Attenuating mutations in the P/C gene of human parainfluenza virus type 1 (HPIV1) vaccine candidates abrogate the inhibition of both induction and signaling of type I interferon (IFN) by wild-type HPIV1

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b RIG-I and mda-5 are activated by viral RNA and stimulate type I interferon production. Laboratory of genetics and physiology 2 (LGP2) shares homology with RIG-I and mda-5 but lacks the CARD domains required for signaling. The V proteins of paramyxoviruses limit interferon induction by binding mda-5 and preventing its activation; however, they do not bind RIG-I and have not been considered inhibitors of RIG-I signaling. Here we uncover a novel mechanism of RIG-I inhibition in which the V protein of parainfluenzavirus type 5 (PIV5; formerly known as simian virus type 5 [SV5]) interacts with LGP2 and cooperatively inhibits induction by RIG-I ligands. A complex between RIG-I and LGP2 is observed in the presence of PIV5-V, and we propose that this complex is refractory to activation by RIG-I ligands. The V proteins from other paramyxoviruses also bind LGP2 and demonstrate LGP2-dependent inhibition of RIG-I signaling. This is significant, because it demonstrates a general mechanism for the targeting of the RIG-I pathway by paramyxoviruses. Virus infection stimulates innate immune responses in the host, among which the production of type I interferon (IFN) plays a critical role in restricting virus replication, via the upregulation of a large number of IFN-stimulated genes, and through the modulation of subsequent adaptive immune responses (reviewed in reference 37). IFN induction is triggered by the recognition of pathogen-associated molecular patterns (PAMPs), principally those associated with viral nucleic acids, which are seen as foreign by cellular pattern recognition receptors (PRRs; reviewed in reference 21). The RNA helicases RIG-I and mda-5 are the two bestcharacterized cytoplasmic PRRs; RIG-I preferentially recognizes RNA molecules with an uncapped 5=-triphosphate in a short region of blunt double-stranded RNA (dsRNA), while mda-5 recognizes longer molecules of dsRNA which need not be 5=-triphosphorylated (13,16,32,33,42,44; reviewed in reference 43). Viral RNA binds to the C-terminal regulatory domain (RD) of RIG-I or mda-5, and it is this region that confers specificity of PAMP recognition, although the central RNA helicase domain may also be involved in binding longer RNA molecules (22,24,28,49,52). RNA binding causes a conformational change which promotes oligomerization and allows interaction of the N-terminal CARD domains of RIG-I or mda-5 with the mitochondrial adapter protein IPS-1 (also known as VISA, MAVS, and CARDIF). This initiates a signaling cascade leading to activation and nuclear translocation of the transcription factors IRF-3 and NF-B, which are needed to turn on transcription of the IFN-␤ gene.While the interactions between synthetic PAMPs and RIG-I or mda-5 have been well characterized, the types of PAMPs recognized by RIG-I and mda-5 during viral infections are less clear. Mice lacking mda-5 showed no IFN induction in response to the picornavirus encephalomyocarditis virus (EMCV) (10, 17), consistent with the generation of a dsRNA replicative intermediate for this virus; RIG-I 0/0 mice...

Section: Discussionmentioning

confidence: 86%

Paramyxovirus V Proteins Interact with the RNA Helicase LGP2 To Inhibit RIG-I-Dependent Interferon Induction

