Nuclear Transport of the Major Capsid Protein Is Essential for Adeno-Associated Virus Capsid Formation

Adeno-associated virus capsids are composed of three proteins, VP1, VP2, and VP3. Although VP1 is necessary for viral infection, it is not essential for capsid formation. The other capsid proteins, VP2 and VP3, are sufficient for capsid formation, but the functional roles of each protein are still not well understood. By analyzing a series of deletion mutants of VP2, we identified a region necessary for nuclear transfer of VP2 and found that the efficiency of nuclear localization of the capsid proteins and the efficiency of virus-like particle (VLP) formation correlated well. To confirm the importance of the nuclear localization of the capsid proteins, we fused the nuclear localization signal of simian virus 40 large T antigen to VP3 protein. We show that this fusion protein could form VLP, indicating that the VP2-specific region located on the N-terminal side of the protein is not structurally required. This finding suggests that VP3 has sufficient information for VLP formation and that VP2 is necessary only for nuclear transfer of the capsid proteins.     

Evidence for pH-Dependent Protease Activity in the Adeno-Associated Virus Capsid

Incubation of highly purified adeno-associated virus (AAV) capsids in vitro at pH 5.5 induced significant autocleavage of capsid proteins at several amino acid positions. No autocleavage was seen at pH 7.5. Examination of other AAV serotypes showed at least two different pH-induced cleavage patterns, suggesting that different serotypes have evolved alternative protease cleavage sites. In contrast, incubation of AAV serotypes with an external protease substrate showed that purified AAV capsid preparations have robust protease activity at neutral pH but not at pH 5.5, opposite to what is seen with capsid protein autocleavage. Several lines of evidence suggested that protease activity is inherent in AAV capsids and is not due to contaminating proteins. Control virus preparations showed no protease activity on external substrates, and filtrates of AAV virus preparations also showed no protease activity contaminating the capsids. Further, N-terminal Edman sequencing identified unique autocleavage sites in AAV1 and AAV9, and mutagenesis of amino acids adjacent to these sites eliminated cleavage. Finally, mutation of an amino acid in AAV2 (E563A) that is in a conserved pH-sensitive structural region eliminated protease activity on an external substrate but did not seem to affect autocleavage. Taken together, our data suggested that AAV capsids have one or more protease active sites that are sensitive to pH induction. Further, it appears that acidic pHs comparable to those seen in late endosomes induce a structural change in the capsid that induces autolytic protease activity. The pH-dependent protease activity may have a role in viral infection.     

Capsid Antibodies to Different Adeno-Associated Virus Serotypes Bind Common Regions

Interactions between viruses and the host antibody immune response are critical in the development and control of disease, and antibodies are also known to interfere with the efficacy of viral vector-based gene delivery. The adeno-associated viruses (AAVs) being developed as vectors for corrective human gene delivery have shown promise in clinical trials, but preexisting antibodies are detrimental to successful outcomes. However, the antigenic epitopes on AAV capsids remain poorly characterized. Cryo-electron microscopy and three-dimensional image reconstruction were used to define the locations of epitopes to which monoclonal fragment antibodies (Fabs) against AAV1, AAV2, AAV5, and AAV6 bind. Pseudoatomic modeling showed that, in each serotype, Fabs bound to a limited number of sites near the protrusions surrounding the 3-fold axes of the T=1 icosahedral capsids. For the closely related AAV1 and AAV6, a common Fab exhibited substoichiometric binding, with one Fab bound, on average, between two of the three protrusions as a consequence of steric crowding. The other AAV Fabs saturated the capsid and bound to the walls of all 60 protrusions, with the footprint for the AAV5 antibody extending toward the 5-fold axis. The angle of incidence for each bound Fab on the AAVs varied and resulted in significant differences in how much of each viral capsid surface was occluded beyond the Fab footprints. The AAV-antibody interactions showed a common set of footprints that overlapped some known receptor-binding sites and transduction determinants, thus suggesting potential mechanisms for virus neutralization by the antibodies.     

