DNA Encapsidation Research Articles

Cis Effects in Adeno-Associated Virus Type 2 Replication

We have utilized deletion mutants of adeno-associated virus (AAV) to investigate which elements of the AAV genome are required in cis for high yields of the wild-type virus in a plasmid transfection assay and in addition whether these elements affect primarily AAV DNA replication or encapsidation. All tested deletions from within the Rep region demonstrated a modest, approximately threefold, decrease in viral production. Deletions within the cap region resulted in markedly less virus. Previous observations suggested that in cells in which recombinant AAV (rAAV) was produced, as in our assay with the helper plasmid pDG, there is a substantial excess of empty capsids. Co-transfections of high- and low-yielding constructs demonstrated that under conditions where Cap is abundant, the constructs with cap deletions did not package efficiently. These observation suggest that the lower yields of rAAV cannot be entirely due to lack of capsids but that elements within the cap region of the wild-type genome are important for efficient encapsidation. The production of virus by the mutants we tested was, however, not consistent with the disruption of a cis-acting packaging signal. Apparently, when Cap is provided "in trans," encapsidation is inefficient. A second observation is that there were equivalent amounts of replicated but unencapsidated viral DNA in cells transfected with each of our constructs. We propose that, in accord with the previously proposed link between DNA replication and encapsidation, the total amount of AAV DNA replication can be limited by the efficiency of encapsidation.

Hamster polyomavirus-derived virus-like particles are able to transfer in vitro encapsidated plasmid DNA to mammalian cells

The authentic major capsid protein 1 (VP1) of hamster polyomavirus (HaPyV) consists of 384 amino acid (aa) residues (42 kDa). Expression from an additional in-frame initiation codon located upstream from the authentic VP1 open reading frame (at position -4) might result in the synthesis of a 388 aa-long, amino-terminally extended VP1 (aa -4 to aa 384; VP1(ext)). In a plasmid-mediated Drosophila Schneider (S2) cell expression system, both VP1 derivatives as well as a VP1(ext) variant with an amino acid exchange of the authentic Met1Gly (VP1(ext-M1)) were expressed to a similar high level. Although all three proteins were detected in nuclear as well as cytoplasmic fractions, formation of virus-like particles (VLPs) was observed exclusively in the nucleus as confirmed by negative staining electron microscopy. The use of a tryptophan promoter-driven Escherichia coli expression system resulted in the efficient synthesis of VP1 and VP1(ext) and formation of VLPs. In addition, establishment of an in vitro disassembly/reassembly system allowed the encapsidation of plasmid DNA into VLPs. Encapsidated DNA was found to be protected against the action of DNase I. Mammalian COS-7 and CHO cells were transfected with HaPyV-VP1-VLPs carrying a plasmid encoding enhanced green fluorescent protein (eGFP). In both cell lines eGFP expression was detected indicating successful transfer of the plasmid into the cells, though at a still low level. Cesium chloride gradient centrifugation allowed the separation of VLPs with encapsidated DNA from "empty" VLPs, which might be useful for further optimization of transfection. Therefore, heterologously expressed HaPyV-VP1 may represent a promising alternative carrier for foreign DNA in gene transfer applications.

In vivo fitness and virulence of a drug-resistant herpes simplex virus 1 mutant

Two important issues regarding a virus mutant that is resistant to an antiviral drug are its ability to replicate in animal hosts (in vivo fitness) relative to other genetic variants, including wild type, and its ability to cause disease. These issues have been investigated for a herpes simplex virus 1 mutant that is resistant to thiourea compounds, which inhibit encapsidation of viral DNA. Following corneal inoculation of mice, the mutant virus replicated very similarly to its wild-type parent in the eye, trigeminal ganglion and brain. The mutant virus was as lethal to mice as its wild-type parent following this route of inoculation. Indeed, it exhibited increased virulence. Thus, unlike most drug-resistant virus mutants, this mutant retained in vivo fitness and virulence.

