Adenovirus structural proteins

Characterization of Adenovirus Structural Proteins for Gene Therapy... 

For SDS-PAGE, adenovirus structural proteins dissociated from 1.0 x 10 particles/mL of virus were loaded onto each well of Novex precast gradient 4% to 20% acrylamide gels and stained with Coomassie Blue. 

The structural proteins of AAV particles have been dissociated with sodium dodecyl sulfate (SDS) or urea and then separated by polyacrylamide gel electrophoresis (Johnson and Hoggan, 1971 Rose et al., 1971). Three proteins were identified with estimated molecular weights of 62-66000 73-80000 and 87-92000, respectively. The smallest polypeptide represented about 80% of the total protein mass while the other two represented approximately 10% each. There was no correspondence in electrophoretic mobility with adenovirus structural proteins except that there was a partial overlap of the major AAV protein and the adenovirus fiber penton. The three AAV capsid proteins were present in all three human serotypes (AAV I-3) and had similar mobilities with the exception that the major protein species of AAV 2 moved slightly faster than the comparable AAV 1 and 3 species. Very minor... 

Larger icosahedral viruses that have been structurally well characterized do not obey the simple quasi-equivalence rule. For example, adenovirus capsids, for which T = 25, are built of 240 hexons (six-coordinated units) that are trimers of the major structural protein, and the 12 pentons consist of a different protein (Burnett 1984). Polyomavirus capsids, for which T = 7, are built of a single major structural protein,... 

Janik, J. E., Huston, M. M., Cho, K. and Rose, J. A. (1989). Efficient synthesis of adeno-associated virus structural proteins requires both adenovirus DNA binding protein and VA I RNA. Virology 168, 320-329. 

Fig. 14.1 The life cycle of coxsackievirus B3. CVB3 starts its life cycle by attaching to its receptor CAR and coreceptor DAF. Internalized virus releases its viral RNA, which can be used as the template for translation of polyprotein or transcription by RNA-dependent RNA polymerase 3D to replicate its genome. The polyprotein is self-cleaved by virus-encoded proteases to release structural proteins and nonstructural proteins. Later, structural proteins and viral RNA will assemble into progeny virions to be released from infected cell. Abbreviations CVB3, coxsackievirus B3 DAF, decay accelerating factor CAR, coxsackievirus and adenovirus receptor 3Dpo1, RNA-dependent RNA polymerase.

The study of the proteome of the recombinant adenovirus type 5 vectors demonstrated an important apphcation of separation techniques in combination with MS methods in the drug discovery process. With completely sequenced adenovirus genome available, this approach provides a chemically well-dehned method of characterization of structural proteins of recombinant adenoviral vectors. The information of protein MWs, tryptic peptide mass mapping, and sequence tags of tryptic peptides derived from HPLC/MS resulted in the identification of 17 adenoviral proteins/polypeptides in the purified virion. The rapid and accurate identification of viral proteins from recombinant adenoviruses in this study is significant since it provides direct evidence of the maturation stage of adenoviruses, which is closely related to viral infectivity and efficacy in gene therapy. 

Although adenovirus is nonenveloped, it is an example of a larger virus ( 150 MDa) with more complex organization (Burnett, 1997). There are 11 structural proteins, 7 of which are capsid proteins, and 4 of which are core proteins. Some of the structures are known at high resolution (see below). The locations of some others have been determined by one of the first combined electron microscopic and X-ray studies. This was a tour... 

Adenovirus contains a 35-kb dsDNA complexed with four adenovirus-encoded proteins (V, VII, m, and terminal), perhaps in a nucleosome-like arrangement (Burnett, 1997). The arrangement has been controversial, as it has been suggested that the nucleoprotein is arranged both with and without overall icosahedral symmetry. Cryo-EM data support the notion that there is perhaps a small degree of icosahedral symmetry enforced by the capsid lattice, but that there is no well-structured overall icosahedral symmetry within the core (Stewart et al, 1991). 

Benson, S. D., Bamford, J. K., Bamford, D. H., and Burnett, R. M. (1999). Viral evolution revealed by bacteriophage PRDl and human adenovirus coat protein structures. Cell 98, 825-833. 

Combining these two approaches, by determining the crystal structure of individual subcomponents and fitting these into the lower resolution maps of intact particles, may offer the best current hope for obtaining high-resolution information about virion structure. An example of this is provided by adenovirus, where the crystal structure of the hexon, formed by three copies of the 100-kDa major structural protein, has been fitted into a low-resolution capsid structure determined by electron cryomicro-scopy to provide a clearer picture of capsid organization and information about the locations of minor capsid proteins (Stewart et al, 1993). A similar strategy has been used to fit the crystal structure of a portion of the HSV-1 major capsid protein (VPS) into an intermediate-resolution map of the capsid (B. Bowman, personal communication). 