Biophysical and Ultrastructural Characterization of Adeno-Associated Virus Capsid Uncoating and Genome Release

We describe biophysical and ultrastructural differences in genome release from adeno-associated virus (AAV) capsids packaging wild-type DNA, recombinant single-stranded DNA (ssDNA), or dimeric, self-complementary DNA (scDNA) genomes. Atomic force microscopy and electron microscopy (EM) revealed that AAV particles release packaged genomes and undergo marked changes in capsid morphology upon heating in physiological buffer (pH 7.2). When different AAV capsids packaging ss/scDNA varying in length from 72 to 123% of wild-type DNA (3.4 to 5.8 kb) were incrementally heated, the proportion of uncoated AAV capsids decreased with genome length as observed by EM. Genome release was further characterized by a fluorimetric assay, which demonstrated that acidic pH and high osmotic pressure suppress genome release from AAV particles. In addition, fluorimetric analysis corroborated an inverse correlation between packaged genome length and the temperature needed to induce uncoating. Surprisingly, scAAV vectors required significantly higher temperatures to uncoat than their ssDNA-packaging counterparts. However, externalization of VP1 N termini appears to be unaffected by packaged genome length or self-complementarity. Further analysis by tungsten-shadowing EM revealed striking differences in the morphologies of ssDNA and scDNA genomes upon release from intact capsids. Computational modeling and molecular dynamics simulations suggest that the unusual thermal stability of scAAV vectors might arise from partial base pairing and optimal organization of packaged scDNA. Our work further defines the biophysical mechanisms underlying adeno-associated virus uncoating and genome release.     

Capsid Mutated Adeno-Associated Virus Delivered to the Anterior Chamber Results in Efficient Transduction of Trabecular Meshwork in Mouse and Rat

Background

Adeno associated virus (AAV) is well known for its ability to deliver transgenes to retina and to mediate improvements in animal models and patients with inherited retinal disease. Although the field is less advanced, there is growing interest in AAV’s ability to target cells of the anterior segment. The purpose of our study was to fully articulate a reliable and reproducible method for injecting the anterior chamber (AC) of mice and rats and to investigate the transduction profiles of AAV2- and AAV8-based capsid mutants containing self-complementary (sc) genomes in the anterior segment of the eye.

Methodology/Principle Findings

AC injections were performed in C57BL/6 mice and Sprague Dawley rats. The cornea was punctured anterior of the iridocorneal angle. To seal the puncture site and to prevent reflux an air bubble was created in the AC. scAAVs expressing GFP were injected and transduction was evaluated by immunohistochemistry. Both parent serotype and capsid modifications affected expression. scAAV2- based vectors mediated efficient GFP-signal in the corneal endothelium, ciliary non-pigmented epithelium (NPE), iris and chamber angle including trabecular meshwork, with scAAV2(Y444F) and scAAV2(triple) being the most efficient.

Conclusions/Significance

This is the first study to semi quantitatively evaluate transduction of anterior segment tissues following injection of capsid-mutated AAV vectors. scAAV2- based vectors transduced corneal endothelium, ciliary NPE, iris and trabecular meshwork more effectively than scAAV8-based vectors. Mutagenesis of surface-exposed tyrosine residues greatly enhanced transduction efficiency of scAAV2 in these tissues. The number of Y-F mutations was not directly proportional to transduction efficiency, however, suggesting that proteosomal avoidance alone may not be sufficient. These results are applicable to the development of targeted, gene-based strategies to investigate pathological processes of the anterior segment and may be applied toward the development of gene-based therapies for glaucoma and acquired or inherited corneal anomalies.     

