The C proteins of human parainfluenza virus type 1 limit double-stranded RNA accumulation that would otherwise trigger activation of MDA5 and protein kinase R

Human parainfluenza virus type 1 (HPIV1) is an important respiratory pathogen in young children, the immunocompromised, and the elderly. We found that infection with wild-type (WT) HPIV1 suppressed the innate immune response in human airway epithelial cells by preventing not only phosphorylation of interferon regulatory factor 3 (IRF3) but also degradation of IκBβ, thereby inhibiting IRF3 and NF-κB activation, respectively. Both of these effects were ablated by a F170S substitution in the HPIV1 C proteins (F170S) or by silencing the C open reading frame [P(C-)], resulting in a potent beta interferon (IFN-β) response. Using murine knockout cells, we found that IFN-β induction following infection with either mutant relied mainly on melanoma-associated differentiation gene 5 (MDA5) rather than retinoic acid-inducible gene I (RIG-I). Infection with either mutant, but not WT HPIV1, induced a significant accumulation of intracellular double-stranded RNA (dsRNA). These mutant viruses directed a marked increase in the accumulation of viral genome, antigenome, and mRNA that was coincident with the accumulation of dsRNA. In addition, the amount of viral proteins was reduced compared to that of WT HPIV1. Thus, the accumulation of dsRNA might be a result of an imbalance in the N protein/genomic RNA ratio leading to incomplete encapsidation. Protein kinase R (PKR) activation and IFN-β induction followed the kinetics of dsRNA accumulation. Interestingly, the C proteins did not appear to directly inhibit intracellular signaling involved in IFN-β induction; instead, their role in preventing IFN-β induction appeared to be in suppressing the formation of dsRNA. PKR activation contributed to IFN-β induction and also was associated with the reduction in the amount of viral proteins. Thus, the HPIV1 C proteins normally limit the accumulation of dsRNA and thereby limit activation of IRF3, NF-κB, and PKR. If C protein function is compromised, as in the case of F170S HPIV1, the resulting PKR activation and reduction in viral protein levels enable the host to further reduce C protein levels and to mount a potent antiviral type I IFN response.     

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

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.     

Down-regulation of paramyxovirus hemagglutinin-neuraminidase glycoprotein surface expression by a mutant fusion protein containing a retention signal for the endoplasmic reticulum.

The human parainfluenza virus type 3 (HPIV3) fusion (F) and hemagglutinin-neuraminidase (HN) glycoproteins are the principal components involved in virion receptor binding, membrane penetration, and ultimately, syncytium formation. While the requirement for both F and HN in this process has been determined from recombinant expression studies, stable physical association of these proteins in coimmunoprecipitation studies has not been observed. In addition, coexpression of other heterologous paramyxovirus F or HN glycoproteins with either HPIV3 F or HN does not result in the formation of syncytia, suggesting serotype-specific protein differences. In this study, we report that simian virus 5 and Sendai virus heterologous HN proteins and measles virus hemagglutinin (H) were found to be down-regulated when coexpressed with HPIV3 F. As an alternative to detecting physical associations of these proteins by coimmunoprecipitation, further studies were performed with a mutant HPIV3 F protein (F-KDEL) lacking a transmembrane anchor and cytoplasmic tail and containing a carboxyl-terminal retention signal for the endoplasmic reticulum (ER). F-KDEL was defective for transport to the cell surface and could down-regulate surface expression of HPIV3 HN and heterologous HN/H proteins from simian virus 5, Sendai virus, and measles virus in coexpression experiments. HN/H down-regulation appeared to result, in part, from an early block to HPIV3 HN synthesis, as well as an instability of the heterologous HN/H proteins within the ER. In contrast, coexpression of F-KDEL with HPIV3 wild-type F or the heterologous receptor-binding proteins, respiratory syncytial virus glycoprotein (G) and vesicular stomatitis virus glycoprotein (G), were not affected in transport to the cell surface. Together, these results support the notion that the reported serotype-specific restriction of syncytium formation may involve, in part, down-regulation of heterologous HN expression.     

