It is shown that Cedar virus does use mRNA editing, but at an unusual sequence and by inserting either an adenine or a guanine nucleotide and generating a previously unknown protein, termed U, that shares selected features with the V and W proteins of other henipaviruses.
Abstract
Highly pathogenic Hendra and Nipah viruses encode accessory P gene products (C, V and W) that antagonize innate immunity and contribute to pathogenicity. Cedar virus (CedV), an apathogenic bat-borne henipavirus, is presumed to lack P gene mRNA editing and therefore is unable to express V and W proteins. Here, we identify CedV peptides originating from a frameshifted P gene open reading frame and demonstrate a previously unrecognized, noncanonical editing site at a homopolymeric adenine tract that introduces single-nucleotide A or G insertion. This mRNA editing produces a protein that we refer to as U protein, whose C-terminal domain shares sequence and predicted structural features with those of the henipavirus V protein. Recombinant CedV mutants defective in mRNA editing were only recoverable by trans-complementation and showed markedly reduced release of infectious virus in cell culture and attenuated replication in mice lacking type I interferon receptor. Our data revise the CedV gene expression models and reveal a noncanonical editing mechanism that supports the production of a U protein critical for efficient infectious virus release. These results expand the fundamental concepts of paramyxovirus gene expression and reveal an unexpected requirement for P-gene editing in efficient infectious-virus production, with implications for the evaluation of potentially high-consequence paramyxoviruses. Author Summary Cedar virus is a close relative of the highly pathogenic Nipah and Hendra viruses but is considered non-pathogenic. Unlike these viruses, Cedar virus was thought to lack a mechanism called P-gene mRNA editing, which allows related viruses to produce additional proteins that support infection. Here, we show that Cedar virus does use mRNA editing, but at an unusual sequence and by inserting either an adenine or a guanine nucleotide. This editing event generates a previously unknown protein, termed U, that shares selected features with the V and W proteins of other henipaviruses. Most strikingly, viruses unable to produce U released far fewer infectious virus, showed abnormal membrane-associated structures, and replicated less efficiently in susceptible mice. These findings revise the current model of Cedar virus gene expression and reveal that mRNA editing can contribute directly to efficient virus production, not only to immune evasion. More broadly, our results highlight the need to search for unconventional editing sites when annotating and assessing newly discovered paramyxoviruses.
Marek's disease virus (MDV), an oncogenic alphaherpesvirus, induces severe immunosuppression and T-cell lymphomas in chickens, posing a major threat to poultry production. Viral morphogenesis in alphaherpesviruses depends on coordinated membrane remodeling processes mediated by conserved viral membrane proteins. Although MDV encodes a homolog of UL20 protein, its functional role remains unknown. In this study, we employed an integrated approach combining bioinformatic analysis, bacterial artificial chromosome (BAC)-based mutagenesis, and ultrastructural characterization to elucidate the role of MDV UL20 in viral replication and morphogenesis. Sequence analysis revealed that MDV UL20 is a conserved four-pass transmembrane protein with a membrane topology similar to that of UL20 homologs in other alphaherpesviruses. To assess its functional relevance, a UL20 deletion mutant (Md5BACΔUL20) was generated, and its biological properties were evaluated in chicken embryonic fibroblasts. Deletion of UL20 completely abolished the production of infectious virus, resulting in the absence of plaque formation and impaired cell-to-cell spread. In contrast, genetic reconstitution of UL20 fully restored viral replication to wild-type levels. Transmission electron microscopy demonstrated that UL20 deletion did not affect nuclear capsid assembly but markedly reduced the number of cytoplasmic capsids, secondary envelopment intermediates, and extracellular virion accumulation. UL20 re-expression assays confirmed that these defects were specifically attributable to the loss of UL20. Collectively, these findings demonstrate that MDV UL20 is dispensable for nuclear capsid formation but essential for cytoplasmic secondary envelopment and infectious virion production, providing new insights into the membrane-associated mechanisms underlying MDV morphogenesis.