一类考虑CTL免疫反应的病毒动力学模型的定性分析

在本文中,主要研究了一类具有CTL免疫反应的病毒动力学的数学模型,对其具有一般形式疾病发生率的模型进行了讨论,通过构造Lyapunov函数,给出了平衡点处的全局稳定性分析.当R₀≤1时,病毒在体内清除;当R₀>1,如果R₁<1,表示病毒最终生存而CTL免疫细胞最终消失;如果R₁>1,病毒和CTL免疫细胞会持续生存.     

Heavy and Light Particles of Adeno-Associated Virus

KB cells coinfected with adenovirus and adeno-associated virus (AAV) yielded two kinds of infectious AAV particles that banded in CsCl at densities of 1.45 and 1.41 g/cm², respectively. The 1.45 band was found to be composed of a heterogeneous group of viral particles that could be subfractionated by velocity sedimentation. The main component from this band had a smaller S value (109) than the main component from the 1.41 band (111S), although both had the same DNA/protein ratio and the same density in metrizamide gradients. Continuous-label experiments showed that early after infection, both components (1.45 and 1.41) were generated in the same amounts, but this was followed by a relative increase in the proportion of the 1.41 component over the 1.45 particles. Pulse-chase analysis failed to demonstrate a precursor-product relationship between these two bands. The slower-sedimenting components from the 1.45 band were unstable in CsCl and were present in a greater proportion early after infection. These particles contained DNA that was enriched for the terminal sequences of the AAV genomes and was accessible to digestion with micrococcal nuclease.     

重组腺相关病毒生产方法研究进展

重组腺相关病毒(rAAV)作为基因治疗的载体,具有感染范围广、能在宿主细胞中长久稳定表达外源基因和非致病性等优点,因此在基因治疗领域倍受青睐。rAAV的大规模制备技术一直是限制其临床广泛应用的瓶颈。近年来,有许多改进rAAV制备工艺的尝试。我们在介绍rAAV载体制备所需各种元件的基础上,对这些方法进行了简要综述。     

Structural Analysis of Sindbis Virus Capsid Mutants Involving Assembly and Catalysis

Sindbis virus core protein (SCP) has been isolated from virus and crystallized. The X-ray crystallographic structure showed that the amino-terminal 113 residues appeared to be either disordered or truncated during crystallization and that the carboxy-terminal residues 114 to 264 had a chymotrypsin-like structure. The carboxy-terminal residues 106 to 264 and 106 to 266 of SCP have now been expressed inEscherichia coli. Most crystal forms of the truncated proteins were isomorphous with those of the virally extracted protein. There are only small structural differences between the truncated recombinant protein and the ordered part of the wild-type virus-extracted protein. Hence,E. coli-expressed SCP can be used to study proteolytic properties and the contribution of SCP to nucleocapsid assembly, interaction with the E2 glycoprotein and interaction with RNA.The same dimer that was found in two different crystal forms of the virus-extracted SCP was present also in some of the crystals of the truncated recombinant protein. The monomer – monomer interface is maintained by two pairs of hydrogen bonds and by hydrophobic interactions. Removal of the hydrogen bonds by single substitutions did not prevent dimer formation. However, a mutation that reduced the hydrophobic contacts did inhibit dimer formation.The wild-type truncated SCP is active inE. coli, as evidenced by proteolytic processing of a series of progressively longer precursors that extend beyond residue 264. Unlike the virus-extracted capsid protein, theE. coli-expressed SCP described here is terminated following the carboxy-terminal residue and, therefore, does not require autocatalysis. Nevertheless, theE. coli-expressed protein folds with the carboxy-terminal tryptophan residue in the specificity pocket. Two crystallographically independent molecules of SCP(106 to 266), which had two additional downstream residues and had the essential S215 mutated to alanine, showed two distinct modes of binding the uncleaved carboxy-terminal residues. These may represent successive steps of binding substrate prior to catalytic cleavage.Refinement of the various crystal structures of SCP showed that the amino-terminal arm from residues 107 to 113 was not disordered, but is associated with neighboring molecules. Residues 108 to 111 bind into a hydrophobic pocket composed primarily of Y180, W247 and F166. It had been shown that the double mutant (Y180S; E183G), with the Y180S substitution in this pocket, produced a large number of non-infectious virions, possibly because of modification in the interaction of the glycoprotein spikes with core proteins. The crystal structure of this double mutant showed that there was a large positional change in the side-chain of W247, which moved into the space created by the replacement of Y180 with serine. These conformational changes may alter the stability of the virion and, thus, regulate its functional requirements during cell entry.     

