404. Roles of Inverted Terminal Repeats (ITRs) and Capsid Proteins from Novel NHP AAVs in rAAV Mediated Gene Transfer

We are reporting the sequences of seven complete genomes of parvovirus B19, which were extracted from human plasma specimens collected in the United States. The seven B19 genome sequences, which are 5,596 nucleotides long and include the inverted terminal repeats (ITRs), share an identity of 96.73%.

Download Full-text

Inverted Terminal Repeats

Encyclopedia of Genetics ◽

10.1006/rwgn.2001.1882 ◽

2001 ◽

pp. 1054

Keyword(s):

Inverted Terminal Repeats ◽

Terminal Repeats

Download Full-text

In Vitro Packaging of Adeno-Associated Virus DNA

Journal of Virology ◽

10.1128/jvi.72.4.3241-3247.1998 ◽

1998 ◽

Vol 72 (4) ◽

pp. 3241-3247 ◽

Cited By ~ 43

Author(s):

Xiaohuai Zhou ◽

Nicholas Muzyczka

Keyword(s):

Virus Particle ◽

Mammalian Cells ◽

Gradient Centrifugation ◽

Marker Gene ◽

Dnase I ◽

Capsid Proteins ◽

Wild Type Virus ◽

Adeno Associated Virus ◽

Terminal Repeats

ABSTRACT 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.

Download Full-text

Cotransfection with adenovirus DNA enhances transcription from linear DNA containing eucaryotic promoters.

Molecular and Cellular Biology ◽

10.1128/mcb.7.3.1063 ◽

1987 ◽

Vol 7 (3) ◽

pp. 1063-1069

Author(s):

M B Vasudevachari ◽

V Natarajan ◽

N P Salzman

Keyword(s):

Rna Synthesis ◽

Chloramphenicol Acetyltransferase ◽

Repeat Sequence ◽

Early Gene ◽

Inverted Terminal Repeat ◽

Adenovirus E1a ◽

293 Cells ◽

Inverted Terminal Repeat Sequence ◽

Inverted Terminal Repeats ◽

Terminal Repeats

Linear DNAs, containing a copy of the adenovirus serotype 2 (Ad2) inverted terminal repeat sequence at each end, replicate in 293 cells when cotransfected with Ad2 DNA (Hay et al., J. Mol. Biol. 175:493-510, 1984). We have linked either the Ad2 IVa2 promoter (IVa2) or major late promoter (MLP) to the chloramphenicol acetyltransferase gene and inserted this DNA into such a plasmid (pARKR) between its two inverted terminal repeats. These recombinant plasmids were linearized and then used to transfect 293 cells in the presence or absence of Ad2 helper DNA. Synthesis of IVa2 and MLP RNAs, and production of chloramphenicol acetyltransferase was increased dramatically when the Ad2 DNA was included. However, unlike the patterns of temporal regulation which are seen during a cycle of virus replication when these genes are contained within the virion, there was no obvious difference in the timing of RNA synthesis from plasmid IVa2 or MLP after cotransfection. When linearized plasmids containing IVa2 and MLP sequences but lacking inverted terminal repeats at their ends (replication deficient plasmids) were used for transfection, an increase in RNA synthesis from IVa2 or MLP was also observed and similarly required cotransfection with Ad2 DNA. When HeLa cells, which do not constitutively express the adenovirus E1a gene, were cotransfected with linearized plasmids and adenovirus DNA that lacks the E1a region (H5dl312), a stimulation of transcription was also observed, although it was less than the level observed with wild-type DNA. The results of the present study demonstrate that an early gene product(s) besides E1a functions in trans to regulate transcription.

Download Full-text

Role for the Adenovirus IVa2 Protein in Packaging of Viral DNA

Journal of Virology ◽

10.1128/jvi.75.21.10446-10454.2001 ◽

2001 ◽

Vol 75 (21) ◽

pp. 10446-10454 ◽

Cited By ~ 59

Author(s):

Wei Zhang ◽

Jonathan A. Low ◽

Joan B. Christensen ◽

Michael J. Imperiale

Keyword(s):

Gene Transfer ◽

Direct Evidence ◽

Helper Virus ◽

Dna Packaging ◽

Chimeric Virus ◽

Viral Dna ◽

293 Cells ◽

Inverted Terminal Repeats ◽

Viral Dna Packaging ◽

Packaging Sequence

ABSTRACT 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.

Download Full-text