Adenovirus has a single promoter for all RNA made late in the cycle of infection. The primary transcripts terminate at five major polyadenylation sites. Each termination site influences the splicing pattern by allowing particular in-trons or intron termini to be present or not. The five sets are not used with equal frequency, with the result that most mRNAs encoding various genes are not the same. This is the primary mechanism for determining the relative amounts of the different structural proteins synthesized late in the adenovirus life cycle. 

One of the main challenges in the development of the adenovirus purification process was the quantitation of the intact viral particles in crude samples, such as the clarified cell lysate, for the optimization of viral recovery at each step. Non-aggre-gated, purified viral samples were easily quantified by the reverse-phase (RP)-HPLC method developed by Lehmberg et al. [42] The RP-HPLC assay quantitates the indi vidual structural proteins with UV absor bance at 214 nm as the basis for the mea surement of the adenovirus concentration... 

More recently, the use of liquid chromatography and tandem MS (LC/MS/MS) has also eased purification and recovery methods. For instance, Vamum and colleagues utilized gel-free two-dimensional capillary LC/MS/MS and Fourier transform ion cyclotron resonance MS to identily and determine the relative abundances of viral and cellular proteins in purified HCMV virions and dense bodies. Analysis of the proteins Irom purified HCMV virion preparations has indicated that the particle contains significantly more viral proteins than previously known. They identified more than 71 HCMV-encoded proteins and 70 host cellular proteins in HCMV virions, which included cellular structural proteins, enzymes, and chaperones [6]. Another study using LC/MS/MS for the adenovirus type 5 pro-teome found a total of 11 protein species from 154 peptides, at a sensitivity of 10 copies per virus and a detection limit of 70 finol for two proteins [36]. 

Methisazone (Fig. 3) is the most active thiosemicarbazone derivative. It inhibits the multiplication of vaccinia and variola viruses, and is utilized in the prophylaxis of smallpox. It blocks the viral infection at a late stage of the process by making the late viral messenger RNA unstable. It thus prevents the synthesis of the structural proteins and the right assembly of the viral particle. Recently it has been found active against several adenoviruses and against encepbalomyocarditis virus which, in contrast to the other sensitive particles, contains RNA. 

The relevance of the examples of activated cellular gene expression that have been reported so far to the life cycle of the virus has also to be established. Adenovirus depends on its hose cell for a large number of products, whose synthesis might be expected to be enhanced soon after infection to prime the cell for the period when massive quantities of viral DNA, late mRNA, and structural proteins are made. At this juncture, it is, however, difficult to appreciate the significance of increased expression of such genes as those encoding P-tubulin and the 70K heat-shock protein. This may reflect our ignorance about many aspects of the virus-cell interaction. Alterna-... 

The first evidence for control of translation in adenovirus-infected cells came when mRNA isolated from infected cells harvested during the late phase was translated in heterologous, cell-free systems, usually derived from reticulocyte lysates such mRNA preparations direct the synthesis of quite substantial quantities of cellular proteins in addition to large amounts of viral structural proteins (see, for example, Anderson et ai, 1974 Lewis et ai, 1975 Paterson et ai, 1977). The mRNA was prepared from cells collected at a time... 

Pettersson, U., and Hdglund, L., 1969, Structural proteins of adenoviruses III. Purification and characterization of the adenovirus type 2 penton antigen. Virology 39 90. 

DNA viruses Viral DNA becomes incorporated into host cell DNA and codes for RNA which in turn codes for the structural proteins and enzymes that the virus required to reproduce itself. (Examples herpesviruses (chickenpox, shingles, cold sores/genital herpes), glandular fever, adenovirus). 

Crawford-MikszaL, Schnurr DP. Analysis of 15 adenovirus hexon proteins reveals the location and structure of seven hypervaiiable regions containing serotype-specific residues. J Virol 1996 70 1836-1844. 

Fig. 1. Schematic drawings of the viruses discussed in this chapter. (A) An icosahe-dral virus with fiber proteins inserted in its pentameric vertices. The gray box denotes domains with known structures for adenovirus, reovirus, and bacteriophage PRD1, in each case containing the head domain and proximal part of the triple /8-spiral shaft domain. (B) Contractile-tailed bacteriophage T4. T4 contains three different fibrous proteins, fibritin connected to the neck, the long (bent) fibers connected to the base plate, and the short fibers also connected to the base plate. Only two of each of the trimeric fibrous proteins are shown for clarity. The gray box denotes domains with known structure for the T4 short fiber.

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