Cloning and Characterization of Adeno-Associated Virus Type 5

Adeno-associated virus type 5 (AAV5) is distinct from other dependovirus serotypes based on DNA hybridization and serological data. To better understand the biology of AAV5, we have cloned and sequenced its genome and generated recombinant AAV5 particles. The single-stranded DNA genome is similar in length and genetic organization to that of AAV2. The rep gene of AAV5 is 67% homologous to AAV2, with the majority of the changes occurring in the carboxyl and amino termini. This homology is much less than that observed with other reported AAV serotypes. The inverted terminal repeats (ITRs) are also unique compared to those of the other AAV serotypes. While the characteristic AAV hairpin structure and the Rep DNA binding site are retained, the consensus terminal resolution site is absent. These differences in the Rep proteins and the ITRs result in a lack of cross-complementation between AAV2 and AAV5 as measured by the production of recombinant AAV particles. Alignment of the cap open reading frame with that of the other AAV serotypes identifies both conserved and variable regions which could affect tissue tropism and particle stability. Comparison of transduction efficiencies in a variety of cells lines and a lack of inhibition by soluble heparin indicate that AAV5 may utilize a distinct mechanism of uptake compared to AAV2.     

Properties of the Adeno-Associated Virus Assembly-Activating Protein

Adeno-associated virus (AAV) capsid assembly requires expression of the assembly-activating protein (AAP) together with capsid proteins VP1, VP2, and VP3. AAP is encoded by an alternative open reading frame of the cap gene. Sequence analysis and site-directed mutagenesis revealed that AAP contains two hydrophobic domains in the N-terminal part of the molecule that are essential for its assembly-promoting activity. Mutation of these sequences reduced the interaction of AAP with the capsid proteins. Deletions and a point mutation in the capsid protein C terminus also abolished capsid assembly and strongly reduced the interaction with AAP. Interpretation of these observations on a structural basis suggests an interaction of AAP with the VP C terminus, which forms the capsid protein interface at the 2-fold symmetry axis. This interpretation is supported by a decrease in the interaction of monoclonal antibody B1 with VP3 under nondenaturing conditions in the presence of AAP, indicative of steric hindrance of B1 binding to its C-terminal epitope by AAP. In addition, AAP forms high-molecular-weight oligomers and changes the conformation of nonassembled VP molecules as detected by conformation-sensitive monoclonal antibodies A20 and C37. Combined, these observations suggest a possible scaffolding activity of AAP in the AAV capsid assembly reaction.     

