The coiled-coil domain of the adenovirus type 5 protein IX is dispensable for capsid incorporation and thermostability

The 14.4-kDa hexon-associated protein IX (pIX) acts as a cement in the capsids of primate adenoviruses and confers a thermostable phenotype. Here we show that deletion of amino acids 100 to 114 of adenovirus type 5 pIX, which eliminates the conserved coiled-coil domain, impairs its capacity to self-associate. However, pIXDelta100-114 is efficiently incorporated into the viral capsid, and the resulting virions are thermostable. Deletion of the central alanine-rich domain, as in pIXDelta60-72, does not impair self-association, incorporation into the capsid, or the thermostable phenotype. These data demonstrate, first, that the self-association of pIX is dispensable for its incorporation into the capsid and generation of the thermostability phenotype and, second, that the increased thermostability results from pIX monomers binding to different hexon capsomers rather than capsid stabilization by pIX multimers.     

Spacers increase the accessibility of peptide ligands linked to the carboxyl terminus of adenovirus minor capsid protein IX

The efficiency and specificity of gene transfer with human adenovirus (hAd)-derived gene transfer vectors would be improved if the native viral tropism could be modified. Here, we demonstrate that the minor capsid protein IX (pIX), which is present in 240 copies in the Ad capsid, can be exploited as an anchor for heterologous polypeptides. Protein IX-deleted hAd5 vectors were propagated in hAd5 helper cells expressing pIX variants, with heterologous carboxyl-terminal extensions of up to 113 amino acids in length. The extensions evaluated consist of alpha-helical spacers up to 75 A in length and to which peptide ligands were fused. The pIX variants were efficiently incorporated into the capsids of Ad particles. On intact particles, the MYC-tagged-pIX molecules were readily accessible to anti-MYC antibodies, as demonstrated by electron microscopic analyses of immunogold-labeled virus particles. The labeling efficiency improved with increasing spacer length, suggesting that the spacers lift and expose the ligand at the capsid surface. Furthermore, we found that the addition of an integrin-binding RGD motif to the pIX markedly stimulated the transduction of coxsackievirus group B and hAd receptor-deficient endothelioma cells, demonstrating the utility of pIX modification in gene transfer. Our data demonstrate that the minor capsid protein IX can be used as an anchor for the addition of polypeptide ligands to Ad particles.     

Engineering of adenovirus vectors containing heterologous peptide sequences in the C terminus of capsid protein IX

The utility of the present generation of adenovirus (Ad) vectors for gene therapy applications could be improved by restricting native viral tropism to selected cell types. In order to achieve modification of Ad tropism, we proposed to exploit a minor component of viral capsid, protein IX (pIX), for genetic incorporation of targeting ligands. Based on the proposed structure of pIX, we hypothesized that its C terminus could be used as a site for incorporation of heterologous peptide sequences. We engineered recombinant Ad vectors containing modified pIX carrying a carboxy-terminal Flag epitope along with a heparan sulfate binding motif consisting of either eight consecutive lysines or a polylysine sequence. Using an anti-Flag antibody, we have shown that modified pIXs are incorporated into virions and display Flag-containing C-terminal sequences on the capsid surface. In addition, both lysine octapeptide and polylysine ligands were accessible for binding to heparin-coated beads. In contrast to virus bearing lysine octapeptide, Ad vector displaying a polylysine was capable of recognizing cellular heparan sulfate receptors. We have demonstrated that incorporation of a polylysine motif into the pIX ectodomain results in a significant augmentation of Ad fiber knob-independent infection of CAR-deficient cell types. Our data suggest that the pIX ectodomain can serve as an alternative to the fiber knob, penton base, and hexon proteins for incorporation of targeting ligands for the purpose of Ad tropism modification.     

