A minimal avian retroviral packaging sequence has a complex structure

Abstract

We have defined a 160-nucleotide region, Mpsi, from the 5' leader region of the Rous sarcoma virus genome that is sufficient to direct the packaging of a heterologous RNA. Mpsi contains the putative O3 stem structure that has previously been shown, and that has been confirmed in this study, to be important for the efficient packaging of avian leukosis-sarcoma virus RNA. Analyses of several O3 stem mutants revealed that other regions within Mpsi can interfere with the proper folding of altered sequences which are predicted to form a wild-type O3 stem.

FIG. 1

Constructs used to assay regions of the genome necessary for ψ function. Portions of the RSV Prague C genome were cloned into pASY185, a pCMVneo derivative. Numbering is from the first nucleotide of the 5′ R region of the RSV Prague C genome (GenBank accession no., J02342). The S1 and S2 inverted repeats are boxed.

FIG. 2

(A) RNase protection analysis to determine the amount of neo RNA in cells. Total cell RNAs were extracted from G418-resistant mass cultures of Q2bn-4D cells transfected with plasmids, as indicated above each lane, and annealed to the antisense neo probe. The sample in lane 7 had no cell RNA added. The sample in lane 8 was not treated with RNase. The arrows show the expected locations of the free probe and the neo-protected band, seen as a doublet. (B) RNase protection analysis to determine the amount of neo RNA packaged in virions released from the cells described in the legend for panel A. Virus was collected, and RNAs were extracted and annealed to the antisense neo probe. The sample in lane 1 had no RNase added. The sample in lane 8 had no viral RNA added.

FIG. 3

(A) The RSV genome showing the location and sequence of the minimal packaging region, Mψ. The S1 and S2 inverted repeats composing the O3 stem, as well as the downstream PS3 region, are underlined in gray. The SacI restriction site and uORF-3 are underlined in black. R, repeated sequence; U5, unique 5′ sequences; gag, gene encoding viral structural proteins; MA, matrix protein; CA, capsid protein; NC, nucleocapsid protein; pol, gene encoding reverse transcriptase and integrase; env, gene encoding glycoproteins; U3, unique 3′ sequences; 5′ ss, 5′ splice donor site; 3′ ss, 3′ splice acceptor site. (B) Predicted secondary structure of Mψ. The M-Fold RNA folding program based on the computer algorithm of Zuker et al. (6, 7, 15) was used. The folding energy obtained was −59.0 kcal. The S1, S2, and PS3 regions are outlined.

FIG. 4

(A) The sequences of O3S1B and O3S2B are shown above and below, respectively, the wt O3 stem. Nucleotides differing from those of the wt sequence are highlighted. (B) RNase protection analysis to determine the amount of neo RNA packaged in virions released from G418-resistant mass cultures of Q2bn-4D cells transfected with plasmids, as indicated above each lane, and annealed to the antisense neo probe. The expected locations of free probe and the protected neo band are indicated by arrows. No RNase was added to the sample in lane 10.

FIG. 5

(A) The sequences of O3S1A and O3S2A are shown above and below, respectively, the wt O3 stem. Nucleotides differing from those of the wt sequence are highlighted. (B) RIPA to determine the amount of viral particles released from G418-resistant mass cultures of Q2bn-4D cells transfected with plasmids, as indicated above each lane, and precipitated with the α-PrB antibody. The expected size of the capsid (CA) band is indicated. (C) RNase protection analysis of the RNA from virus released from cells described for panel B. The expected sizes of the free probe and the protected neo probe are indicated by arrows. The sample in lane 1 had no RNase added. The sample in lane 2 had no viral RNA added.

FIG. 6

(A) The sequence of O3S1S2A+PS3. The sequences that differ from that of the wt O3 stem are highlighted. The PS3 region is outlined, and the sequences mutated in O3S1S2A+PS3 are indicated. (B) The predicted base pairing between S1 and PS3 in the O3S1S2A mutant. The nucleotide changes in the O3S1S2A+PS3 mutant are shown in boldface below the PS3 sequence. (C) RNase protection analysis of the RNA from virus released from G418-resistant mass cultures of Q2bn-4D cells transfected with plasmids, as indicated above each lane, and annealed to the neo probe. The expected sizes of the free probe and the protected neo probe are indicated by arrows. The sample in lane 7 had no viral RNA added.

FIG. 6

(A) The sequence of O3S1S2A+PS3. The sequences that differ from that of the wt O3 stem are highlighted. The PS3 region is outlined, and the sequences mutated in O3S1S2A+PS3 are indicated. (B) The predicted base pairing between S1 and PS3 in the O3S1S2A mutant. The nucleotide changes in the O3S1S2A+PS3 mutant are shown in boldface below the PS3 sequence. (C) RNase protection analysis of the RNA from virus released from G418-resistant mass cultures of Q2bn-4D cells transfected with plasmids, as indicated above each lane, and annealed to the neo probe. The expected sizes of the free probe and the protected neo probe are indicated by arrows. The sample in lane 7 had no viral RNA added.