In Vitro Packaging of Adeno-Associated Virus DNA

We have developed an in vitro procedure for packaging of recombinant adeno-associated virus (AAV). By using AAV replicative-form DNA as the substrate, it is possible to synthesize an infectious AAV particle in vitro that can be used to transfer a marker gene to mammalian cells. The packaging procedure requires the presence of both the AAV Rep and capsid proteins. Two kinds of in vitro products can be formed which facilitate DNA transfer. Both are resistant to heat and have a density in cesium chloride gradients that is indistinguishable from that of the in vivo-synthesized wild-type virus. This indicates that the particles formed have the appropriate protein-to-DNA ratio and a structure that shares the heat resistance of mature AAV particles. The two types of particles can be distinguished by their sensitivity to chloroform and DNase I treatment. The chloroform-resistant product is, by several criteria, an authentic AAV particle. In addition to having the correct density and being resistant to treatment with chloroform, DNase I, and heat, this particle is efficiently synthesized only if the AAV genome contains intact terminal repeats, which are known to be required for AAV packaging. It is also precipitated by a monoclonal antibody that recognizes mature virus particles but not bound by an antibody that recognizes monomeric or denatured capsid proteins. The chloroform-resistant species is not made when aphidicolin is present in the reaction mixture, suggesting that active DNA replication is required for in vitro packaging. In contrast, the chloroform-sensitive product has several features that suggest it is an incompletely assembled virus particle. It is sensitive to DNase I, does not require the presence of AAV terminal repeats, and is capable of transferring DNA that is theoretically too large to package. Sucrose gradient centrifugation of the in vitro-synthesized products reveals that the particles have sedimentation values between 60S and 110S, which is consistent with partially assembled and mature AAV particles. The in vitro packaging procedure should be useful for studying the mechanism by which a human icosahedral DNA virus particle is assembled, and it may be useful for producing recombinant AAV for gene therapy. The chloroform-sensitive particle may also be useful for transferring DNA that is too large to be packaged in mature recombinant AAV.     

Testing an Electrostatic Interaction Hypothesis of Hepatitis B Virus Capsid Stability by Using an In Vitro Capsid Disassembly/Reassembly System

