Immature herpes simplex virus (HSV) capsids, like those of all herpesviruses, consist of two protein shells. The outer shell comprises 150 hexons, each composed of six copies of VP5, and 11 pentons, each containing five copies of VP5 (23, 29, 47). One vertex of fivefold symmetry is composed of 12 copies of the protein encoded by the U_L6 gene and serves as the portal through which DNA is inserted (22, 39). The pentons and hexons are linked together by 320 triplexes composed of two copies of the U_L18 gene product, VP23, and one copy of the U_L38 gene product, VP19C (23). Each triplex arrangement has two arms contacting neighboring VP5 subunits (47). The internal shell of the capsid consists primarily of more than 1,200 copies of the scaffold protein ICP35 (VP22a) and a smaller number of protease molecules encoded by the U_L26 open reading frame, which self-cleaves to form VP24 and VP21 derived from the amino and carboxyl termini, respectively (11, 12, 19, 25; reviewed in reference 31). The outer shell is virtually identical in the three capsid types found in HSV-infected cells, termed types A, B, and C (5, 6, 7, 29, 43, 48). It is believed that all three are derived from the immature procapsid (21, 38). Type C capsids contain DNA in place of the internal shell, type B capsids contain both shells, and type A capsids consist only of the outer shell (15, 16). Cleavage of viral DNA to produce type C capsids requires not only the portal protein, but all of the major capsid proteins and the products of the U_L15, U_L17, U_L28, U_L32, and U_L33 genes (2, 4, 10, 18, 26, 28, 35, 46). Only C capsids go on to become infectious virions (27).The outer capsid shell contains minor capsid proteins encoded by the U_L25 and U_L17 open reading frames (1, 17, 20). These proteins are located on the external surface of the viral capsid (24, 36, 44) and are believed to form a heterodimer arranged as a linear structure, termed the C capsid-specific complex (CCSC), located between pentons and hexons (41). This is consistent with the observation that levels of pU_L25 are increased in C capsids as opposed to in B capsids (30). On the other hand, other studies have indicated that at least some U_L17 and U_L25 proteins (pU_L17 and pU_L25, respectively) associate with all capsid types, and pU_L17 can associate with enveloped light particles, which lack capsid and capsid proteins but contain a number of viral tegument proteins (28, 36, 37). How the U_L17 and U_L25 proteins attach to capsids is not currently known, although the structure of the CCSC suggests extensive contact with triplexes (41). It is also unclear when pU_L17 and pU_L25 become incorporated into the capsid during the assembly pathway. Less pU_L25 associates with pU_L17(−) capsids, suggesting that the two proteins bind capsids either cooperatively or sequentially, although this could also be consequential to the fact that less pU_L25 associates with capsids lacking DNA (30, 36).Both pU_L25 and pU_L17 are necessary for proper nucleocapsid assembly, but their respective deletion generates different phenotypes. Deletion of pU_L17 precludes DNA packaging and induces capsid aggregation in the nuclei of infected cells, suggesting a critical early function (28, 34), whereas deletion of pU_L25 precludes correct cleavage or retention of full-length cleaved DNA within the capsid (8, 20, 32), thus suggesting a critical function later in the assembly pathway.The current studies were undertaken to determine how pU_L17 and pU_L25 associate with capsids by studying their interaction and localization in the presence and absence of other capsid proteins.