Childs

Randall

Goodbourn

2012

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119

“…To date, the HPIV1 C proteins have not been as extensively studied as those of SeV. However, the HPIV1 C proteins, like the SeV C proteins, play a role in evasion of host innate immunity through inhibition of type I IFN production and signaling (8,65). Type I IFN was not detected during infection with wild-type (wt) HPIV1 in A549 cells, a human epithelial lung carcinoma cell line, but was induced during infection with a recombinant HPIV1 (rHPIV1) mutant bearing an F170S amino acid substitution in C, designated rHPIV1-C F170S (65).…”

mentioning

confidence: 99%

“…However, the HPIV1 C proteins, like the SeV C proteins, play a role in evasion of host innate immunity through inhibition of type I IFN production and signaling (8,65). Type I IFN was not detected during infection with wild-type (wt) HPIV1 in A549 cells, a human epithelial lung carcinoma cell line, but was induced during infection with a recombinant HPIV1 (rHPIV1) mutant bearing an F170S amino acid substitution in C, designated rHPIV1-C F170S (65). Wt HPIV1, but not the rHPIV1-C F170S mutant, inhibited the antiviral state induced by type I IFN, most likely due to inhibition of STAT1 nuclear translocation in human lung cells (8,65).…”

mentioning

confidence: 99%

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

Bartlett

Cruz

Esker

et al. 2008

Self Cite

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.

“…For native PAGE analysis of IRF3 monomers and dimers, LLC-MK2 cells were lysed in buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% IGEPAL CA-360 (Sigma-Aldrich), 50 mM NaF, 5 mM Na 3 VO 4 , and a Complete miniprotease inhibitor tablet (Roche). Native PAGE was performed using 7% polyacrylamide gels and Tris-glycine running buffer (Bio-Rad, Hercules, CA) containing 0.2% sodium deoxycholate in the cathode chamber (60). Gels were first prerun for 30 min at 40 mA prior to electrophoresis of lysates at 25 mA.…”

Section: Methodsmentioning

confidence: 99%

“…VSV-GFPinfected cells were cultured under 0.8% methylcellulose overlay prepared in MEM and supplemented with 50 g/ml gentamicin sulfate and 4 mM L-glutamine. Plates were read for GFP expression 48 h after VSV-GFP infection using a Typhoon 8600 scanner (Molecular Dynamics), and VSV-GFP-positive foci were counted (60).…”

Section: Methodsmentioning

confidence: 99%

Identification of Human Parainfluenza Virus Type 2 (HPIV-2) V Protein Amino Acid Residues That Reduce Binding of V to MDA5 and Attenuate HPIV-2 Replication in Nonhuman Primates

Schaap-Nutt¹,

Higgins²,

Amaro-Carambot³

et al. 2011

Self Cite

Human parainfluenza virus type 2 (HPIV-2), an important pediatric respiratory pathogen, encodes a V protein that inhibits type I interferon (IFN) induction and signaling. Using reverse genetics, we attempted the recovery of a panel of V mutant viruses that individually contained one of six cysteine-to-serine (residues 193, 197, 209, 211, 214, and 218) substitutions, one of two paired charge-to-alanine (R175A/R176A and R205A/ K206A) substitutions, or a histidine-to-phenylalanine (H174F) substitution. This mutagenesis was performed using a cDNA-derived HPIV-2 virus that expressed the V and P coding sequences from separate mRNAs. Of the cysteine substitutions, only C193S, C214S, and C218S yielded viable virus, and only the C214S mutant replicated well enough for further analysis. The H174F, R175A/R176A, and R205A/K206A mutants were viable and replicated well. The H174F and R205A/K206A mutants did not differ from the wild-type (WT) V in their ability to physically interact with MDA5, a cytoplasmic sensor of nonself RNA that induces type I IFN. Like WT HPIV-2, these mutants inhibited IFN-␤ induction and replicated efficiently in African green monkeys (AGMs). In contrast, the C214S and R175A/R176A mutants did not bind MDA5 efficiently, did not inhibit interferon regulatory factor 3 (IRF3) dimerization or IFN-␤ induction, and were attenuated in AGMs. These findings indicate that V binding to MDA5 is important for HPIV-2 virulence in nonhuman primates and that some V protein residues involved in MDA5 binding are not essential for efficient HPIV-2 growth in vitro. Using a transient expression system, 20 additional mutant V proteins were screened for MDA5 binding, and the region spanning residues 175 to 180 was found to be essential for this activity.