To test a previously coined “charge balance hypothesis” of human hepatitis B virus (HBV) capsid stability, we established an in vitro disassembly and reassembly system using bacterially expressed HBV capsids. Capsid disassembly can be induced by micrococcal nuclease digestion of encapsidated RNA. HBV core protein (HBc) mutants containing various amounts of arginine were constructed by serial truncations at the C terminus. Capsids containing smaller amounts of arginine (HBc 149, 154, and 157) remained intact after micrococcal nuclease digestion by native gel electrophoresis. Capsids containing larger amounts of arginine (HBc 159, 164, 169, and 171) exhibited reduced and more diffuse banding intensity and slightly upshifted mobility (HBc 159 and 164). Capsids containing the largest amounts of arginine (HBc 173, 175, and 183), as well as HBc 167, exhibited no detectable banding signal, indicating loss of capsid integrity or stability. Interestingly, capsid reassembly can be induced by polyanions, including oligonucleotides, poly-glutamic acid, and nonbiological polymer (polyacrylic acid). In contrast, polycations (polylysine and polyethylenimine) and low-molecular-weight anions (inositol triphosphate) induced no capsid reassembly. Results obtained by gel assay were confirmed by electron microscopy. Reassembled capsids comigrated with undigested parental capsids on agarose gels and cosedimented with undigested capsids by sucrose gradient ultracentrifugation. Taken together, the results indicate that HBV capsid assembly and integrity depend on polyanions, which probably can help minimize intersubunit charge repulsion caused mainly by arginine-rich domain III or IV in close contact. The exact structure of polyanions is not important for in vitro capsid reassembly. A large amount of independent experimental evidence for this newly coined “electrostatic interaction hypothesis” is discussed.Chronic infection with hepatitis B virus (HBV) leads to the development of cirrhosis and hepatocellular carcinoma (6, 31, 36). HBV core protein (HBc) consists of the assembly domain (HBc amino acids 1 to 149) at the N terminus and the arginine-rich domain (ARD) at the C terminus (HBc amino acids 150 to 183) (33, 34). Escherichia coli-expressed HBc can spontaneously self-assemble into 28-nm capsid particles with a spherical appearance indistinguishable from that of human liver-derived capsid particles (7). Such capsid particles have been shown to package RNAs transcribed in E. coli (5, 8, 11, 28, 37). The four-helix bundle structure of HBV capsid particles is based on cryo-electron microscopy and X-ray crystallography using C-terminally truncated HBc (34, 38). At present, there is no known structure at the C terminus of HBc capsids (34, 41). HBc amino acids 150 to 183 contains four stretches of clustering arginine residues (ARD-I, -II, -III, and -IV) (Fig. ​(Fig.1).1). When the C-terminal domain of hepadnaviral core protein was serially truncated, a viral replication defect was observed (4, 19, 22, 27, 39). To date, it remains to be elucidated why the C terminus is so important for diverse biological activities, including RNA encapsidation and DNA replication. To address this issue, we proposed previously a so-called “charge balance hypothesis” (22), which highlights the importance of adequate electrostatic interactions between positive charge (basic residues) from HBc and negative charge from encapsidated RNA or DNA.Open in a separate windowFIG. 1.Effects of micrococcal nuclease treatment on HBV nucleocapsids with serially truncated C termini of core proteins. A series of HBV core expression vectors with different lengths and arginine contents were constructed in pET-Blue-1. The truncations are illustrated in the top panel, and the four ARDs (ARD-I, -II, -III, and -IV) are underlined. All constructs self-assembled into capsids when expressed in E. coli. The capsids run as a distinct band on 1% native agarose gels and can be stained with EtBr (upper panel) and Coomassie blue (middle panel). Untreated controls without micrococcal nuclease were incubated with the same buffer and conditions as for micrococcal nuclease-digested samples. In the upper panel, note the loss of the EtBr signal when the encapsidated RNAs were digested by micrococcal nuclease. In the middle panel, we observed three different groups of mutants with three different CBS patterns. Group 1 mutants, including HBc mutants 149, 154, and 157, exhibited no significant change in CBS banding pattern before or after micrococcal nuclease digestion. In group 2 capsids 159, 164, 169, and 171 (#), the CBS banding pattern became more diffuse, less intense, and slightly (yet reproducibly) upshifted. * indicates the near-complete loss of CBS banding in group 3 capsids 167, 173, 175, and 183. To confirm that this loss of CBS probably results from a loss of structural integrity of the capsid particle, rather than from a loss of the core protein per se, aliquots of samples were also run on denaturing SDS-PAGE (bottom panel).The first clue that such an electrostatic interaction could play an important role in RNA encapsidation is from the study of an engineered mutant, HBc 164. Despite its reduced arginine content relative to the wild-type (WT) full-length HBc 183 (Fig. ​(Fig.1),1), HBc mutant 164 can encapsidate, as efficiently as WT HBV, both 3.5-kb pregenomic RNA (pgRNA) and a spliced 2.2-kb subgenomic RNA (sgRNA) (19, 22). When WT HBV nucleocapsids (capsids) were treated with micrococcal nuclease, the encapsidated 3.5-kb pgRNA and its reverse-transcribing RNA template were resistant to micrococcal nuclease treatment. In contrast, when mutant 164 capsids were treated with micrococcal nuclease, the encapsidated 3.5-kb RNA was highly nuclease sensitive, while the encapsidated 2.2-kb sgRNA appeared to be nuclease resistant (19, 22).To further elucidate the mechanism behind this phenomenon, we hypothesized that this result could be due to an abnormal or less stable capsid structure generated by a charge imbalance (insufficient positive charge or excessive negative charge) when arginine-deficient mutant 164 encapsidates the full-length 3.5-kb pgRNA. In contrast, a more normal or stable capsid structure can be generated when arginine-deficient mutant 164 encapsidates the 2.2-kb sgRNA (with a reduced negative charge content relative to the 3.5-kb pgRNA) (22). In addition to RNA encapsidation and capsid stability, electrostatic interaction could also play a role in HBV DNA synthesis and genome maturation. For example, when the truncated C terminus of HBc 164 was progressively restored, the core-associated viral DNA gradually increased in both size and signal intensity (22).In this study, we designed an in vitro capsid disassembly/reassembly experiment which is complementary to the in vivo approach in tissue culture (22; P. K. Chua, F. M. Tang, J. Y. Huang, C. S. Suen, and C. Shih, submitted for publication). We investigated the relationship between the arginine content of HBV capsids and the encapsidated nucleic acid in maintaining capsid stability in vitro. In addition, we demonstrated that HBV capsids that are disassembled by depletion of encapsidated RNA can be efficiently reassembled in the presence of exogenous nucleic acid and non-nucleic acid polyanions but not in the presence of polycations or low-molecular-weight (low-MW) anions. Capsid disassembly induced by RNA digestion appears to be related to intersubunit charge repulsion between ARD-III or ARD-IV in close contact.     