Mutations in the temperature‐sensitive murine cytomegalovirus (MCMV) mutants tsm5 and tsm30: A study of genes involved in immune evasion, DNA packaging and processing, and DNA replication

A murine cytomegalovirus (MCMV) temperature-sensitive (ts) mutant, tsm5, of the K181 (Birmingham) strain, showed approximately 10-fold and approximately 10,000-fold reductions in yields at the permissive (33 degrees C) and non-permissive temperature (40 degrees C), respectively. It did not replicate to detectable levels in any tissue of 1-week-old Balb/c mice for up to 21 days following i.p. inoculation with 4 x 10(3) pfu although it did replicate, albeit with considerably delayed kinetics, in SCID mice. tsm5 expressed all kinetic classes of transcript (immediate-early, early and late) both in vitro at the non-permissive temperature and in vivo. To identify mutations contributing to this phenotype, chimaeric viruses produced from overlapping cosmids generated from tsm5 and the Smith strain of MCMV were examined. A virus, Smith/tsm5DGIK, comprising the central conserved region of the tsm5 genome, was not attenuated at 33 or 37 degrees C but was ts at 40 degrees C, although not to the same extent as tsm5. In contrast to tsm5, this chimaeric virus replicated to similar levels as parental viruses in adult BALB/c mice. These results suggested that genes contributing to reduced replication at 33 degrees C and lack of replication in vivo are located at the ends of the tsm5 genome while those contributing to the ts phenotype are located in the central conserved region of the genome. Sequencing of some immune evasion genes known to be located at the 3' or 5' ends of the MCMV genome showed that no mutations were present in ORFs m04, m06, M33, M37, m38.5, m144, m152, or m157 although mutations were found in M27 (A658S) and M36Ex1 (V54I). tsm5 made few capsids at 40 degrees C and these lacked DNA. DNA synthesis was significantly reduced in tsm5-infected cells at 40 degrees C although DNA cleavage occurred with close to wt efficiency. Sequencing of the herpesvirus conserved cis-acting elements, pac1 and pac2, and genes involved in DNA packaging and cleavage located in the central core region of the genome identified few point mutations. Two were identified that alter the encoded protein in tsm5 ORFs M98 (P324S) and M56 (G439R). Furthermore, a point mutation (C890Y) was identified in M70, the primase. Another mutant, tsm30, which is also defective in DNA packaging and processing, has a point mutation in M52 (D494N). Thus, a number of mutations have been identified in tsm5 that suggests that it is defective in genes involved in immune evasion, DNA replication and DNA encapsidation.

Reconstitution of Herpes Simplex Virus Type 1 Nuclear Capsid Egress In Vitro

Newly assembled herpesvirus capsids travel from the nucleus to the plasma membrane by a mechanism that is poorly understood. Furthermore, the contribution of cellular proteins to this egress has yet to be clarified. To address these issues, an in vitro nuclear egress assay that reproduces the exit of herpes simplex virus type 1 (HSV-1) capsids from nuclei isolated from infected cells was established. As expected, the assay has all the hallmarks of intracellular transport assays, namely, a dependence on time, energy, and temperature. Surprisingly, it is also dependent on cytosol and was slightly enhanced by infected cytosol, suggesting an implication of both host and viral proteins in the process. The capsids escaped these nuclei by budding through the inner nuclear membrane, accumulated as enveloped capsids between the two nuclear membranes, and were released in cytosol exclusively as naked capsids, exactly as in intact cells. This is most consistent with the view that the virus escapes by crossing the two nuclear membranes rather than through nuclear pores. Unexpectedly, nuclei isolated at the nonpermissive temperature from cells infected with a U(L)26 thermosensitive protease mutant (V701) supported capsid egress. Although electron microscopy, biochemical, and PCR analyses hinted at a likely reconstitution of capsid maturation, DNA encapsidation could not be confirmed by a traditional SQ test. This assay should prove very useful for identification of the molecular players involved in HSV-1 nuclear egress.

