Systematic mutagenesis of Polerovirus protein P0 reveals distinct and overlapping amino acid functions in Nicotiana glutinosa.

The protein P0 serves as the viral suppressor of RNA silencing (VSR) for poleroviruses, but elicits the hypersensitive response (HR) in specific Nicotiana species. We subjected P0 proteins from turnip yellows virus (P0Tu) and potato leafroll virus (P0PL) to serial deletion and performed extensive site-directed mutagenesis of P0Tu. Most deletions of the N-terminus and many substitution mutations disrupted both HR elicitation and VSR activity. Two conserved blocks of amino acid residues were found to be associated with HR. A double lysine to arginine substitution in HR-specific block 1 caused P0Tu to elicit a more robust HR. Conversely, deletion or mutation of block 2 in the C-terminus preserved VSR activity, but impaired HR elicitation, allowing virus escape from Nicotiana glutinosa resistance when expressed in the heterologous potato virus X vector. Our observations suggest that P0 residues responsible for suppressing RNA silencing and eliciting HR have overlapping, but distinct functions.

Non-canonical nematode endogenous retroviruses resulting from RNA virus glycoprotein gene capture by a metavirus.

Reverse-transcribing retroviruses exist as horizontally transmitted infectious agents or vertically transmitted endogenous retroviruses (ERVs) resident in eukaryotic genomes, and they are phylogenetically related to the long terminal repeat (LTR) class of retrotransposons. ERVs and retrotransposons are often distinguished only by the presence or absence of a gene encoding the envelope glycoprotein (env). Endogenous elements of the virus family Metaviridae include the insect-restricted Errantivirus genus of ERVs, for which some members possess env, and the pan-eukaryotic Metavirus genus that lacks an envelope glycoprotein gene. Here we report a novel Nematoda endogenous retrovirus (NERV) clade with core retroviral genes arranged uniquely as a continuous gag-env-pro-pol ORF. Reverse transcriptase sequences were phylogenetically related to metaviruses, but envelope glycoprotein sequences resembled those of the Nyamiviridae and Chrysoviridae RNA virus families, suggesting env gene capture during host cell infection by an RNA virus. NERVs were monophyletic, restricted to the nematode subclass Chromadoria, and included additional ORFs for a small hypothetical protein or a large Upf1-like RNA-dependent AAA-ATPase/helicase indicative of viral transduction of a host gene. Provirus LTR identity, low copy number, ORF integrity and segregation of three loci in Meloidogyne incognita, taken together with detection of NERV transcriptional activity, support potential infectivity of NERVs, along with their recent emergence and integration. Altogether, NERVs constitute a new and distinct Metaviridae lineage demonstrating retroviral evolution through sequential heterologous gene capture events.

GGERV20, a recently integrated, segregating endogenous retrovirus in Gallus gallus.

Endogenous retroviruses (ERVs) are widespread in vertebrate genomes. The recent availability of whole eukaryotic genomes has enabled their characterization in many organisms, including Gallus gallus (red jungle fowl), the progenitor of the domesticated chicken. Our bioinformatics analysis of a G. gallus ERV previously designated GGERV20 identified 35 proviruses with complete long terminal repeats (LTRs) and gag-pol open reading frames (ORFs) in the Genome Reference Consortium Chicken Build 6a, of which 8 showed potential for translation of functional retroviral polyproteins, including the integrase and reverse transcriptase enzymes. No elements were discovered with an env gene. Fifteen loci had LTR sequences with 100 % identity, indicative of recent integration. Chicken embryo fibroblast RNA-seq datasets showed reads representing the entire length of the GGERV20 provirus, supporting their potential for expressing viral proteins. To investigate the possibility that GGERV20 elements may not be fixed in the genome, we assessed the integration status of five loci in a meat-type chicken. PCRs targeting a GGERV20 locus on G. gallus chromosome one (GGERV201-1) reproducibly amplified both LTRs and the preintegration state, indicating that the bird from which the DNA was sampled was hemizygous at this locus. The four other loci examined only produced the preintegration state amplicons. These results reveal that GGERV20 is not fixed in the G. gallus population, and taken together with the lack of mutations seen in several provirus LTRs and their transcriptional activity, suggest that GGERV20 retroviruses have recently been and continue to be active in the chicken genome.

Elicitation of hypersensitive responses in Nicotiana glutinosa by the suppressor of RNA silencing protein P0 from poleroviruses.

