Mutations in the C, D, and V open reading frames of human parainfluenza virus type 3 attenuate replication in rodents and primates

Human parainfluenza virus type 3 (HPIV3) is a single-stranded negative-sens e RNA virus belonging to the Respirovirus genus of the Paramyxoviridae fami ly in the order Mononegavirales. The P gene encodes at least four proteins, including the C protein, which is expressed from an open reading frame (OR F) that overlaps the P ORF, and the D protein, which is encoded when the P ORF is fused to the D ORF by transcriptional editing. The P mRNA also conta ins a third ORF for the V protein, although it is unclear how or whether th is ORF is accessed. We have used recombinant DNA technology to recover five mutant viruses that either interrupt or alter the C, D, and V ORFs. In one mutant virus, rC-KO, expression of the C protein was abrogated by changing the start codon from methionine to threonine and introducing two stop codo ns at amino acid positions 7 and 26 of the C ORF. Tn a second mutant virus, rF164S, a point mutation was introduced into the C ORF changing amino acid position 164 from phenylalanine (F) to serine (S), which corresponds to th e F170S mutation described in the C protein of Sendai virus (Itoh et ai., J . Gen. Virol. 78, 3207-3215). rC-KO was significantly attenuated in vitro a nd in vivo (rodents and primates), whereas rF164S was attenuated only in vi vo. Interestingly, the rF164S mutant was more attenuated in the upper than in the lower respiratory tract of hamsters and monkeys. This pattern is the converse of that seen with temperature-sensitive attenuating mutations, an d thus inclusion of this novel mutation in a recombinant live-attenuated va ccine candidate might prove useful in reducing residual virulence in the up per respiratory tract. Both rC-KO and rF164S conferred protection against c hallenge with wild-type HPIV3. In three other viruses, the D and V ORFs wer e interrupted singly or in combination. Although interruption of the D and V ORFs individually did not affect virus replication in vitro or in vivo, i nterruption of both together attenuated replication in vivo. These results indicate that the C, D, and V proteins of HPIV3 each has a role in virus re plication in vitro, in vivo, or both, and define mutations that might be us eful for the development of a vaccine against HPIV3.