The DNA Translocating ATPase of Bacteriophage T4 Packaging Motor

In double-stranded DNA bacteriophages the viral DNA is translocated into an empty prohead shell by a powerful ATP-driven motor assembled at the unique portal vertex. Terminases consisting of two to three packaging-related ATPase sites are central to the packaging mechanism. But the nature of the key translocating ATPase, stoichiometry of packaging motor, and basic mechanism of DNA encapsidation are poorly understood. A defined phage T4 packaging system consisting of only two components, proheads and large terminase protein (gp17; 70 kDa), is constructed. Using the large expanded prohead, this system packages any linear double-stranded DNA, including the 171 kb T4 DNA. The small terminase protein, gp16 (18 kDa), is not only not required but also strongly inhibitory. An ATPase activity is stimulated when proheads, gp17, and DNA are actively engaged in the DNA packaging mode. No packaging ATPase was stimulated by the N-terminal gp17-ATPase mutants, K166G (Walker A), D255E (Walker B), E256Q (catalytic carboxylate), D255E-E256D and D255E-E256Q (Walker B and catalytic carboxylate), nor could these sponsor DNA encapsidation. Experiments with the two gp17 domains, N-terminal ATPase domain and C-terminal nuclease domain, suggest that terminase association with the prohead portal and communication between the domains are essential for ATPase stimulation. These data for the first time established an energetic linkage between packaging stimulation of N-terminal ATPase and DNA translocation. A core pathway for the assembly of functional DNA translocating motor is proposed. Since the catalytic motifs of the N-terminal ATPase are highly conserved among > 200 large terminase sequences analyzed, these may represent common themes in phage and herpes viral DNA translocation.

Modulation of the Viral ATPase Activity by the Portal Protein Correlates with DNA Packaging Efficiency

DNA packaging in tailed bacteriophages and herpesviruses requires assembly of a complex molecular machine at a specific vertex of a preformed procapsid. As in all these viruses, the DNA translocation motor of bacteriophage SPP1 is composed of the portal protein (gp6) that provides a tunnel for DNA entry into the procapsid and of the viral ATPase (gp1-gp2 complex) that fuels DNA translocation. Here we studied the cross-talk between the components of the motor to control its ATP consumption and DNA encapsidation. We showed that gp6 embedded in the procapsid structure stimulated more than 10-fold the gp2 ATPase activity. This stimulation, which was significantly higher than the one conferred by isolated gp6, depended on the presence of gp1. Mutations in different regions of gp6 abolished or decreased the gp6-induced stimulation of the ATPase. This effect on gp2 activity was observed both in the presence and in the absence of DNA and showed a strict correlation with the efficiency of DNA packaging into procapsids containing the mutant portals. Our results demonstrated that the portal protein has an active control over the viral ATPase activity that correlates with the performance of the DNA packaging motor.

The L4 22-Kilodalton Protein Plays a Role in Packaging of the Adenovirus Genome

Packaging of the adenovirus (Ad) genome into a capsid is absolutely dependent upon the presence of a cis-acting region located at the left end of the genome referred to as the packaging domain. The functionally significant sequences within this domain consist of at least seven similar repeats, referred to as the A repeats, which have the consensus sequence 5' TTTG-N(8)-CG 3'. In vitro and in vivo binding studies have demonstrated that the adenovirus protein IVa2 binds to the CG motif of the packaging sequences. In conjunction with IVa2, another virus-specific protein binds to the TTTG motifs in vitro. The efficient formation of these protein-DNA complexes in vitro was precisely correlated with efficient packaging activity in vivo. We demonstrate that the binding activity to the TTTG packaging sequence motif is the product of the L4 22-kDa open reading frame. Previously, no function had been ascribed to this protein. Truncation of the L4 22-kDa protein in the context of the viral genome did not reduce viral gene expression or viral DNA replication but eliminated the production of infectious virus. We suggest that the L4 22-kDa protein, in conjunction with IVa2, plays a critical role in the recognition of the packaging domain of the Ad genome that leads to viral DNA encapsidation. The L4 22-kDa protein is also involved in recognition of transcription elements of the Ad major late promoter.