Generation of recombinant human parainfluenza virus type 1 vaccine candidates by importation of temperature-sensitive and attenuating mutations from heterologous paramyxoviruses

Human parainfluenza virus type 1 (HPIV1) is a significant cause of respiratory tract disease in infants and young children for which a vaccine is needed. In the present study, we sought to attenuate HPIV1 by the importation of one or more known attenuating point mutations from heterologous paramyxoviruses into homologous sites in HPIV1. The introduced mutations were derived from three attenuated paramyxoviruses: (i) HPIV3cp45, a live-attenuated HPIV3 vaccine candidate containing multiple attenuating mutations; (ii) the respiratory syncytial virus cpts530 with an attenuating mutation in the L polymerase protein; and (iii) a murine PIV1 (MPIV1) attenuated by a mutation in the accessory C protein. Recombinant HPIV1 (rHPIV1) mutants bearing a single imported mutation in C, any of three different mutations in L, or a pair of mutations in F exhibited a 100-fold or greater reduction in replication in the upper or lower respiratory tract of hamsters. Both temperature-sensitive (ts) (mutations in the L and F proteins) and non-ts (the mutation in the C protein) attenuating mutations were identified. rHPIV1 mutants containing a combination of mutations in L were generated that were more attenuated than viruses bearing the individual mutations, showing that the systematic accretion of mutations can yield progressive increases in attenuation. Hamsters immunized with rHPIV1 mutants bearing one or two mutations developed neutralizing antibodies and were resistant to challenge with wild-type HPIV1. Thus, importation of attenuating mutations from heterologous viruses is an effective means for rapidly identifying mutations that attenuate HPIV1 and for generating live-attenuated HPIV1 vaccine candidates.     

Differential Regulation of Type I Interferon and Epidermal Growth Factor Pathways by a Human Respirovirus Virulence Factor

A number of paramyxoviruses are responsible for acute respiratory infections in children, elderly and immuno-compromised individuals, resulting in airway inflammation and exacerbation of chronic diseases like asthma. To understand the molecular pathogenesis of these infections, we searched for cellular targets of the virulence protein C of human parainfluenza virus type 3 (hPIV3-C). We found that hPIV3-C interacts directly through its C-terminal domain with STAT1 and GRB2, whereas C proteins from measles or Nipah viruses failed to do so. Binding to STAT1 explains the previously reported capacity of hPIV3-C to block type I interferon signaling, but the interaction with GRB2 was unexpected. This adaptor protein bridges Epidermal Growth Factor (EGF) receptor to MAPK/ERK pathway, a signaling cascade recently found to be involved in airway inflammatory response. We report that either hPIV3 infection or transient expression of hPIV3-C both increase cellular response to EGF, as assessed by Elk1 transactivation and phosphorylation levels of ERK1/2, 40S ribosomal subunit protein S6 and translation initiation factor 4E (eIF4E). Furthermore, inhibition of MAPK/ERK pathway with U0126 prevented viral protein expression in infected cells. Altogether, our data provide molecular basis to explain the role of hPIV3-C as a virulence factor and determinant of pathogenesis and demonstrate that Paramyxoviridae have evolved a single virulence factor to block type I interferon signaling and to boost simultaneous cellular response to growth factors.     

The structural and accessory proteins M,ORF 4a,ORF 4b,and ORF 5 of Middle East respiratory syndrome coronavirus (MERS-CoV) are potent interferon antagonists

The newly emerged Middle East respiratory syndrome coronavirus (MERS-CoV) is a highly pathogenic respiratory virus with pathogenic mechanisms that may be driven by innate immune pathways. The goal of this study is to characterize the expression of the structural (S, E, M, N) and accessory (ORF 3, ORF 4a, ORF 4b, ORF 5) proteins of MERS-CoV and to determine whether any of these proteins acts as an interferon antagonist. Individual structural and accessory protein-coding plasmids with an N-terminal HA tag were constructed and transiently transfected into cells, and their native expression and subcellular localization were assessed using Wes tern blotting and indirect immunofluorescence. While ORF 4b demonstrated majorly nuclear localization, all of the other proteins demonstrated cytoplasmic localization. In addition, for the first time, our experiments revealed that the M, ORF 4a, ORF 4b, and ORF 5 proteins are potent interferon antagonists. Further examination revealed that the ORF 4a protein of MERS-CoV has the most potential to counteract the antiviral effects of IFN via the inhibition of both the interferon production (IFN-β promoter activity, IRF-3/7 and NF-κB activation) and ISRE promoter element signaling pathways. Together, our results provide new insights into the function and pathogenic role of the structural and accessory proteins of MERS-CoV.