DeltaPhage--a novel helper phage for high-valence pIX phagemid display

Phage display has been instrumental in discovery of novel binding peptides and folded domains for the past two decades. We recently reported a novel pIX phagemid display system that is characterized by a strong preference for phagemid packaging combined with low display levels, two key features that support highly efficient affinity selection. However, high diversity in selected repertoires are intimately coupled to high display levels during initial selection rounds. To incorporate this additional feature into the pIX display system, we have developed a novel helper phage termed DeltaPhage that allows for high-valence display on pIX. This was obtained by inserting two amber mutations close to the pIX start codon, but after the pVII translational stop, conditionally inactivating the helper phage encoded pIX. Until now, the general notion has been that display on pIX is dependent on wild-type complementation, making high-valence display unachievable. However, we found that DeltaPhage does facilitate high-valence pIX display when used with a non-suppressor host. Here, we report a side-by-side comparison with pIII display, and we find that this novel helper phage complements existing pIX phagemid display systems to allow both low and high-valence display, making pIX display a complete and efficient alternative to existing pIII phagemid display systems.     

Redesign of retrovirus packaging cell lines to avoid recombination leading to helper virus production.

Retrovirus vectors can be made in the absence of helper virus by using retrovirus packaging cell lines. Helper-free virus is critical for a variety of gene transfer studies. The most useful packaging cell lines contain helper virus DNA from which the signal required for packaging of the viral RNA genome into virions has been deleted. However, we showed that the ability to package virus is conferred at very low frequency to cells infected with virus from these packaging cell lines, presumably by low-frequency transmission of the deleted virus genome. In addition, these packaging cell lines can interact with some retroviral vectors to yield replication-competent virus. We constructed packaging cell lines containing helper virus DNA that had several alterations in addition to deletion of the packaging signal. The new packaging cells retained the useful features of previously available lines but did not yield helper virus after introduction of any of the vectors tested, and transfer of the packaging function was not detected.     

A system for efficient generation of adenovirus protein IX-producing helper cell lines

BACKGROUND: The adenovirus 14.3 kDa hexon-associated protein IX (pIX) functions in the viral capsid as 'cement' and assembles the hexons in stable groups-of-nine (GONs). Although viruses lacking pIX do not form GONs, and are less heat-stable than wild-type (wt) viruses, they can be propagated with the same kinetics and yields as the wt viruses. To facilitate 'pseudotyping' of adenoviral vectors we have set up an efficient system for the generation of pIX-producing helper cell lines. METHODS: With a lentiviral pIX-expression cassette, monoclonal and polyclonal helper cell lines were generated, which express wt or modified pIX genes at levels equivalent to wt HAdV-5 infected cells. The incorporation efficiency into pIX gene deleted viruses was examined by Western analysis, immuno-affinity electron microscopy, and heat-stability assays. RESULTS: Immuno-affinity electron microscopy on viruses lacking the pIX gene demonstrated that more than 96% of the particles contain pIX protein in their capsids after propagation on the pIX-expressing helper cell lines. In addition, the pIX level in the helper cells was sufficient to generate heat-stable particles. Finally, the ratio between pIX and fiber was equivalent to that found in wt particles. The pIX-producing cell lines are very stable, demonstrating that pIX is not toxic to cells. CONCLUSION: These data demonstrate that lentivirus vectors can be used for the establishment of pIX-complementing helper cell lines.     

The Subgenus-Specific C-Terminal Region of Protein IX Is Located on the Surface of the Adenovirus Capsid

We have investigated the antigenicity of the C- and N-terminal halves of pIX of human adenovirus types 2 and 3 (Ad2 and Ad3) as well as their orientations in virions. We found that only the C-terminal halves of Ad2 pIX and Ad3 pIX reacted in a subgenus-specific manner by enzyme-linked immunosorbent assay and immunoblot analysis. Based on immunoelectron microscopy experiments, pIX in viral capsids appears to be positioned such that the C-terminal part of pIX constitutes the surface domain whereas the N terminus of the protein makes up the internal domain in icosahedral Ad capsids.     

Palindromic structure and polypeptide expression of 36 kilobase pairs of heterogeneous Epstein-Barr virus (P3HR-1) DNA.

