Structural Proteins of Adenovirus-Associated Viruses

Rose, James A.; Maizel, Jacob V.; Inman, John K.; Shatkin, Aaron J.

doi:10.1128/jvi.8.5.766-770.1971

Cited by 170 publications

(73 citation statements)

References 26 publications

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“…7) we have proposed are approximately 2.3 kb in length and code for proteins of 85 and 86 kilodaltons. These values agree reasonably well with the observed molecular weights of 85,000 to 90,000 for VP1 (9,10,35,50,62). Needless to say, other explanations for mutation responsible for the lip phenotype are possible.…”

Section: Discussionsupporting

confidence: 86%

“…can code for a protein of approximately 63 kilodaltons (504 amino acids) and presumably is expressed from the most abundant spliced mRNA (2.3 kb) synthesized during AAV infections. Comparison of the amino acid composition of the hypothetical protein coded by this region with the amino acid composition and size (60 to 67 kilodaltons) of the major AAV capsid protein, VP3, indicates that they are the same (62,66). In vitro translation experiments, however, indicate that the 2.3-kb mRNA is capable of synthesizing not only VP3, but VP1 (85 to 90 kilodaltons) and VP2 (72 to 73 kilodaltons) as well (35).…”

mentioning

confidence: 98%

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Genetics of adeno-associated virus: isolation and preliminary characterization of adeno-associated virus type 2 mutants

Hermonat¹,

Labow²,

Wright³

et al. 1984

J Virol

279

186

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We constructed insertion and deletion mutants with mutations within the adeno-associated virus (AAV) sequences of the infectious recombinant plasmid pSM620. Studies of these mutants revealed at least three AAV phenotypes. Mutants with mutations between 11 and 42 map units were partially or completely defective for rescue and replication of the AAV sequences from the recombinant plasmids (rep mutants). The mutants could be complemented by mutants with replication-positive phenotypes. The protein(s) that is affected in rep mutants has not been identified, but the existence of the rep mutants proves that at least one AAV-coded protein is required for viral DNA replication. Also, the fact that one of the rep mutant mutations maps within the AAV intron suggests that the intron sequences code for part of a functional AAV protein. Mutants with mutations between 63 and 91 map units synthesized normal amounts of AAV duplex DNA but could not generate singlestranded virion DNA (cap mutants). The cap phenotype could be complemented by rep mutants and is probably due to a defect in the major AAV capsid protein, VP3. This suggests that a preformed capsid or precursor is required for the accumulation of single-stranded AAV progeny DNA. Mutants with mutations between 48 and 55 map units synthesized normal amounts of AAV single-stranded and duplex DNA but produced substantially lower yields of infectious virus particles than wild-type AAV (lip mutants). The lip phenotype is probably due to a defect in the minor capsid protein, VP1, and suggests the existence of an additional (as yet undiscovered) AAV mRNA. Evidence is also presented for recombination between mutant AAV genomes during lytic growth.

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Section: Discussionsupporting

confidence: 86%

mentioning

confidence: 98%

Genetics of adeno-associated virus: isolation and preliminary characterization of adeno-associated virus type 2 mutants

Hermonat¹,

Labow²,

Wright³

et al. 1984

J Virol

279

186

View full text Add to dashboard Cite

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“…Cells expressing the transgene appear blue when stained with X-gal. Rose et al (1971) established that AAV2 particles are composed of Vp1, Vp2, and Vp3 at a 1:1:20 ratio (Rose et al, 1971). In an effort to determine if class II and class III mutant virions maintained this ratio, Western blots were performed on the cesium chloride-purified virus.…”

Section: Electron Microscopy Of Class II and Class Iii Mutantsmentioning

confidence: 99%

“…After translation, the capsid proteins migrate to the nucleus and assemble into progeny virions. The virions are composed of 60 subunits with a stoichiometry of 1:1:20 for Vp1, Vp2, and Vp3, respectively (Rose et al, 1971). The ratio of virion capsid subunits correlates with in vivo expression levels seen during productive infection.…”