Herpes Simplex Virus Capsid Structure: DNA Packaging Protein UL25 Is Located on the External Surface of the Capsid near the Vertices

UL25 is one of seven herpes simplex virus-encoded proteins involved specifically in DNA encapsidation. Its role appears to be to stabilize the capsid so that DNA is prevented from escaping once it has entered. To clarify the function of UL25, we have examined capsids with the goal of defining where it is located. Analysis of trypsin-treated capsids showed that UL25 is sensitive to cleavage like other proteins such as the major capsid and portal proteins that are exposed on the capsid surface. Internal proteins such as the scaffolding protein and protease were not affected under the same experimental conditions. Capsids were also examined by electron microscopy after staining with gold-labeled antibody specific for UL25. Images of stained capsids demonstrated that most labeled sites (71% in C capsids) were at capsid vertices, and most stained C capsids had label at more than one vertex. A quantitative immunoblotting method showed that the capsid contents of UL25 were 56, 20, and 75 copies per capsid in A, B, and C capsids, respectively. Finally, soluble UL25 protein was found to bind in vitro to purified capsids lacking it. The amount of bound UL25 corresponded to the amount present in B capsids, and bound UL25 was found by immunoelectron microscopy to be located predominantly at the capsid vertices. The results are interpreted to suggest that five UL25 molecules are found at or near each of the capsid vertices, where they are exposed on the capsid surface. Exposure on the surface is consistent with the view that UL25 is added to the capsid as DNA is packaged or during late stages of the packaging process.

The Capsid-Associated UL25 Protein of the Alphaherpesvirus Pseudorabies Virus Is Nonessential for Cleavage and Encapsidation of Genomic DNA but Is Required for Nuclear Egress of Capsids

Homologs of the UL25 gene product of herpes simplex virus (HSV) have been identified in all three subfamilies of the Herpesviridae. However, their exact function during viral replication is not yet known. Whereas earlier studies indicated that the UL25 protein of HSV-1 is not required for cleavage of newly replicated viral DNA but is necessary for stable encapsidation (A. R. McNab, P. Desai, S. Person, L. Roof, D. R. Thompson, W. W. Newcomb, J. C. Brown, and F. L. Homa, J. Virol. 72:1060-1070, 1998), viral DNA packaging has recently been demonstrated to occur in the absence of UL25, although at significantly decreased levels compared to wild-type HSV-1 (N. Stow, J. Virol. 75:10755-10765 2001). To clarify the functional role of UL25 we analyzed the homologous protein of the alphaherpesvirus pseudorabies virus (PrV). PrV UL25 was found to be essential for viral replication, as a mutant virus lacking the UL25 protein required UL25-expressing cells for productive propagation. In the absence of the UL25 protein, newly replicated PrV DNA was cleaved and DNA-containing C-type capsids were detected in infected cell nuclei. However, although capsids were frequently found in close association with the inner nuclear membrane, nuclear egress was not observed. Consequently, no capsids were found in the cytoplasm, resulting in an inhibition of virion morphogenesis. In contrast, the formation of capsidless enveloped tegument structures (L particles) in the cytoplasm was readily observed. Thus, our data demonstrate that the PrV UL25 protein is not essential for cleavage and encapsidation of viral genomes, although both processes occur more efficiently in the presence of the protein. However, the presence of the PrV UL25 protein is a prerequisite for nuclear egress. By immunoelectron microscopy, we detected UL25-specific label on DNA-containing C capsids but not on other intranuclear immature or defective capsid forms. Thus, the PrV UL25 protein may represent the hitherto missing trigger that allows primary envelopment preferably of DNA-filled C capsids.