Plant disease resistance (R) proteins that confer resistance to viruses recognize viral gene products with diverse functions, including viral suppressors of RNA silencing (VSRs). The P0 protein from poleroviruses is a VSR that targets the ARGONAUTE1 (AGO1) protein for degradation, thereby disrupting RNA silencing and antiviral defences. Here, we report resistance against poleroviruses in Nicotiana glutinosa directed against Turnip yellows virus (TuYV) and Potato leafroll virus (PLRV). The P0 proteins from TuYV (P0(T) (u) ), PLRV (P0(PL) ) and Cucurbit aphid-borne yellows virus (P0(CA) ) were found to elicit a hypersensitive response (HR) in N. glutinosa accession TW59, whereas other accessions recognized P0(PL) only. Genetic analysis showed that recognition of P0(T) (u) by a resistance gene designated RPO1 (Resistance to POleroviruses 1) is inherited as a dominant allele. Expression of P0 from a Potato virus X (PVX) expression vector transferred recognition to the recombinant virus on plants expressing RPO1, supporting P0 as the unique Polerovirus factor eliciting resistance. The induction of HR required a functional P0 protein, as P0(T) (u) mutants with substitutions in the F-box motif that abolished VSR activity were unable to elicit HR. We surmised that the broad P0 recognition seen in TW59 and the requirement for the F-box protein motif could indicate detection of P0-induced AGO1 degradation and disruption of RNA silencing; however, other viral silencing suppressors, including the PVX P25 that also causes AGO1 degradation, failed to elicit HR in N. glutinosa. Investigation of P0 elicitation of RPO1 could provide insight into P0 activities within the cell that trigger resistance.

Prototype endogenous avian retroviruses of the genus Gallus.

Ancient endogenous retroviruses (ERVs), designated endogenous avian retrovirus (EAVs), are present in all Gallus spp. including the chicken, and resemble the modern avian sarcoma and leukosis viruses (ASLVs). The EAVs comprise several distinct retroviruses, including EAV-0, EAV-E51 and EAV-HP, as well as a putative member previously named the avian retrotransposon of chickens (ART-CH). Thus far, only the EAV-HP elements have been well characterized. Here, we determined sequences of representative EAV-0 and EAV-E51 proviruses by cloning and data mining of the 2011 assembly of the Gallus gallus genome. Although the EAV-0 elements are primarily deleted in the env region, we identified two complete EAV-0 env genes within the G. gallus genome and prototype elements sharing identity with an EAV-E51-related clone previously designated EAV-E33. Prototype EAV-0, EAV-E51 and EAV-E33 gag, pol and env gene sequences used for phylogenetic analysis of deduced proteins showed that the EAVs formed three distinct clades, with EAV-0 sharing the last common ancestor with the ASLVs. The EAV-E51 clade showed the greatest level of divergence compared with other EAVs or ASLVs, suggesting that these ERVs represented exogenous retroviruses that evolved and integrated into the germline over a long period of time. Moreover, the degree of divergence between the chicken and red jungle fowl EAV-E51 sequences suggested that they were more ancient than the other EAVs and may have diverged through mutations that accumulated post-integration. Finally, we showed that the ART-CH elements were chimeric defective ERVs comprising portions of EAV-E51 and EAV-HP rather than authentic retrotransposons.

The cyst nematode SPRYSEC protein RBP-1 elicits Gpa2- and RanGAP2-dependent plant cell death.

Plant NB-LRR proteins confer robust protection against microbes and metazoan parasites by recognizing pathogen-derived avirulence (Avr) proteins that are delivered to the host cytoplasm. Microbial Avr proteins usually function as virulence factors in compatible interactions; however, little is known about the types of metazoan proteins recognized by NB-LRR proteins and their relationship with virulence. In this report, we demonstrate that the secreted protein RBP-1 from the potato cyst nematode Globodera pallida elicits defense responses, including cell death typical of a hypersensitive response (HR), through the NB-LRR protein Gpa2. Gp-Rbp-1 variants from G. pallida populations both virulent and avirulent to Gpa2 demonstrated a high degree of polymorphism, with positive selection detected at numerous sites. All Gp-RBP-1 protein variants from an avirulent population were recognized by Gpa2, whereas virulent populations possessed Gp-RBP-1 protein variants both recognized and non-recognized by Gpa2. Recognition of Gp-RBP-1 by Gpa2 correlated to a single amino acid polymorphism at position 187 in the Gp-RBP-1 SPRY domain. Gp-RBP-1 expressed from Potato virus X elicited Gpa2-mediated defenses that required Ran GTPase-activating protein 2 (RanGAP2), a protein known to interact with the Gpa2 N terminus. Tethering RanGAP2 and Gp-RBP-1 variants via fusion proteins resulted in an enhancement of Gpa2-mediated responses. However, activation of Gpa2 was still dependent on the recognition specificity conferred by amino acid 187 and the Gpa2 LRR domain. These results suggest a two-tiered process wherein RanGAP2 mediates an initial interaction with pathogen-delivered Gp-RBP-1 proteins but where the Gpa2 LRR determines which of these interactions will be productive.