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The structures of murine sarcoma virus (MuSV) tsllO viral RNA and intracellular RNA present in MuSV tsllO-infected cells (6m2 cells) have been examined by S1 nuclease analysis. A previous study involving heteroduplex analysis of MuSV tsllO viral RNAs hybridized to wild-type DNA revealed the presence of two MuSV tsllO RNAs, 4.0 and 3.5 kilobases (kb) in length, containing overlapping central deletions relative to wild-type MuSV 124 viral RNA (Junghans et al., J. Mol. Biol. 161:229-255, 1982). Here we show that the deletion (termed Al) in the 4.0-kb RNA has a 5' border located at about nucleotide 2409 (using the numbering system of Van Beveren et al., Cell 27:97-108, 1981), a position 63 bases upstream of the junction of the p30 and plO coding sequences. The 3' border of the Al deletion is found 1,473 bases downstream at approximately nucleotide 3883, 10 nucleotides downstream of the first mos gene initiation codon. In the 3.5kb MuSV tsllO RNA, the 5' border of the deleted central region (termed A2) is located in a splice consensus donor site at approximately nucleotide 2017, 330 bases downstream from the junction of the p12 and p30 coding sequences, and extends about 1,915 bases in the downstream direction to nucleotide 3935, found in a splice consensus acceptor site about 55 nucleotides downstream of the first mos gene initiation codon and 30 bases upstream of the second initiation codon. No alteration of polyadenylate addition sites was observed in either MuSV tsllO RNA species, as compared with MuSV 349 RNA. The observation that the 5' and 3' borders of the deletion in the 3.5-kb RNA are within in-frame splice donor and acceptor sites suggests strongly that the 3.5-kb RNA is derived from the 4.0-kb RNA by a temperature-sensitive splice mechanism. Data presented here show unequivocally that formation of the 3.5-kb MuSV tsllO RNA from which the P85gag-mos polypeptide is translated is temperature sensitive. At 33°C, with S1 analysis, the 3.5-kb RNA is found readily in 6m2 cells. Within 4 h of a shift to 39°C, however, only trace amounts of this RNA can be found. Moreover, reshifting 6m2 cells to 33°C permits the reappearance of the 3.5-kb RNA at its original level.

ChimeriVax-dengue (DEN) viruses are live attenuated vaccine candidates. They are constructed by replacing the premembrane (prM) and envelope (E) genes of the yellow fever (YF) 17D virus vaccine with the corresponding genes from wild-type DEN viruses (serotypes 1-4) isolated from humans. In this study, the growth kinetics of ChimeriVax-DEN1-4 and parent viruses (wild-type DEN-1-4 and YF 17D) were assessed in human myeloid dendritic cells (DCs) and in three hepatic cell lines (HepG2, Huh7, and THLE-3). In DC, ChimeriVax-DEN-1-4 showed similar growth kinetics to their parent viruses, wild-type DEN virus (propagated in Vero cells), or YF 17D virus (peak titers ~3-4.5 log(10) plaque-forming units (PFU)/mL at 48-72 hours post-infection). Parent wild-type DEN-1-4 viruses derived from C6/36 mosquito cells did not show any growth at a multiplicity of infection of 0.1 in DCs, except for DEN-2 virus, which grew to a modest titer of 2.5 log(10) PFU/mL at 48 hours post-infection. ChimeriVax-DEN1-4 grew to significantly lower titers (2-5 log(10) PFU/mL) than YF 17D virus in hepatic cell lines THLE-3 and HepG2, but not in Huh7 cells. These experiments suggest that ChimeriVax-DEN1-4 viruses replicate similarly to YF-VAX in DCs, but at a lower level than YF 17D virus in hepatic cell lines. The lack of growth of chimeric viruses in human hepatic cells suggests that these viruses may be less hepatotropic than YF 17D virus vaccine in humans.

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A live chimeric vaccine virus against Japanese encephalitis (JE), ChimeriVax-JE, was used to define methods for optimal, random insertion of foreign immunologic determinants into flavivirus glycoproteins. The conserved M2e peptide of influenza A virus was randomly inserted into the yellow fever-specific NS1 glycoprotein of ChimeriVax-JE. A technique combining plaque purification with immunostaining yielded a recombinant virus that stably expressed M2e at NS1-236 site. The site was found permissive for other inserts. The insertion inhibited NS1 dimerization in vitro, which had no significant effect on virus replication in vitro and immunogenicity in vivo. Two different NS1-specific monoclonal antibodies and a polyclonal antibody efficiently recognized only the NS1 protein dimer, but not monomer. Adaptation of the virus to Vero cells resulted in two amino acid changes upstream from the insert which restored NS1 dimerization. Immunized mice developed high-titer M2e-specific antibodies predominantly of the IgG2A isotype indicative of a Th1-biased response.

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Numerous viruses of the Flaviviridae family, including dengue, yellow fever, Japanese encephalitis, and West Nile, cause significant disease in humans and animals. The structure and function of the molecular components of the flavivirus envelope are therefore of significant interest. To our knowledge, a membrane (M) protein mutation which affects the pH at which flavivirus particles are inactivated in vitro has never been reported. Here we show that substitution of proline for glutamine at residue M5 (MQ5P) of a Japanese encephalitis-yellow fever chimera (ChimeriVax-JE) increases its acid sensitivity in vitro by 0.3 pH units (i.e., increases the pH at which virus titer is reduced by 50% from 6.08 to 6.38). In addition, growth kinetics of this mutant virus are accelerated in Vero cells, while neurovirulence and neuroinvasiveness measured in a mouse model are unaffected. A possible interpretation of these observations is that M can modulate the envelope (E) protein function during cell infection.

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