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.     

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.     

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.     

In Vitro Dynamic Visualization Analysis of Fluorescently Labeled Minor Capsid Protein IX and Core Protein V by Simultaneous Detection

Oncolytic adenoviruses represent a promising therapeutic medicine for human cancer therapy, but successful translation into human clinical trials requires careful evaluation of their viral characteristics. While the function of adenovirus proteins has been analyzed in detail, the dynamics of adenovirus infection remain largely unknown due to technological constraints that prevent adequate tracking of adenovirus particles after infection. Fluorescence labeling of adenoviral particles is one new strategy designed to directly analyze the dynamic processes of viral infection in virus-host cell interactions. We hypothesized that the double labeling of an adenovirus with fluorescent proteins would allow us to properly analyze intracellular viruses and the fate of viral proteins in a live analysis of an adenovirus as compared to single labeling. Thus, we generated a fluorescently labeled adenovirus with both a red fluorescent minor capsid protein IX (pIX) [pIX monomeric red fluorescent protein 1 (mRFP1)] and a green fluorescent minor core protein V (pV) [pV enhanced green fluorescent protein (EGFP)], resulting in Ad5-IX-mRFP1-E3-V-EGFP. The fluorescent signals for pIX-mRFP1 and pV-EGFP were detected within 10 min in living cells. However, a growth curve analysis of Ad5-IX-mRFP1-E3-V-EGFP showed an approximately 150-fold reduced production of the viral progeny at 48 h postinfection as compared to adenovirus type 5. Interestingly, pIX-mRFP1 and pV-EGFP were initially localized in the cytoplasm and nucleolus, respectively, at 18 h postinfection. These proteins were observed in the nucleus during the late stage of infection, and relocalization of the proteins was observed in an adenoviral-replication-dependent manner. These results indicate that simultaneous detection of adenoviruses using dual-fluorescent proteins is suitable for real-time analysis, including identification of infected cells and monitoring of viral spread, which will be required for a complete evaluation of oncolytic adenoviruses.     

Nuclear import of moloney murine leukemia virus DNA mediated by adenovirus preterminal protein is not sufficient for efficient retroviral transduction in nondividing cells

Moloney murine leukemia virus (MoMLV)-derived vectors require cell division for efficient transduction, which may be related to an inability of the viral DNA-protein complex to cross the nuclear membrane. In contrast, adenoviruses (Ad) can efficiently infect nondividing cells. This property may be due to the presence of multiple nuclear translocation signals in a number of Ad proteins, which are associated with the incoming viral genomes. Of particular interest is the Ad preterminal protein (pTP), which binds alone or in complex with the Ad polymerase to specific sequences in the Ad inverted terminal repeat. The goal of this study was to test whether coexpression of pTP with retroviral DNA carrying pTP-binding sites would facilitate nuclear import of the viral preintegration complex and transduction of quiescent cells. In preliminary experiments, we demonstrated that the karyophylic pTP can coimport plasmid DNA into the nuclei of growth-arrested cells. Retroviral transduction studies were performed with G(1)/S-arrested LTA cells or stationary-phase human primary fibroblasts. These studies demonstrated that pTP or pTP-Ad polymerase conferred nuclear import of retroviral DNA upon arrested cells when the retrovirus vector contained the corresponding binding motifs. However, pTP-mediated nuclear translocation of MoMLV DNA in nondividing cells was not sufficient for stable transduction. Additional cellular factors activated during S phase or DNA repair synthesis were required for efficient retroviral integration.     

Conserved leucines in N-terminal heptad repeat HR1 of envelope fusion protein F of group II nucleopolyhedroviruses are important for correct processing and essential for fusogenicity

The heptad repeat (HR), a conserved structural motif of class I viral fusion proteins, is responsible for the formation of a six-helix bundle structure during the envelope fusion process. The insect baculovirus F protein is a newly found budded virus envelope fusion protein which possesses common features to class I fusion proteins, such as proteolytic cleavage and the presence of an N-terminal open fusion peptide and multiple HR domains on the transmembrane subunit F(1). Similar to many vertebrate viral fusion proteins, a conserved leucine zipper motif is predicted in this HR region proximal to the fusion peptide in baculovirus F proteins. To facilitate our understanding of the functional role of this leucine zipper-like HR1 domain in baculovirus F protein synthesis, processing, and viral infectivity, key leucine residues (Leu209, Leu216, and Leu223) were replaced by alanine (A) or arginine (R), respectively. By using Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) as a pseudotype expression system, we demonstrated that all mutant F proteins incorporated into budded virus, indicating that leucine substitutions did not affect intercellular trafficking of F. Furin-like protease cleavage was not affected by any of the leucine substitutions; however, the disulfide bridging and N-linked glycosylation patterns were partly altered. Single substitutions in HR1 showed that the three leucine residues were critical for F fusogenicity and the rescue of AcMNPV infectivity. Our results support the view that the leucine zipper-like HR1 domain is important to safeguard the proper folding, glycosylation, and fusogenicity of baculovirus F proteins.     