Separate Basic Region Motifs within the Adeno-Associated Virus Capsid Proteins Are Essential for Infectivity and Assembly

Adeno-associated virus (AAV) is gaining momentum as a gene therapy vector for human applications. However, there remain impediments to the development of this virus as a vector. One of these is the incomplete understanding of the biology of the virus, including nuclear targeting of the incoming virion during initial infection, as well as assembly of progeny virions from structural components in the nucleus. Toward this end, we have identified four basic regions (BR) on the AAV2 capsid that represent possible nuclear localization sequence (NLS) motifs. Mutagenesis of BR1 ((120)QAKKRVL(126)) and BR2 ((140)PGKKRPV(146)) had minor effects on viral infectivity ( approximately 4- and approximately 10-fold, respectively), whereas BR3 ((166)PARKRLN(172)) and BR4 ((307)RPKRLN(312)) were found to be essential for infectivity and virion assembly, respectively. Mutagenesis of BR3, which is located in Vp1 and Vp2 capsid proteins, does not interfere with viral production or trafficking of intact AAV capsids to the nuclear periphery but does inhibit transfer of encapsidated DNA into the nucleus. Substitution of the canine parvovirus NLS rescued the BR3 mutant to wild-type (wt) levels, supporting the role of an AAV NLS motif. In addition, rAAV2 containing a mutant form of BR3 in Vp1 and a wt BR3 in Vp2 was found to be infectious, suggesting that the function of BR3 is redundant between Vp1 and Vp2 and that Vp2 may play a role in infectivity. Mutagenesis of BR4 was found to inhibit virion assembly in the nucleus of transfected cells. This affect was not completely due to the inefficient nuclear import of capsid subunits based on Western blot analysis. In fact, aberrant capsid foci were observed in the cytoplasm of transfected cells, compared to the wild type, suggesting a defect in early viral assembly or trafficking. Using three-dimensional structural analysis, the lysine- and arginine-to-asparagine change disrupts hydrogen bonding between these basic residues and adjacent beta strand glutamine residues that may prevent assembly of intact virions. Taken together, these data support that the BR4 domain is essential for virion assembly. Each BR was also found to be conserved in serotypes 1 to 11, suggesting that these regions are significant and function similarly in each serotype. This study establishes the importance of two BR motifs on the AAV2 capsid that are essential for infectivity and virion assembly.

Core labeling of adenovirus with EGFP

The study of adenovirus could greatly benefit from diverse methods of virus detection. Recently, it has been demonstrated that carboxy-terminal EGFP fusions of adenovirus core proteins Mu, V, and VII properly localize to the nucleus and display novel function in the cell. Based on these observations, we hypothesized that the core proteins may serve as targets for labeling the adenovirus core with fluorescent proteins. To this end, we constructed various chimeric expression vectors with fusion core genes (Mu-EGFP, V-EGFP, preVII-EGFP, and matVII-EGFP) while maintaining expression of the native proteins. Expression of the fusion core proteins was suboptimal using E1 expression vectors with both conventional CMV and modified (with adenovirus tripartite leader sequence) CMV5 promoters, resulting in non-labeled viral particles. However, robust expression equivalent to the native protein was observed when the fusion genes were placed in the deleted E3 region. The efficient Ad-wt-E3-V-EGFP and Ad-wt-E3-preVII-EGFP expression vectors were labeled allowing visualization of purified virus and tracking of the viral core during early infection. The vectors maintained their viral function, including viral DNA replication, viral DNA encapsidation, cytopathic effect, and thermostability. Core labeling offers a means to track the adenovirus core in vector targeting studies as well as basic adenovirus virology.

The Endonuclease Domain of Bacteriophage Terminases Belongs to the Resolvase/Integrase/Ribonuclease H Superfamily: A BIOINFORMATICS ANALYSIS VALIDATED BY A FUNCTIONAL STUDY ON BACTERIOPHAGE T5

Bacteriophage terminases are essential molecular motors involved in the encapsidation of viral DNA. They are hetero-multimers whose large subunit encodes both ATPase and endonuclease activities. Although the ATPase domain is well characterized from sequence and functional analysis, the C-terminal region remains poorly defined. We describe sequence-structure comparisons of the endonuclease region of various bacteriophages that revealed new sequence similarities shared by this region and the Holliday junction resolvase RuvC and to a lesser extent the HIV integrase and the ribonuclease H. Extensive sequence comparison and motif refinement led to a common signature of terminases and resolvases with three conserved acidic residues engaged in catalytic activity. Sequence analyses were validated by in vivo and in vitro functional assays showing that the nuclease activity of the endonuclease domain of bacteriophage T5 terminase was abolished by mutation of any of the three predicted catalytic aspartates. Overall, these data suggest that the endonuclease domains of terminases operate autonomously and that they adopt a fold similar to that of resolvases and share the same divalent cation-dependent enzymatic mechanism.