Among the Epstein-Barr virions (EBV) produced by the P3HR-1 (HR-1) cell line are a defective subpopulation with rearranged viral DNA designated heterogeneous DNA (het DNA). These defective virions are responsible for the capacity of HR-1 virus to induce early antigen in Raji c cells and for trans activation of latent EBV in X50-7 cells. Virions with het DNA are independent replicons which pass horizontally from cell to cell rather than being partitioned vertically. We analyzed the structure and defined several polypeptide products of het DNA to understand these remarkable biologic properties. A 36-kilobase-pair (kbp) stretch of het DNA was cloned (as two EcoRI fragments of 20 and 16 kbp) from virions released from a cellular subclone of HR-1 cells. The unusual aspect of the 20-kbp fragment was the linkage of sequences of BamHI-M and BamHI-B', which are not adjacent on the standard EBV genome. The 16-kbp fragment was a palindrome in which at least two additional recombinations on each side of the palindrome had linked regions of the standard EBV genome which are not normally contiguous. The 20-kbp het DNA fragment was attached to at least one and possibly both ends of the 16-kbp het DNA fragment. We identified antigenic polypeptides produced in COS-1 cells after gene transfer of various cloned het DNA fragments. The 20-kbp fragment encoded a cytoplasmic antigen of about 95 kilodaltons (kDa). The 16-kbp fragment encoded antigens located in the nucleus, nuclear membrane, and cytoplasm. These were represented by several polypeptides, the most prominent of which were about 55, 52, and 36 kDa. The 36-kDa polypeptide was localized to a 2.7-kbp BamHI fragment which had homology to standard BamHI-W and BamHI-Z. Another polypeptide of 50 kDa found in the nucleus was mapped to the 7.1-kbp BamHI het DNA fragment which spans the EcoRI site linking the 20- and 16-kbp fragments of het DNA. Thus, HR-1 het DNA encodes several discrete polypeptide products, one or more of which could be responsible for the unusual biologic properties of the virus. The composition, regulation, and ultimately the expression of some of these products relative to standard EBV is probably altered by the genomic rearrangements of het DNA.     

Packaging capacity and stability of human adenovirus type 5 vectors.

Adenovirus vectors are extensively used for high-level expression of proteins in mammalian cells and are receiving increasing attention for their potential use as live recombinant vaccines and as transducing viruses for use in gene therapy. Although it is commonly argued that one of the chief advantages of adenovirus vectors is their relative stability, this has not been thoroughly investigated. To examine the genetic stability of adenovirus type 5 vectors and in particular to examine the relationship between genetic stability and genome size, adenovirus vectors were constructed with inserts of 4.88 (herpes simplex virus type 1 gB), 4.10 (herpes simplex virus type 1 gB), or 3.82 (LacZ) kb combined with a 1.88-kb E3 deletion or with a newly generated 2.69-kb E3 deletion. The net excess of DNA over the wild-type (wt) genome size ranged from 1.13 to 3.00 kb or 3.1 to 8.3%. Analysis of these vectors during serial passage in tissue culture revealed that when the size exceeded 105% of the wt genome length by approximately 1.2 kb (4.88-kb insert combined with a 1.88-kb deletion), the resulting vector grew very poorly and underwent rapid rearrangement, resulting in loss of the insert after only a few passages. In contrast, vectors with inserts resulting in viral DNA close to or less than a net genome size of 105% of that of the wt grew well and were relatively stable. In general, viruses with genomes only slightly above 105% of that of the wt were unstable and the rapidity with which rearrangement occurred correlated with the size of the insert. These findings suggest that there is a relatively tight constraint on the amount of DNA which can be packaged into virions and that exceeding the limit results in a sharply decreased rate of virus growth. The resultant strong selection for variants which have undergone rearrangement, generating smaller genomes, is manifested as genetic instability of the virus population.     

Ectodomain of coxsackievirus and adenovirus receptor genetically fused to epidermal growth factor mediates adenovirus targeting to epidermal growth factor receptor-positive cells

Human adenovirus (Ad) is extensively used for a variety of gene therapy applications. However, the utility of Ad vectors is limited due to the low efficiency of Ad-mediated gene transfer to target cells expressing marginal levels of the Ad fiber receptor. Therefore, the present generation of Ad vectors could potentially be improved by modification of Ad tropism to target the virus to specific organs and tissues. The fact that coxsackievirus and adenovirus receptor (CAR) does not play any role in virus internalization, but functions merely as the virus attachment site, suggests that the extracellular part of CAR might be utilized to block the receptor recognition site on the Ad fiber knob domain. We proposed to design bispecific fusion proteins formed by a recombinant soluble form of truncated CAR (sCAR) and a targeting ligand. In this study, we derived sCAR genetically fused with human epidermal growth factor (EGF) and investigated its ability to target Ad infection to the EGF receptor (EGFR) overexpressed on cancer cell lines. We have demonstrated that sCAR-EGF protein is capable of binding to Ad virions and directing them to EGFR, thereby achieving targeted delivery of reporter gene. These results show that sCAR-EGF protein possesses the ability to effectively retarget Ad via a non-CAR pathway, with enhancement of gene transfer efficiency.     

