Spontaneous and engineered deletions in the 3' noncoding region of tick-borne encephalitis virus: construction of highly attenuated mutants of a flavivirus

Abstract

The flavivirus genome is a positive-strand RNA molecule containing a single long open reading frame flanked by noncoding regions (NCR) that mediate crucial processes of the viral life cycle. The 3' NCR of tick-borne encephalitis (TBE) virus can be divided into a variable region that is highly heterogeneous in length among strains of TBE virus and in certain cases includes an internal poly(A) tract and a 3'-terminal conserved core element that is believed to fold as a whole into a well-defined secondary structure. We have now investigated the genetic stability of the TBE virus 3' NCR and its influence on viral growth properties and virulence. We observed spontaneous deletions in the variable region during growth of TBE virus in cell culture and in mice. These deletions varied in size and location but always included the internal poly(A) element of the TBE virus 3' NCR and never extended into the conserved 3'-terminal core element. Subsequently, we constructed specific deletion mutants by using infectious cDNA clones with the entire variable region and increasing segments of the core element removed. A virus mutant lacking the entire variable region was indistinguishable from wild-type virus with respect to cell culture growth properties and virulence in the mouse model. In contrast, even small extensions of the deletion into the core element led to significant biological effects. Deletions extending to nucleotides 10826, 10847, and 10870 caused distinct attenuation in mice without measurable reduction of cell culture growth properties, which, however, were significantly restricted when the deletion was extended to nucleotide 10919. An even larger deletion (to nucleotide 10994) abolished viral viability. In spite of their high degree of attenuation, these mutants efficiently induced protective immune responses even at low inoculation doses. Thus, 3'-NCR deletions represent a useful technique for achieving stable attenuation of flaviviruses that can be included in the rational design of novel flavivirus live vaccines.

FIG. 1

Schematic representation of the 3′ NCR of TBE virus Neudoerfl. Numbers correspond to the full-length genomic sequence (GenBank accession no. U27495). Shaded areas depict sequence elements that were described previously (15, 31): R1, R2, R3, direct repeats; polyA, internal poly(A) sequence, here 49 residues long, as it is in the infectious cDNA clone of TBE virus; IR, inverted repeat; PU, homopurine box; PY, homopyrimidine box; PR, pyrimidine-rich box. The positions of spontaneous 3′-NCR deletions observed during cell culture growth (BHK), in suckling-mouse brain stock solutions (s.m.b.), and in virus isolated from adult mouse brains are aligned below the schematic. For the exact positions of the deletion boundaries, refer to Tables 1 and 2. The bottom section shows the engineered deletions which extend from nucleotide 10378 to the positions as given in the corresponding plasmid name. A boxed A represents the 6-nucleotide AgeI recognition sequence which was retained in the construction procedure.

FIG. 2

RNA secondary-structure predictions of the 3′ NCRs of the TBE virus deletion mutants. Sequences are shown starting with the first nucleotide following the stop codon, which is also the first nucleotide of an AgeI restriction site (Fig. 1). At the top is the structure of the 3′ NCR as present in mutant R-Nd/3′Δ10795, corresponding exactly to the previously published structure of the TBE 3′-NCR core element (27). The borders of the individual engineered deletions are indicated by the corresponding nucleotide numbers. Positions of the sequence elements R3, IR, PU, PY, and PR (compare Fig. 1) are indicated by triangles next to the sequence. Base-pairing elements are denoted by Roman numerals (I to X), MS depicts a multiloop-stem, and A1 and A2 are the known conserved flavivirus 3′-terminal structures. Below are structures as predicted for the truncated sequences present in the various deletion mutants. Wild-type structural elements are shown in boldface type; sequences in undefined or nonauthentic folding patterns are shown in normal type. A truncated form of stem VII is shown in parentheses. The two sequences carrying the largest deletions may adopt two alternative structures in which the energetically favored ones (energy differences are shown) lack some of the wild-type base-pairing elements.

FIG. 3

Growth curves of wild-type and mutant TBE virus strains determined on primary chicken embryo cells infected at an m.o.i. of 1. The values plotted were derived from duplicate experiments. ▪, Neudoerfl; ▴, R-Nd/3′Δ10795; ▵, R-Nd/3′Δ10826; ○, R-Nd/3′Δ10847; •, R-Nd/3′Δ10870; □, R-Nd/3′Δ10919.