Recombinant human parainfluenza virus type 1 (rHPIV1) was modified to create rHPIV1-P(C؊), a virus in which expression of the C proteins (C, C, Y1, and Y2) was silenced without affecting the amino acid sequence of the P protein. Infectious rHPIV1-P(C؊) was readily recovered from cDNA, indicating that the four C proteins were not essential for virus replication. Early during infection in vitro, rHPIV1-P(C؊) replicated as efficiently as wild-type (wt) HPIV1, but its titer subsequently decreased coincident with the onset of an extensive cytopathic effect not observed with wt rHPIV1. rHPIV1-P(C؊) infection, but not wt rHPIV1 infection, induced caspase 3 activation and nuclear fragmentation in LLC-MK2 cells, identifying the HPIV1 C proteins as inhibitors of apoptosis. In contrast to wt rHPIV1, rHPIV1-P(C؊) and rHPIV1-C F170S , a mutant encoding an F170S substitution in C, induced interferon (IFN) and did not inhibit IFN signaling in vitro. However, only rHPIV1-P(C؊) induced apoptosis. Thus, the anti-IFN and antiapoptosis activities of HPIV1 were separable: both activities are disabled in rHPIV1-P(C؊), whereas only the anti-IFN activity is disabled in rHPIV1-C F170S . In African green monkeys (AGMs), rHPIV1-P(C؊) was considerably more attenuated than rHPIV1-C F170S , suggesting that disabling the anti-IFN and antiapoptotic activities of HPIV1 had additive effects on attenuation in vivo. Although rHPIV1-P(C؊) protected against challenge with wt HPIV1, its highly restricted replication in AGMs and in primary human airway epithelial cell cultures suggests that it might be overattenuated for use as a vaccine. Thus, the C proteins of HPIV1 are nonessential but have anti-IFN and antiapoptosis activities required for virulence in primates.

A novel recombinant human parainfluenza virus type 1 (rHPIV1), rHPIV1-C+P, in which the overlapping open reading frames of the C and P genes were separated in order to introduce mutations into the C gene without affecting P, was generated. Infectious rHPIV1-C+P was readily recovered and replicated as efficiently as HPIV1 wild-type (wt) in vitro and in African green monkeys (AGMs). rHPIV1-C+P expressed increased levels of C protein and, surprisingly, activated the type I IFN and apoptosis responses more strongly than HPIV1 wt. rHPIV1-C+P provided a useful backbone for recovering an attenuated P/C gene mutation, (Δ84-85), which was previously unrecoverable, likely due to detrimental effects of the deletion on the P protein. rHPIV1-C Δ84-85 +P and an additional mutant, rHPIV1-C Δ169-170 +P, were found to replicate to similar titers in vitro and to activate the type I IFN and apoptosis responses to a similar degree as rHPIV1-C+P. rHPIV1-C Δ84-85 +P was found to be highly attenuated in AGMs, and all viruses were immunogenic and effective in protecting AGMs against challenge with HPIV1 wt. rHPIV1-C Δ84-85 +P will be investigated as a potential liveattenuated vaccine candidate for HPIV1.

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