Nature of the nuclear inclusions formed by PQBP1, a protein linked to neurodegenerative polyglutamine diseases

PQBP1, for polyglutamine tract-binding protein-1, has been linked to progressive neurodegenerative diseases, such as spinocerebellar ataxia, that are caused by the expansion of a polyglutamine repeat in a key regulatory protein. The overexpression of PQBP1 results in the formation of nuclear inclusions, reminiscent of the protein aggregates that are detected in polyglutamine diseases. We show here that the occurrence of PQBP1-induced nuclear inclusions is dramatically increased by the co-expression of the pre-mRNA splicing factor SIPP1, a protein ligand of PQBP1. These nuclear inclusions did not co-localise with nuclear structures such as nucleoli, coiled bodies, PML bodies, speckles and stress bodies, and were not associated with (in)active chromatin or with nucleic acids. Site-directed mutagenesis showed that the facilitation in the formation of the nuclear inclusions required multiple independent interaction sites between SIPP1 and PQBP1. Moreover, the nuclear inclusions were highly dynamic and their formation did not require energy. Our data suggest that the SIPP1-PQBP1-induced nuclear inclusions are distinct from the protein aggregates that are associated with polyglutamine diseases and represent dynamic nucleoplasmic heteropolymers of SIPP1 and PQBP1.     

Protein crystals in Adenovirus type 5-infected cells: requirements for intranuclear crystallogenesis, structural and functional analysis

Intranuclear crystalline inclusions have been observed in the nucleus of epithelial cells infected with Adenovirus serotype 5 (Ad5) at late steps of the virus life cycle. Using immuno-electron microscopy and confocal microscopy of cells infected with various Ad5 recombinants modified in their penton base or fiber domains, we found that these inclusions represented crystals of penton capsomers, the heteromeric capsid protein formed of penton base and fiber subunits. The occurrence of protein crystals within the nucleus of infected cells required the integrity of the fiber knob and part of the shaft domain. In the knob domain, the region overlapping residues 489-492 in the FG loop was found to be essential for crystal formation. In the shaft, a large deletion of repeats 4 to 16 had no detrimental effect on crystal inclusions, whereas deletion of repeats 8 to 21 abolished crystal formation without altering the level of fiber protein expression. This suggested a crucial role of the five penultimate repeats in the crystallisation process. Chimeric pentons made of Ad5 penton base and fiber domains from different serotypes were analyzed with respect to crystal formation. No crystal was found when fiber consisted of shaft (S) from Ad5 and knob (K) from Ad3 (heterotypic S5-K3 fiber), but occurred with homotypic S3K3 fiber. However, less regular crystals were observed with homotypic S35-K35 fiber. TB5, a monoclonal antibody directed against the Ad5 fiber knob was found by immunofluorescence microscopy to react with high efficiency with the intranuclear protein crystals in situ. Data obtained with Ad fiber mutants indicated that the absence of crystalline inclusions correlated with a lower infectivity and/or lower yields of virus progeny, suggesting that the protein crystals might be involved in virion assembly. Thus, we propose that TB5 staining of Ad-infected 293 cells can be used as a prognostic assay for the viability and productivity of fiber-modified Ad5 vectors.     

Properties of polyglutamine expansion in vitro and in a cellular model for Huntington's disease.

Eight neurodegenerative diseases have been shown to be caused by the expansion of a polyglutamine stretch in specific target proteins that lead to a gain in toxic property. Most of these diseases have some features in common. A pathological threshold of 35-40 glutamine residues is observed in five of the diseases. The mutated proteins (or a polyglutamine-containing subfragment) form ubiquitinated aggregates in neurons of patients or mouse models, in most cases within the nucleus. We summarize the properties of a monoclonal antibody that recognizes specifically, in a Western blot, polyglutamine stretches longer than 35 glutamine residues with an affinity that increases with polyglutamine length. This indicates that the pathological threshold observed in five diseases corresponds to a conformational change creating a pathological epitope, most probably involved in the aggregation property of the carrier protein. We also show that a fragment of a normal protein carrying 38 glutamine residues is able to aggregate into regular fibrils in vitro. Finally, we present a cellular model in which the induced expression of a mutated full-length huntingtin protein leads to the formation of nuclear inclusions that share many characteristics with those observed in patients: those inclusions are ubiquitinated and contain only an N-terminal fragment of huntingtin. This model should thus be useful in studying a processing step that is likely to be important in the pathogenicity of mutated huntingtin.     

The adenovirus L4 100-kilodalton protein is necessary for efficient translation of viral late mRNA species.