The Geminiviridae family encompasses over 500 species of economically important plant viruses. A new geminivirus species named parsley yellow leaf curl virus (PYLCV) was first identified in 2020 in Southeastern Iran. The PYLCV genome, comprising 2,779 nucleotides, exhibits a pairwise sequence identity of less than 66% with other geminiviruses and encodes a divergent V2 protein that lacks sequence similarity to known proteins in GenBank. This study explores the functions of this V2 through various analyses. An assessment of the V2 amino acid composition indicates a higher prevalence of acidic residues. By expressing N-terminal and C-terminal GFP fusions (GFP-V2 and V2-GFP) in Nicotiana benthamiana, we demonstrate that V2 localizes to the cytoplasm, nucleus, endoplasmic reticulum, and the Cajal body. Notably, PYLCV V2 suppressed local PTGS and delayed systemic PTGS in GFP-based silencing assays and restored GFP transcript accumulation in a systemic 16c-TGS assay, indicating suppression of transcriptional silencing. Co-immunoprecipitation assays further showed that PYLCV V2 interacts with NbAGO4-1 and with itself, but not detectably with NbSGS3 or NbHDA6, supporting a potential role for PYLCV V2 in targeting the AGO4-mediated transcriptional silencing pathway. Ectopic expression of V2 via a PVX vector induced necrotic lesions with HR-like features, together with systemic mosaic patterns and severe leaf curling in upper leaves. Importantly, PYLCV V2 retains core V2-like biological activities despite extensive primary-sequence divergence, suggesting that functional conservation may rely on structural or biophysical determinants rather than linear sequence similarity. Our findings demonstrate that the divergent V2 protein from PYLCV has the capacity to function as a viral suppressor of RNA silencing (VSR) and as a potential symptom determinant, suggesting relevant roles in viral pathogenicity analogous to those of other geminiviral V2 proteins.
Hasan Zeitooni, L. Medina-Puche, Rosa Lozano-Durán et al.· BMC Plant Biology· 0 citations
Marek's disease virus (MDV) is a highly contagious and oncogenic avian alphaherpesvirus that causes severe economic losses on the global poultry industry. MicroRNAs (miRNAs), a class of endogenous noncoding RNAs typically 20-25 nucleotides in length, are crucial regulators of post-transcriptional genes expression. Our previous study found that MDV-encoded miR-M6-5p suppresses viral replication in cells. However, the underlying molecular mechanisms remain incompletely understood. In this study, we identified the viral UL42 transcript, bioinformatically predicted to encode a subunit of the viral DNA polymerase, as a direct target of miR-M6-5p. Through constructing a UL42-null MDV mutant, we demonstrated that the deletion of UL42 completely abolished viral replication in cells by severely impairing MDV DNA polymerase activity. Furthermore, we demonstrated that miR-M6-5p negatively regulates viral DNA polymerase activity, as its overexpression suppressed whereas its knockout enhanced enzymatic activity. Thus, miR-M6-5p-mediated downregulation of UL42 impairs viral DNA polymerase activity, thereby restricting MDV replication. These findings provide novel mechanistic insights into how MDV exploits its own miRNAs to modulate viral replication via the core DNA replication machinery.
Linyi Zhou, Rui Wang, Jing Cheng et al.· International Journal of Bio...· 0 citations
Abstract SARS-CoV-2 manipulates host gene expression through multiple mechanisms, including disruption of RNA processing. Here, we identify a novel function of the viral non-structural protein 14 (NSP14) in inducing N7-methylguanosine (m7G) modification in the internal sequences of host mRNA. We demonstrate that NSP14 catalyzes the conversion of GTP to m7GTP, which is subsequently incorporated into mRNA by RNA polymerase II, resulting in widespread internal m7G modification. This activity is dependent on NSP14’s N7-methyltransferase (N7-MTase) domain, and the NSP10–NSP14 interaction increases cellular m7G levels primarily by increasing NSP14 protein abundance. NSP14-induced m7G modification is conserved across alpha-, beta-, and gamma-coronaviruses. Mechanistically, we show that this RNA modification is associated with altered splicing, particularly in genes regulating genome stability, RNA metabolism, and nuclear processes. Importantly, using SARS-CoV-2 infection models, we show that viral replication is associated with increased cellular m7G signal, supporting the relevance of this pathway during infection. Inhibition of NSP14 N7-MTase or RNA polymerase II reduces SARS-CoV-2 replication, consistent with a model in which NSP14-induced m7G modification may contribute to viral replication. Our findings reveal a previously unrecognized epitranscriptomic mechanism and suggest that NSP14-induced m7G modification may contribute to the remodeling of host gene expression during coronavirus infection.
