Synthesis of adeno-associated virus structural proteins requires both alternative mRNA splicing and alternative initiations from a single transcript.

The three adeno-associated virus type 2 (AAV2) structural proteins (A, B, and C) are specified by transcripts generated from the most-rightward promoter (p40). Protein C (60 kilodaltons [kDa]), the most abundantly produced, is entirely contained within B (72 kDa) which, in turn, is contained within A (90 kDa). Although neither of the known structures of p40 transcripts, an unspliced 2.6-kilobase (kb) RNA and a spliced 2.3-kb RNA, possesses an AUG-initiated open reading frame that accounts for the synthesis of proteins A and B, recent evidence indicates that B is initiated by a unique eucaryotic initiation codon (ACG) (S.P. Becerra, J.A. Rose, M. Hardy, B. Baroudy, and C.W. Anderson, Proc. Natl. Acad. Sci. USA 82:7919-7923, 1985). In the present study, we analyzed the in vitro translation of AAV capsid proteins from synthetic transcripts and the in vivo expression of AAV mRNA and capsid proteins in 293 cells transfected with AAV DNA constructs. The results demonstrated that AAV transcripts contain only one functional 5' splice donor site, that synthesis of capsid proteins from the unspliced 2.6-kb transcript is very inefficient, that transcripts without the intervening sequence (IVS) (i.e., the 2.3-kb RNA) do not produce protein A but effectively synthesize proteins B and C, and that protein A is actively synthesized from transcripts which contain the last 34 bases of the IVS. Protein A initiates within this 34-base segment in reading frame 1, apparently with the AUG codon at nucleotide 2203, and then elongates into the B and C open reading frame. Because A is inefficiently synthesized from the 2.6-kb transcript, we conclude that an effective A transcript is generated by alternative splicing and that the alternative 3' acceptor site may lie at nucleotide 2200 within a context of...CAG]GTA. The levels of B and C produced by a synthetic transcript devoid of the IVS suggest that the known 2.3-kb RNA is the main source of these proteins and indicate that this single RNA species expresses both proteins by alternative use of their respective initiation codons.

Transcription and translation in the autonomous parvovirus KRV.

We describe here the isolation and partial characterization of at least five viral specific RNAs synthesized in the rat nephroma (RN) cell after infection with the parvovirus KRV. The RNAs have the approximate lengths of 4.7, 3.4, 3.0, 1.25, and 0.95 kilobases (kb) and are probably the functional messages. The 4.7-kb RNA would represent a transcript of 95 to 100% of the viral genome. The most abundant message, about 3.0 kb, represents over 50% of the viral genome and probably codes in the reticulocyte transcribing system for the most abundant viral protein (MW 68,000) which is the main viral capsid protein. This abundant RNA, on the basis of R loop data, has an origin of transcription about 0.38 to 0.42 map units from the 3' end of the single-stranded KRV genome.

Synthesis of mRNA by the parvovirus KRV in isolated nuclei.

We have synthesized vial-specific transcripts in nuclei isolated from cells infected with the parvovirus Kilham rat virus. Radioactive vial-specific RNA synthesized in the nuclei was extracted, hybridized to viral DNA, incubated in glyoxal, and electrophoresed in an agarose gel. At least five viral-specific RNAs were detected containing 4.6 Kb (25-26S), 3.2 Kb (major species, 20-22S), 2.9 Kb, 1.3 Kb, and 1.0 Kb pairs. The 4.6 Kb RNA represents a transcript of 95-100% of the viral genome. The major 3.2 and 2.9 Kb RNAs represent a transcript of 50-60% of the viral genome. Over 65% of the viral-specific RNA in the isolated nuclei is polyadenylylated on the 3' terminus. The 5' terminus of the RNA is capped in vitro by the sequence m7G(5')ppp(5')A. Incorporation of [beta-32p]ATP into the 5' cap sequence suggests that initiation of viral RNA synthesis may occur in the infected isolated nuclei.

Nucleotide sequence of the self-priming 3' terminus of the single-stranded DNA extracted from the parvovirus Kilham rat virus.

The parvovirus genome is a linear, single-stranded DNA molecule with double-stranded hairpin termini. The 3' terminus can serve in vitro as a self-primer for the synthesis of a double-stranded viral DNA intermediate. We have sequenced the nucleotides in the 3' terminus and propose a model for the secondary structure of the terminus and the in vitro origin of replication for the complementary viral DNA strand.

In vitro synthesis of double-stranded DNA from the Kilham rat virus single-stranded DNA genome.

Double-stranded, full-length linear DNA was synthesized in vitro by using single-stranded linear DNA as a self-priming template from the parvovirus Kilham rat virus and Escherichia coli DNA polymerase "large fragment" as the polymerizing enzyme. To ascertain the order of the synthesis of the cleavage fragments and to assess the accuracy of the in vitro synthesis, restriction endonuclease cleavage sites with known recognition sequences were mapped on the DNA. Comparing the cleavage pattern of the synthesized DNA with that of double-stranded viral DNA isolated from infected cells confirms that the in vitro synthesis produces a faithful copy of the viral single-stranded genome. Electron micrographs of the in vitro product reveal it to be a double-stranded linear molecule.

Studies on the replication of KRV single-stranded linear DNA.

The autonomous parvovirus, Kilham rat virus (KRV), is composed of three structural proteins (Salzman & White, 1970) and a single molecule of DNA. The DNA molecule is linear and single-stranded (ss). It is believed to have both 3' and 5'-terminal palindromic sequences (Salzman, 1977). Replication of the ss DNA may involve double-stranded (ds) DNA intermediates of one unit length as found in the virion or multiple length concatamers (Salzman & White, 1973; Gunther & May, 1976; Lavelle & Li, 1977). We have attempted to determine when the synthesis of KRV ds DNA can be determined in synchronized infected cells and the structure of the ds DNA molecules.