When screening a number of adenovirus type 5 (Ad5) temperature-sensitive mutants for defects in viral gene expression, we observed that H5ts1-infected 293 cells accumulated reduced levels of newly synthesized viral late proteins. Pulse-labeling and pulse-chase experiments were used to establish that the late proteins synthesized in H5ts1-infected cells under nonpermissive conditions were as stable as those made in Ad5-infected cells. H5ts1-infected cells contained normal levels of viral late mRNAs. Because these observations implied that translation of viral mRNA species was defective in mutant virus-infected cells, the association of viral late mRNAs with polyribosomes was examined during the late phase of infection at a nonpermissive temperature. In Ad5-infected cells, the majority of the viral L2, L3, L4, pIX, and IVa2 late mRNA species were polyribosome bound. By contrast, these same mRNA species were recovered from H5ts1-infected cells in fractions nearer the top of polyribosome gradients, suggesting that initiation of translation was impaired. During the late phase of infection, neither the polyribosome association nor the translation of most viral early mRNA species was affected by the H5ts1 mutation. This lesion, mapped by marker rescue to the L4 100-kilodalton (kDa) nonstructural protein, has been identified as a single base pair substitution that replaces Ser-466 of the Ad5 100-kDa protein with Pro. A set of temperature-independent revertants of H5ts1 was isolated and characterized. Either true reversion of the H5ts1 mutation or second-site mutation of Pro-466 of the H5ts1 100-kDa protein to Thre, Leu, or His restored both temperature-independent growth and the efficient synthesis of viral late proteins. We therefore conclude that the Ad5 L4 100-kDa protein is necessary for efficient initiation of translation of viral late mRNA species during the late phase of infection.     

Integrating Adenovirus–Adeno-Associated Virus Hybrid Vectors Devoid of All Viral Genes

Recently, we demonstrated that inverted repeat sequences inserted into first-generation adenovirus (Ad) vector genomes mediate precise genomic rearrangements resulting in vector genomes devoid of all viral genes that are efficiently packaged into functional Ad capsids. As a specific application of this finding, we generated adenovirus-adeno-associated virus (AAV) hybrid vectors, first-generation Ad vectors containing AAV inverted terminal repeat sequences (ITRs) flanking a reporter gene cassette inserted into the E1 region. We hypothesized that the AAV ITRs present within the hybrid vector genome could mediate the formation of rearranged vector genomes (DeltaAd.AAV) and stimulate transgene integration. We demonstrate here that DeltaAd.AAV vectors are efficiently generated as by-products of first-generation adenovirus-AAV vector amplification. DeltaAd.AAV genomes contain only the transgene flanked by AAV ITRs, Ad packaging signals, and Ad ITRs. DeltaAd.AAV vectors can be produced at a high titer and purity. In vitro transduction properties of these deleted hybrid vectors were evaluated in direct comparison with first-generation Ad and recombinant AAV vectors (rAAVs). The DeltaAd.AAV hybrid vector stably transduced cultured cells with efficiencies comparable to rAAV. Since cells transduced with DeltaAd.AAV did not express cytotoxic viral proteins, hybrid viruses could be applied at very high multiplicities of infection to increase transduction rates. Southern analysis and pulsed-field gel electrophoresis suggested that DeltaAd.AAV integrated randomly as head-to-tail tandems into the host cell genome. The presence of two intact AAV ITRs was crucial for the production of hybrid vectors and for transgene integration. DeltaAd.AAV vectors, which are straightforward in their production, represent a promising tool for stable gene transfer in vitro and in vivo.     

Development of intranuclear inclusions of human polyomavirus JC. Capsid proteins are assembled into virions at the PML nuclear bodies

Human polyomavirus JC (JCV) is a causative agent for progressive multifocal leukoencephalopathy, a fatal demyelinating disorder. The viruses form intranuclear viral inclusions in infected oligodendrocytes. The outer capsid of JCV is thought to be composed of 360 molecules of major capsid protein VP1, and minor capsid proteins VP2 and VP3 in an appropriate ratio. However, the regulatory mechanisms of gene expression for the capsid proteins, their nuclear transport, and formation of viral inclusions are not well understood. We have recently clarified the following regarding the mechanism underlying JCV virion assembly; (i) major and minor capsid proteins are synthesized from messenger RNAs, the expression ratio of which is determined by alternative splicing, (ii) messenger RNAs for the major and minor capsid proteins are polycistronic, and their translation occurs downstream of the regulatory protein, agnoprotein, (iii) major and minor capsid proteins are translocated to the nucleus in a cooperative manner and accumulate at the dot-shaped intranuclear structures called promyelocytic leukemia nuclear bodies (PML-NBs), (iv) efficient viral replication can occur at the PML-NBs, where capsid assembly is likely to be associated with viral DNA replication. PML-NBs are the sites for expression of important nuclear functions for the host cells. The finding that the target of JCV infection is the PML-NB may contribute greatly to our understanding of the mechanism underlying cellular degeneration, which occurs after the formation of intranuclear viral inclusions.     

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.