C Proteins: Controllers of Orderly Paramyxovirus Replication and of the Innate Immune Response

Particles of many paramyxoviruses include small amounts of proteins with a molecular weight of about 20 kDa. These proteins, termed “C”, are basic, have low amino acid homology and some secondary structure conservation. C proteins are encoded in alternative reading frames of the phosphoprotein gene. Some viruses express nested sets of C proteins that exert their functions in different locations: In the nucleus, they interfere with cellular transcription factors that elicit innate immune responses; in the cytoplasm, they associate with viral ribonucleocapsids and control polymerase processivity and orderly replication, thereby minimizing the activation of innate immunity. In addition, certain C proteins can directly bind to, and interfere with the function of, several cytoplasmic proteins required for interferon induction, interferon signaling and inflammation. Some C proteins are also required for efficient virus particle assembly and budding. C-deficient viruses can be grown in certain transformed cell lines but are not pathogenic in natural hosts. C proteins affect the same host functions as other phosphoprotein gene-encoded proteins named V but use different strategies for this purpose. Multiple independent systems to counteract host defenses may ensure efficient immune evasion and facilitate virus adaptation to new hosts and tissue environments.

Structural Insight into the Interaction of Sendai Virus C Protein with Alix To Stimulate Viral Budding

The Sendai virus (SeV), belonging to the Respirovirus genus of the family Paramyxoviridae , harbors an accessory protein, named C protein, which facilitates the viral pathogenicity in mice. In addition, the C protein is known to stimulate the budding of virus-like particles through the binding to the host ALG-2 interacting protein X (Alix), a component of the endosomal sorting complexes required for transport (ESCRT) machinery. However, siRNA-mediated gene knockdown studies suggested that neither Alix nor C protein are related to the SeV budding. In the present study, we determined the crystal structure of a complex comprising of the C -terminal half of the C protein (Y3) and the Bro1 domain of Alix at a resolution of 2.2 Å, to investigate the role of the association in the SeV budding. The structure revealed that a novel consensus sequence, LxxW, which is conserved among the Respirovirus C proteins, is important for the Alix-binding. SeV possessing a mutated C protein with a reduced Alix-binding affinity showed impaired virus production, which correlated with the binding affinity. Infectivity analysis showed a 160-fold reduction at 12 h post-infection compared with non-mutated virus, while C protein competes with CHMP4, one subunit of the ESCRT-III complex, on the binding to Alix. Altogether, these results highlight the critical role of C protein in the SeV budding. IMPORTANCE Human parainfluenza virus type I (hPIV1) is a respiratory pathogen affecting in young children, immunocompromised patients, and the elderly, with no available vaccines or antiviral drugs. Sendai virus (SeV), a murine counterpart of hPIV1, has been extensively studied to determine the molecular and biological properties of hPIV1. These viruses possess a multifunctional accessory protein, C protein, which is essential for stimulating the viral reproduction, however, its role in budding remains controversial. In the present study, the crystal structure of the C -terminal half of the SeV C protein associated with the Bro1 domain of Alix, a component of a cell membrane modulating machinery ESCRT, was elucidated. Based on the structure, we designed mutated C proteins with different binding affinity to Alix, and showed that the interaction between C and Alix is vital for the viral budding. These findings provide new insights into the development of a new antiviral drugs against hPIV1.

C Protein is Essential for Canine Distemper Virus Virulence and Pathogenicity in Ferrets