Targeted and shielded adenovectors for cancer therapy

Conditionally replicative adenovirus (CRAd) vectors are novel vectors with utility as virotherapy agents for alternative cancer therapies. These vectors have already established a broad safety record in humans and overcome some of the limitations of non-replicative adenovirus (Ad) vectors. In addition, one potential problem with these vectors, attainment of tumor or tissue selectivity has widely been addressed. However, two confounding problems limiting efficacy of these drug candidates remains. The paucity of the native Ad receptor on tumor tissues, and host humoral response due to pre-existing titers of neutralizing antibodies against the vector itself in humans have been highlighted in the clinical context. The well-characterized CRAd, AdΔ24-RGD, is infectivity enhanced, thus overcoming the lack of coxsackievirus and adenovirus receptor (CAR), and this agent is already rapidly progressing towards clinical translation. However, the perceived host humoral response potentially will limit gains seen from the infectivity enhancement and therefore a strategy to blunt immunity against the vector is required. On the basis of this caveat a novel strategy, termed shielding, has been developed in which the genetic modification of a virion capsid protein would provide uniformly shielded Ad vectors. The identification of the pIX capsid protein as an ideal locale for genetic incorporation of shielding ligands to conceal the Ad vector from pre-existing neutralizing antibodies is a major progression in the development of shielded CRAds. Preliminary data utilizing an Ad vector with HSV-TK fused to the pIX protein indicates that a shield against neutralizing antibodies can be achieved. The utility of various proteins as shielding molecules is currently being addressed. The creation of AdΔ24S-RGD, an infectivity enhanced and shielded Ad vector will provide the next step in the development of clinically and commercially feasible CRAds that can be dosed multiple times for maximum effectiveness in the fight against cancers in humans.This article is a symposium paper from the Annual Meeting of the "International Society for Cell and Gene Therapy of Cancer", held in Shenzhen, China, on 9–11 December 2005.     

Evaluation of adenoviral vectors by flow cytometry

Biological assays for adenoviral gene therapy vectors have included conventional procedures initially developed to detect wild-type adenoviruses. Standard virological assays to quantitate adenoviruses rely on the virus to infect and replicate in the host cell until a cytopathic effect is observed. The appearance of plaques, colonies of rounded, enlarged cells containing infectious virions, usually takes 2 to 3 weeks to reach an endpoint. We describe a flow cytometric bioassay for adenovirus which shortens the time from when the infection takes place to the time that biological titer is determined. A fluorescent focus-forming assay was one of the first rapid adenoviral bioassays developed. Virus titer was determined using fluorescence immunocytochemistry to detect adenovirus proteins and microscopy to count fluorescent foci in cultures of adenovirus-infected cells. In this study, we describe a flow cytometric assay performed on cells stained for adenovirus hexon capsid protein, where virus titer is determined based on the dose-dependent appearance of hexon-positive cells. Adenovirus hexon detection in infected cells can provide data to determine virus titer, inducible promoter function in vector-complementing cells, and vector replication in complementation-deficient cells.     

Role for the adenovirus IVa2 protein in packaging of viral DNA

Although it has been demonstrated that the adenovirus IVa2 protein binds to the packaging domains on the viral chromosome and interacts with the viral L1 52/55-kDa protein, which is required for viral DNA packaging, there has been no direct evidence demonstrating that the IVa2 protein is involved in DNA packaging. To understand in greater detail the DNA packaging mechanisms of adenovirus, we have asked whether DNA packaging is serotype or subgroup specific. We found that Ad7 (subgroup B), Ad12 (subgroup A), and Ad17 (subgroup D) cannot complement the defect of an Ad5 (subgroup C) mutant, pm8001, which does not package its DNA due to a mutation in the L1 52/55-kDa gene. This indicates that the DNA packaging systems of different serotypes cannot interact productively with Ad5 DNA. Based on this, a chimeric virus containing the Ad7 genome except for the inverted terminal repeats and packaging sequence from Ad5 was constructed. This chimeric virus replicates its DNA and synthesizes Ad7 proteins, but it cannot package its DNA in 293 cells or 293 cells expressing the Ad5 L1 52/55-kDa protein. However, this chimeric virus packages its DNA in 293 cells expressing the Ad5 IVa2 protein. These results indicate that the IVa2 protein plays a role in viral DNA packaging and that its function is serotype specific. Since this chimeric virus cannot package its own DNA, but produces all the components for packaging Ad7 DNA, it may be a more suitable helper virus for the growth of Ad7 gutted vectors for gene transfer.     