Structural Insights into Adeno-Associated Virus Serotype 5

The adeno-associated viruses (AAVs) display differential cell binding, transduction, and antigenic characteristics specified by their capsid viral protein (VP) composition. Toward structure-function annotation, the crystal structure of AAV5, one of the most sequence diverse AAV serotypes, was determined to 3.45-Å resolution. The AAV5 VP and capsid conserve topological features previously described for other AAVs but uniquely differ in the surface-exposed HI loop between βH and βI of the core β-barrel motif and have pronounced conformational differences in two of the AAV surface variable regions (VRs), VR-IV and VR-VII. The HI loop is structurally conserved in other AAVs despite amino acid differences but is smaller in AAV5 due to an amino acid deletion. This HI loop is adjacent to VR-VII, which is largest in AAV5. The VR-IV, which forms the larger outermost finger-like loop contributing to the protrusions surrounding the icosahedral 3-fold axes of the AAVs, is shorter in AAV5, creating a smoother capsid surface topology. The HI loop plays a role in AAV capsid assembly and genome packaging, and VR-IV and VR-VII are associated with transduction and antigenic differences, respectively, between the AAVs. A comparison of interior capsid surface charge and volume of AAV5 to AAV2 and AAV4 showed a higher propensity of acidic residues but similar volumes, consistent with comparable DNA packaging capacities. This structure provided a three-dimensional (3D) template for functional annotation of the AAV5 capsid with respect to regions that confer assembly efficiency, dictate cellular transduction phenotypes, and control antigenicity.     

A Comparative Analysis of Constitutive Promoters Located in Adeno-Associated Viral Vectors

The properties of constitutive promoters within adeno-associated viral (AAV) vectors have not yet been fully characterized. In this study, AAV vectors, in which enhanced GFP expression was directed by one of the six constitutive promoters (human β-actin, human elongation factor-1α, chicken β-actin combined with cytomegalovirus early enhancer, cytomegalovirus (CMV), simian virus 40, and herpes simplex virus thymidine kinase), were constructed and introduced into the HCT116, DLD-1, HT-1080, and MCF-10A cell lines. Quantification of GFP signals in infected cells demonstrated that the CMV promoter produced the highest GFP expression in the six promoters and maintained relatively high GFP expression for up to eight weeks after infection of HCT116, DLD-1, and HT-1080. Exogenous human CDKN2A gene expression was also introduced into DLD-1 and MCF-10A in a similar pattern by using AAV vectors bearing the human β-actin and the CMV promoters. The six constitutive promoters were subsequently placed upstream of the neomycin resistance gene within AAV vectors, and HCT116, DLD-1, and HT-1080 were infected with the resulting vectors. Of the six promoters, the CMV promoter produced the largest number of G418-resistant colonies in all three cell lines. Because AAV vectors have been frequently used as a platform to construct targeting vectors that permit gene editing in human cell lines, we lastly infected the three cell lines with AAV-based targeting vectors against the human PIGA gene in which one of the six promoters regulate the neomycin resistance gene. This assay revealed that the CMV promoter led to the lowest PIGA gene targeting efficiency in the investigated promoters. These results provide a clue to the identification of constitutive promoters suitable to express exogenous genes with AAV vectors, as well as those helpful to conduct efficient gene targeting using AAV-based targeting vectors in human cell lines.