Paramyxoviruses, including members of the genus Morbillivirus, express accessory proteins with ancillary functions during viral replication. One of these, the C protein, is expressed from an alternate open reading frame (ORF) located in the P gene. The measles virus (MeV) C protein has been implicated in modulation of interferon signaling, but has more recently been shown to play a vital role in regulation of viral transcription and replication, preventing the excessive production of double-stranded RNA. Failure to do so, as seen with C-deficient MeV, leads to early activation of innate immune responses resulting in restriction of viral replication and attenuation in the host. One puzzling aspect of morbillivirus C protein biology has been the finding that a C-deficient canine distemper virus (CDV) generated with a similar mutagenesis strategy displayed no attenuation in ferrets, an animal model commonly used to evaluate CDV pathogenesis. To resolve how virus lacking this protein could maintain virulence, we re-visited the CDV C protein and found that truncated C proteins are expressed from the CDV gene using alternative downstream start codons even when the first start codon was disrupted. We introduced an additional point mutation abrogating expression of these truncated C proteins. A new CDV with this mutation was attenuated in vitro and led to increased activation of protein kinase R. It was also strongly attenuated in ferrets, inducing only mild disease in infected animals, thus replicating the phenotype of C-deficient MeV. Our results demonstrate the crucial role of morbillivirus C proteins in pathogenesis. IMPORTANCE The measles (MeV) and canine distemper viruses (CDV) express accessory proteins that regulate the host immune response and enhance replication. The MeV C protein is critical in preventing the generation of excess immunostimulatory double-stranded RNA. C protein-deficient MeV is strongly attenuated compared to wild-type virus, whereas CDV with a similarly disrupted C open reading frame is fully pathogenic. Here we show that CDV can compensate the disrupting mutations by expression of truncated, but apparently functional C proteins from several alternative start codons. We generated a new recombinant CDV that does not express these truncated C protein. This virus was attenuated both in cell culture and in ferrets, and finally resolves the paradox of the MeV and CDV C proteins, showing that both in fact have similar functions important for viral pathogenesis.

Lamin A/C and the Immune System: One Intermediate Filament, Many Faces

Nuclear envelope lamin A/C proteins are a major component of the mammalian nuclear lamina, a dense fibrous protein meshwork located in the nuclear interior. Lamin A/C proteins regulate nuclear mechanics and structure and control cellular signaling, gene transcription, epigenetic regulation, cell cycle progression, cell differentiation, and cell migration. The immune system is composed of the innate and adaptive branches. Innate immunity is mediated by myeloid cells such as neutrophils, macrophages, and dendritic cells. These cells produce a rapid and nonspecific response through phagocytosis, cytokine production, and complement activation, as well as activating adaptive immunity. Specific adaptive immunity is activated by antigen presentation by antigen presenting cells (APCs) and the cytokine microenvironment, and is mainly mediated by the cellular functions of T cells and the production of antibodies by B cells. Unlike most cell types, immune cells regulate their lamin A/C protein expression relatively rapidly to exert their functions, with expression increasing in macrophages, reducing in neutrophils, and increasing transiently in T cells. In this review, we discuss and summarize studies that have addressed the role played by lamin A/C in the functions of innate and adaptive immune cells in the context of human inflammatory and autoimmune diseases, pathogen infections, and cancer.

Interactions of Destruxin A with Silkworms’ Arginine tRNA Synthetase and Lamin-C Proteins

Destruxin A (DA), a cyclodepsipeptidic mycotoxin produced by entomopathogenic fungus Metarhizium anisopliae, has good insecticidal activity and potential to be a new pesticide. However, the mechanism of action is still obscure. Our previous experiments showed that DA was involved in regulation of transcription and protein synthesis and suggested that silkworms’ arginine tRNA synthetase (BmArgRS), Lamin-C Proteins (BmLamin-C) and ATP-dependent RNA helicase PRP1 (BmPRP1) were candidates of DA-binding proteins. In this study, we employed bio-layer interferometry (BLI), circular dichroism (CD), cellular thermal shift assay (CETSA), and other technologies to verify the interaction of DA with above three proteins in vitro and in vivo. The results of BLI indicated that BmArgRS and BmLamin-C were binding-protein of DA with KD value 5.53 × 10−5 and 8.64 × 10−5 M, but not BmPRP1. These interactions were also verified by CD and CETSA tests. In addition, docking model and mutants assay in vitro showed that BmArgRS interacts with DA at the pocket including Lys228, His231, Asp434 and Gln437 in its enzyme active catalysis region, while BmLamin-C binds to DA at His524 and Lys528 in the tail domain. This study might provide new insight and evidence in illustrating molecular mechanism of DA in breaking insect.