Immunological Reactivity of Antisera Prepared Against the Sodium Dodecyl Sulfate-Treated Structural Polypeptides of Adenovirus-Associated Virus

The preparation of antisera to the three purified sodium dodecyl sulfate (SDS)-treated polypeptide components (VP1, VP2, VP3) of adenovirus-associated virus (AAV) type 3H is described. In immunofluorescence tests (FA), these antisera stained heat-stable antigens with distinct morphologies in cells co-infected with either adenovirus or herpes simplex virus. Kinetic studies of antigen formation showed that VP1 antiserum first stained the cytoplasm (14 hr) and later (by 18 hr) stained both cytoplasmic and intranuclear areas. VP2 antiserum stained only discrete intranuclear areas, and VP3 antiserum stained nearly the entire nucleus. All three VP antigens appeared at about the 14th hr postinfection, about 2 hr prior to the appearance of whole virion antigen. The VP antisera cross-reacted in FA with AAV types 1 and 2 (all at one-eighth of the homologous titer), but did not react with other parvoviruses, i.e., rat virus, hemadsorbing enteric virus of calves, minute virus of mice, or H-1 virus. These non-neutralizing antisera reacted specifically with SDS-treated AAV virion antigens in complement fixation and immunodiffusion tests, and antiserum prepared against SDS-treated helper adenovirus structural polypeptides reacted with adenovirus polypeptide antigens. All antisera to SDS-treated polypeptides were specific for new antigens revealed on the dissociated peptides and did not react with whole virions, whereas whole-virion antisera did not cross-react with the polypeptide antigens. These findings suggest that antigens unique to the polypeptides of AAV are revealed by SDS treatment and that these antigens can be detected in cells prior to the folding of the polypeptides into the molecular configuration they possess as virion subunits. These results also indicate that at least one AAV polypeptide component is synthesized in the cell cytoplasm.     

Foamy virus vectors.

Human foamy virus (HFV) is a retrovirus of the spumavirus family. We have constructed vectors based on HFV that encode neomycin phosphotransferase and alkaline phosphatase. These vectors are able to transduce a wide variety of vertebrate cells by integration of the vector genome. Unlike vectors based on murine leukemia virus, HFV vectors are not inactivated by human serum, and they transduce stationary-phase cultures more efficiently than murine leukemia virus vectors. These properties, as well as their large packaging capacity, make HFV vectors promising gene transfer vehicles.     

Cre levels limit packaging signal excision efficiency in the Cre/loxP helper-dependent adenoviral vector system

Helper-dependent (HD) adenovirus vectors devoid of all viral coding sequences have a large cloning capacity and provide long-term transgene expression in vivo with negligible toxicity, making them attractive vectors for gene therapy. Currently, the most efficient means of producing HD vectors involves coinfecting 293 cells expressing Cre with the HD vector and a helper virus bearing a packaging signal flanked by loxP sites. Cre-mediated packaging signal excision renders the helper virus genome unpackageable but still able to replicate and provide helper functions for HD vector propagation. Typically, helper virus contamination is < or =1% pre- and < or =0.1% postpurification by CsCl banding. While these contamination levels are low, further reduction is desirable. However, this objective has not been realized since the Cre/loxP system was first developed. This lack of progress is due, at least in part, to our lack of understanding of the origins of the contaminating helper virus, thus rendering its reduction or elimination difficult to achieve. This study was designed to investigate the possible sources of contaminating helper virus persisting during HD vector amplification. The results revealed that Cre is limiting in helper virus-infected Cre-expressing 293 cells, thereby permitting helper viruses to escape packaging signal excision and propagate. The results of this study should provide a foundation for developing rational strategies to further reduce or possibly eliminate the contaminating helper virus.