Section: Introductionmentioning

confidence: 99%

Insertional Mutagenesis of AAV2 Capsid and the Production of Recombinant Virus

1999

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The structural genes of adeno-associated virus serotype 2 (AAV2) have been altered by linker insertional mutagenesis in order to define critical components of virion assembly and infectivity. An in-frame restriction site linker was inserted across the capsid coding domain of a recombinant plasmid. After complementation in vivo, recombinant AAV2 viruses were generated and assayed for capsid production, packaging, transduction, heparin agarose binding, and morphology. Three classes of capsid mutants where identified. Class I mutants expressed structural proteins but were defective in virion assembly. Class II mutants generated intact virions that protected the viral genome from DNase, but failed to infect target cells. The majority of these mutants bound the heparin affinity matrix, suggesting that attachment to the AAV primary receptor was not rate limiting. One class II mutant, H2634, assembled virions and bound heparin using only Vp3, indicating that this subunit is responsible for mediating AAV receptor attachment. Finally, class III mutants assembled virions, encapsidated DNA, and infected target cells. Infectivity of these mutants ranged from 5 to 100% of that of the wild-type, demonstrating for the first time the ability to alter capsid proteins without interfering with infectivity. These AAV virions with altered capsid subunits will provide critical templates for manipulating AAV vectors for cell-specific gene delivery in vivo. In summary, the AAV capsid variants described here will facilitate further study of virus assembly, entry, and infection, as well as advance the development of this versatile vector system.

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“…Mutations that prevent the synthesis of the small Rep proteins Rep52 and Rep40 lead to a strongly reduced level of single-stranded DNA (ssDNA) accumulation, suggesting that they play a role in DNA packaging (17,26). The AAV cap gene located in the right half of the AAV genome codes for three capsid proteins, VP1, VP2, and VP3, which are present in the virion in a 1:1:10 stoichiometry (32,33,52). Pulse-chase experiments support the view that the single-stranded AAV genomes are packaged into preformed capsids (42).…”

mentioning

confidence: 99%

Subcellular compartmentalization of adeno-associated virus type 2 assembly

Wistuba

Kern

Weger

et al. 1997

J Virol

181

102

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Using immunofluorescence and in situ hybridization techniques, we studied the intracellular localization of adeno-associated virus type 2 (AAV-2) Rep proteins, VP proteins, and DNA during the course of an AAV-2/ adenovirus type 2 coinfection. In an early stage, the Rep proteins showed a punctate distribution pattern over the nuclei of infected cells, reminiscent of replication foci. At this stage, no capsid proteins were detectable. At later stages, the Rep proteins were distributed more homogeneously over the nuclear interior and finally became redistributed into clusters slightly enriched at the nuclear periphery. During an intermediate stage, they also appeared at an interior part of the nucleolus for a short period, whereas most of the time the nucleoli were Rep negative. AAV-2 DNA colocalized with the Rep proteins. All three capsid proteins were strongly enriched in the nucleolus in a transient stage of infection, when the Rep proteins homogeneously filled the nucleoplasm. Thereafter, they became distributed over the whole nucleus and colocalized in nucleoplasmic clusters with the Rep proteins and AAV-2 DNA. While VP1 and VP2 strongly accumulated in the nucleus, VP3 was almost equally distributed between the nucleus and cytoplasm. Capsids, visualized by a conformationspecific antibody, were first detectable in the nucleoli and then spread over the whole nucleoplasm. This suggests that nucleolar components are involved in initiation of capsid assembly whereas DNA packaging occurs in the nucleoplasm. Expression of a transfected full-length AAV-2 genome followed by adenovirus infection showed all stages of an AAV-2/adenovirus coinfection, whereas after expression of the cap gene alone, capsids were restricted to the nucleoli and did not follow the nuclear redistribution observed in the presence of the whole AAV-2 genome. Coexpression of Rep proteins released the restriction of capsids to the nucleolus, suggesting that the Rep proteins are involved in nuclear redistribution of AAV capsids during viral infection. Capsid formation was dependent on the concentration of expressed capsid protein.

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Structural Proteins of Adenovirus-Associated Viruses

Cited by 170 publications

References 26 publications

Genetics of adeno-associated virus: isolation and preliminary characterization of adeno-associated virus type 2 mutants

Genetics of adeno-associated virus: isolation and preliminary characterization of adeno-associated virus type 2 mutants

Insertional Mutagenesis of AAV2 Capsid and the Production of Recombinant Virus

Subcellular compartmentalization of adeno-associated virus type 2 assembly

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