Dynamic Monitoring of Oncolytic Adenovirus In Vivo by Genetic Capsid Labeling

Conditionally replicative adenoviruses represent a promising strategy to address the limited efficacy and safety issues associated with conventional cancer treatment. Despite rapid translation into human clinical trials and demonstrated safety, the fundamental properties of oncolytic adenovirus replication and spread and host-vector interactions in vivo have not been completely evaluated. We developed a noninvasive dynamic monitoring system to detect adenovirus replication. We constructed capsid-labeled E1/E3-deleted and wild-type adenoviruses (Ad-wt) by fusing the minor capsid protein IX with red fluorescent proteins mRFP1 and tdimer2(12), resulting in Ad-IX-mRFP1, Ad-IX-tdimer2(12), and Ad-wt-IX-mRFP1. Virus DNA replication, encapsidation, cytopathic effect, thermostability, and binding to primary receptor (coxsackie adenovirus receptor) were analyzed using real-time quantitative polymerase chain reaction, cell viability (MTS) assay, and fluorescence microscopy. Athymic mice (n = 4) carrying xenograft tumors that were derived from A549 lung adenocarcinoma cells were intratumorally inoculated with Ad-wt-IX-mRFP1, and adenovirus replication was dynamically monitored with a fluorescence noninvasive imaging system. Correlations between fluorescence signal intensity and viral DNA synthesis and replication were calculated using Pearson's correlation coefficient (r). The red fluorescence label had little effect on viral DNA replication, encapsidation, cytopathic effect, thermostability, and coxsackie adenovirus receptor binding. The fluorescent signal correlated with viral DNA synthesis and infectious progeny production both in vitro and in vivo (in A549 cells, r = .99 and r = .65; in tumors, r = .93 and r = .92, respectively). The replication efficiency of Ad-wt-IX-mRFP1 in vivo was variable, and replication and viral spreading and persistence were limited, consistent with clinical observations. Genetic capsid labeling provides a promising approach for the dynamic assessment of oncolytic adenovirus function in vivo.

1066. A Novel Class of Miniature Stabilized Double-Stranded AAV (msdsAAV) Vectors for the In Vivo Expression of Short Hairpin RNAs

Top of pageAbstract Double-stranded adeno-associated viral (dsAAV) vectors are currently emerging as a powerful tool for the in vitro and in vivo expression of short hairpin RNA (shRNA), to induce RNAi-mediated gene silencing. In these vectors, the shRNA expression cassette is typically flanked by a full-length AAV-2 ITR (inverted terminal repeat) and a mutant form devoid of the terminal resolution site (trs), blocking the AAV DNA replication cycle and creating the ds phenotype. However, two inherent drawbacks have prevented the clinical evaluation of dsAAVs thus far: 1) the near sequence identity of the mutant and intact ITRs promotes their frequent recombination, resulting in repair of the mutation or in partial ITR deletion, and thus loss of the ds phenotype; and 2) the usually small size of shRNA expression cassettes, and the presumed lower DNA packaging limit of AAV capsids, require the additional and undesirable encapsidation of stuffer DNA. Here, we present an improved dsAAV vector design which overcomes both limitations and thus represents a better candidate for clinical evaluation. The major advance was replacement of the mutant AAV-2 ITR with a trs-deleted AAV-4 repeat, sharing 80% on protein level). Notably, both double-stranded vectors (sds/msds) were significantly more potent than an identical dose of a conventional single-stranded AAV-8 expressing the same shRNA. In conclusion, msdsAAVs represent a novel class of powerful vectors for persistent and robust in vivo shRNA expression, which should be particularly interesting for clinical development due to the uniqueabsence of stuffer DNA sequences.

