Three Amino Acid Substitutions in the L Protein of the Human Parainfluenza Virus Type 3 cp 45 Live Attenuated Vaccine Candidate Contribute to Its Temperature-Sensitive and Attenuation Phenotypes

Abstract

Studies were initiated to define the genetic basis of the temperature-sensitive (ts), cold adaptation (ca), and attenuation (att) phenotypes of the human parainfluenza virus type 3 (PIV3) cp45 live attenuated vaccine candidate. Genetic data had previously suggested that the L polymerase protein of cp45, which contains three amino acid substitutions at positions 942, 992, and 1558, contributed to its temperature sensitivity (R. Ray, M. S. Galinski, B. R. Heminway, K. Meyer, F. K. Newman, and R. B. Belshe, J. Virol. 70:580–584, 1996; A. Stokes, E. L. Tierney, C. M. Sarris, B. R. Murphy, and S. L. Hall, Virus Res. 30:43–52, 1993). To study the individual and aggregate contributions that these amino acid substitutions make to the ts, att, and ca phenotypes of cp45, seven PIV3 recombinant viruses (three single, three double, and one triple mutant) representing all possible combinations of the three amino acid substitutions were recovered from full-length antigenomic cDNA and analyzed for their ts, att, and caphenotypes. None of the seven mutant recombinant PIVs was cold adapted. The substitutions at L protein amino acid positions 992 and 1558 each specified a 105-fold reduction in plaque formation in cell culture at 40°C, whereas the substitution at position 942 specified a 300-fold reduction. Thus, each of the three mutations contributes individually to the ts phenotype. The triple recombinant which possesses an L protein with all three mutations was almost as temperature sensitive as cp45, indicating that these mutations are the major contributors to the ts phenotype ofcp45. The three individual mutations in the L protein each contributed to restricted replication in the upper or lower respiratory tract of hamsters, and this likely contributes to the observed stability of the ts and att phenotypes ofcp45 during replication in vivo. Importantly, the recombinant virus possessing L protein with all three mutations was as restricted in replication as was the cp45 mutant in both the upper and lower respiratory tracts of hamsters, indicating that the L gene of the cp45 virus is a major attenuating component of this candidate vaccine.

Role of fusion protein cleavage site in the virulence of Newcastle disease virus

“…For instance, mutations in the leader region of human parainfluenza virus 3 (hPIV3) vaccine candidates have been shown to specify attenuation phenotype [21]. Besides the envelope protein genes affecting neurovirulence, there have been reports of several viruses, like the human parainfluenza virus [22], polio virus [23], measles virus [24], corona virus [25], bunya virus [26] and Sindbis virus [27], in which alterations in the internal protein genes affect virus tropism and virulence.…”

A Single Amino Acid Substitution in the Viral Polymerase Creates a Temperature-Sensitive and Attenuated Recombinant Bovine Parainfluenza Virus Type 3

“…4). These three amino acid substitutions, Y942H, L992F, T1558I, in the L protein of hPIV3 have been shown to result in temperature-sensitive and attenuation phenotypes when present alone or in combination (Skiadopoulos et al, 1998). None of the three point mutations present in cp45 hPIV3 were close in primary sequence to the I1103V change found in r-bPIV3.…”

Human parainfluenza virus type 3 expressing the respiratory syncytial virus pre-fusion F protein modified for virion packaging yields protective intranasal vaccine candidates

“…The goal of the present study was to evaluate the level of attenuation, immunogenicity, protective efficacy, and stability of unmodified rHPIV3 JS strain expressing the DS-Cav1 form of RSV F, with and without TMCT modification. In subsequent studies, we will add additional attenuating mutations as needed to achieve an optimal balance of attenuation and immunogenicity, using attenuating mutations that have been identified and characterized previously [26][27][28].…”