Human Parainfluenza Virus Type 1 C Proteins Are Nonessential Proteins That Inhibit the Host Interferon and Apoptotic Responses and Are Required for Efficient Replication in Nonhuman Primates

Abstract

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.

Attenuated Human Parainfluenza Virus Type 1 Expressing Ebola Virus Glycoprotein GP Administered Intranasally Is Immunogenic in African Green Monkeys

“…One aspect of this study was to increase safety by using an attenuated virus instead of a wt virus as was previously used in rHPIV3/EboGP. The C Δ170 mutation in HPIV1 was designed in previous studies to confer stability against deattenuation and to confer substantial attenuation in NHPs (15), and it thus should have increased safety in humans. Another aspect of this study was to explore the possibility of increasing GP immunogenicity by increasing its packaging in the vector particle by TMCT modification.…”

“…We previously developed a non-temperature-sensitive attenuating mutation, called C Δ170 , that consists of a 6-nucleotide deletion in the overlapping P and C ORFs (16,17). This mutation reduces the ability of C proteins to inhibit the host type I interferon response and apoptosis (15,18,19), resulting in viral attenuation. We previously evaluated HPIV1 as a vaccine vector for expressing the respiratory syncytial virus (RSV) fusion (F) glycoprotein and showed that the HPIV1-C Δ170 backbone was the better of two attenuated backbones (17).…”

“…The HPIV1 genome consists of 6 genes encoding the nucleoprotein (N), phosphoprotein (P/C), internal matrix protein (M), fusion glycoprotein (F), hemagglutinin-neuraminidase glycoprotein (HN), and the large polymerase protein (L) (14). The P gene carries an additional overlapping open reading frame expressing the C proteins, which antagonize host interferon and apoptosis responses (15). We previously developed a non-temperature-sensitive attenuating mutation, called C Δ170 , that consists of a 6-nucleotide deletion in the overlapping P and C ORFs (16,17).…”

Host specificity of the anti-interferon and anti-apoptosis activities of parainfluenza virus P/C gene products

“…rSeVhP infection induces significant cytopathogenicity due to apoptosis in murine cells compared with SeV Several studies have suggested that the accessory proteins of the P/C/V genes of various paramyxoviruses are able to antagonize cell death and apoptotic activity (Bartlett et al, 2008a;He et al, 2002;Kato et al, 1997). To determine the effect and specificity of the P/C gene products in cell death, we infected MLg2908 and A549 cells with the viruses and observed the cytopathic effect (CPE).…”

“…Regulation of virally induced apoptosis is another viral mechanism of evading host protection. The V and C proteins of SeV, as well as HPIV-1 C protein, have been shown to be important elements in eliminating apoptotic cell death (Bartlett et al, 2008a;Kato et al, 1997;Koyama et al, 2003).…”