Impact of Capsid Conformation and Rep-Capsid Interactions on Adeno-Associated Virus Type 2 Genome Packaging

Single-stranded genomes of adeno-associated virus (AAV) are packaged into preformed capsids. It has been proposed that packaging is initiated by interaction of genome-bound Rep proteins to the capsid, thereby targeting the genome to the portal of encapsidation. Here we describe a panel of mutants with amino acid exchanges in the pores at the fivefold axes of symmetry on AAV2 capsids with reduced packaging and reduced Rep-capsid interaction. Mutation of two threonines at the rim of the fivefold pore nearly completely abolished Rep-capsid interaction and packaging. This suggests a Rep-binding site at the highly conserved amino acids at or close to the pores formed by the capsid protein pentamers. A different mutant (P. Wu, W. Xiao, T. Conlon, J. Hughes, M. Agbandje-McKenna, T. Ferkol, T. Flotte, and N. Muzyczka, J. Virol. 74:8635-8647, 2000) with an amino acid exchange at the interface of capsid protein pentamers led to a complete block of DNA encapsidation. Analysis of the capsid conformation of this mutant revealed that the pores at the fivefold axes were occupied by VP1/VP2 N termini, thereby preventing DNA introduction into the capsid. Nevertheless, the corresponding capsids had more Rep proteins bound than wild-type AAV, showing that correct Rep interaction with the capsid depends on a defined capsid conformation. Both mutant types together support the conclusion that the pores at the fivefold symmetry axes are involved in genome packaging and that capsid conformation-dependent Rep-capsid interactions play an essential role in the packaging process.

Liver Transduction with Recombinant Adeno-Associated Virus Is Primarily Restricted by Capsid Serotype Not Vector Genotype

We and others have recently reported highly efficient liver gene transfer with adeno-associated virus 8 (AAV-8) pseudotypes, i.e., AAV-2 genomes packaged into AAV-8 capsids. Here we studied whether liver transduction could be further enhanced by using viral DNA packaging sequences (inverted terminal repeats [ITRs]) derived from AAV genotypes other than 2. To this end, we generated two sets of vector constructs carrying expression cassettes embedding a gfp gene or the human factor IX (hfIX) gene flanked by ITRs from AAV genotypes 1 through 6. Initial in vitro analyses of gfp vector DNA replication, encapsidation, and cell transduction revealed a surprisingly high degree of interchangeability among the six genotypes. For subsequent in vivo studies, we cross-packaged the six hfIX variants into AAV-8 and infused mice via the portal vein with doses of 5 x 10(10) to 1.8 x 10(12) particles. Notably, all vectors expressed comparably high plasma hFIX levels within a dose cohort over the following 6 months, concurrent with the finding of equivalent vector DNA copy numbers per cell. Partial hepatectomies resulted in approximately 80% drops of hFIX levels and vector DNA copy numbers in all groups, indicating genotype-independent persistence of predominantly episomal vector DNA. Southern blot analyses of total liver DNA in fact confirmed the presence of identical and mostly nonintegrated molecular vector forms for all genotypes. We conclude that, unlike serotypes, AAV genotypes are not critical for efficient hepatocyte transduction and can be freely substituted. This corroborates our current model for AAV vector persistence in the liver and provides useful information for the future design and application of recombinant AAV.

Quantitation of adenovirus type 5 empty capsids

Adenovirus empty capsids are immature intermediates that lack DNA and viral core proteins. Highly purified preparations of empty and full capsids were generated by subjecting purified adenovirus preparations to repeated cesium chloride gradient separations. PAGE results revealed that empty capsids contain at least five bands that correspond to proteins absent from the mature virus proteome. Peptide mapping by matrix-assisted laser desorption/ionization time-of-flight MS revealed that three of these bands correspond to varying forms of L1 52/55 kDa, a protein involved in the encapsidation of the viral DNA. One band at around 31 kDa was found to include precursors to proteins VI and VIII. These precursors correspond to proteins that have not been cleaved by the adenovirus-encoded protease and are not present in the mature full capsids. The precursor to protein VIII (pVIII), a capsid cement protein, is used in this study as a marker in reverse-phased HPLC (RP-HPLC) analyses of adenovirus for the quantitation of empty capsids. A novel calculation method applied to the integration of RP-HPLC chromatograms allowed for the generation of a percentage empty capsid value in a given adenovirus preparation. The percentage empty capsid values generated to date by this method show a high degree of precision and good agreement with a cesium chloride gradient/SDS–PAGE quantitation method of empty capsids. The advantage of this method lies in the accurate, precise, and rapid generation of the percentage of empty capsids in a given purified virus preparation without relying on tedious and time-consuming cesium chloride gradient separations and extractions.

Herpes simplex virus 1 immediate-early and early gene expression during reactivation from latency under conditions that prevent infectious virus production.

The program of gene expression exhibited by herpes simplex virus during productive infection of cultured cells is well established; however, less is known about the regulatory controls governing reactivation from latency in neurons. One difficulty in examining gene regulation during reactivation lies in distinguishing between events occurring in initial reactivating cells versus events occurring in secondarily infected cells. Thus, two inhibitors were employed to block production of infectious virus: acyclovir, which inhibits viral DNA synthesis, and WAY-150138, which permits viral DNA synthesis but inhibits viral DNA encapsidation. Latently infected murine ganglia were explanted in the presence of either inhibitor, and then amounts of RNA, DNA, or infectious virus were quantified. In ganglia explanted for 48 h, the levels of five immediate-early and early RNAs did not exhibit meaningful differences between acyclovir and WAY-150138 treatments when analyzed by in situ hybridization or quantitative reverse transcription-PCR. However, comparative increases in viral DNA and RNA content in untreated ganglia suggested that virus was produced before 48 h postexplant. This was confirmed by the detection of infectious virus as early as 14 h postexplant. Together, these results suggest that high levels of viral gene expression at 48 h postexplant are due largely to the production of infectious virus and subsequent spread through the tissue. These results lead to a reinterpretation of previous results indicating a role for DNA replication in immediate-early and early viral gene expression; however, it remains possible that viral gene expression is regulated differently in neurons than in cultured cells.

Parvovirus uncoating in vitro reveals a mechanism of DNA release without capsid disassembly and striking differences in encapsidated DNA stability

The uncoating mechanism of parvoviruses is unknown. Their capsid robustness and increasing experimental data would suggest an uncoating mechanism without capsid disassembly. We have developed an in vitro system to detect and quantify viral DNA externalization and applied the assay on two parvoviruses with important differences in capsid structure, human B19 and minute virus of mice (MVM). Upon briefly treating the capsids to increasing temperatures, the viral genome became accessible in its full-length in a growing proportion of virions. Capsid disassembly started at temperatures above 60 °C for B19 and 70 °C for MVM. For both viruses, the externalization followed an all-or-nothing mechanism, without transitions exposing only a particular genomic region. However, the heat-induced DNA accessibility was remarkably more pronounced in B19 than in MVM. This difference was also evident under conditions mimicking endosomal acidification (pH 6.5 to 5), which triggered the externalization of B19-DNA but not of MVM-DNA. The externalized ssDNA was a suitable template for the full second-strand synthesis. Immunoprecipitation with antibodies against conformational epitopes and quantitative PCR revealed that the DNA externalized by heat was mostly dissociated from its capsid, however, the low pH-induced DNA externalization of B19 was predominantly capsid-associated. These results provide new insights into parvovirus uncoating suggesting a mechanism by which the full-length viral genome is released without capsid disassembly. The remarkable instability of the encapsidated B19 DNA, which is easily released from its capsid, would also explain the faster heat inactivation of B19 when compared to other parvoviruses.