- Volume 90, Issue 3, 2009

Two sets of infectious cDNA clones of raspberry bushy dwarf virus (RBDV) have been constructed, enabling either the synthesis of infectious RNA transcripts or the delivery of infectious binary plasmid DNA by infiltration of Agrobacterium tumefaciens. In whole plants and in protoplasts, inoculation of RBDV RNA1 and RNA2 transcripts led to a low level of infection, which was greatly increased by the addition of RNA3, a subgenomic RNA coding for the RBDV coat protein (CP). Agroinfiltration of RNA1 and RNA2 constructs did not produce a detectable infection but, again, inclusion of a construct encoding the CP led to high levels of infection. Thus, RBDV replication is greatly stimulated by the presence of the CP, a mechanism that also operates with ilarviruses and alfalfa mosaic virus, where it is referred to as genome activation. Mutation to remove amino acids from the N terminus of the CP showed that the first 15 RBDV CP residues are not required for genome activation. Other experiments, in which overlapping regions at the CP N terminus were fused to the monomeric red fluorescent protein, showed that sequences downstream of the first 48 aa are not absolutely required for genome activation.

Ten beet virus Q (BVQ) strains from six different countries were sequenced to characterize the readthrough (RT) domain of the coat protein (CP). The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this paper are FM244643–FM244652. With three nucleotide additions of 5, 285 and 1 nt, the common RT of 76 kDa was found to be longer than the single reference available to date (35 kDa). It is hypothesized that multiple inoculation cycles on Chenopodium quinoa were responsible for these three deletions in the C-terminal part of the BVQ RNA-2 previously described. Two putative transmembrane domains, TM1 and TM2, were predicted in the consensus amino acid sequence of the ten BVQ strains, and the putative BVQ TM2 was aligned with that of potato mop-top virus.

Beet necrotic yellow vein virus (BNYVV) A type isolates E12 and S8, originating from areas where resistance-breaking had or had not been observed, respectively, served as starting material for studying the influence of sequence variations in BNYVV RNA 3 on virus accumulation in partially resistant sugar beet varieties. Sub-isolates containing only RNAs 1 and 2 were obtained by serial local lesion passages; biologically active cDNA clones were prepared for RNAs 3 which differed in their coding sequences for P25 aa 67, 68 and 129. Sugar beet seedlings were mechanically inoculated with RNA 1+2/RNA 3 pseudorecombinants. The origin of RNAs 1+2 had little influence on virus accumulation in rootlets. E12 RNA 3 coding for V67C68Y129 P25, however, enabled a much higher virus accumulation than S8 RNA 3 coding for A67H68H129 P25. Mutants revealed that this was due only to the V67 ‘G U U’ codon as opposed to the A67 ‘G C U’ codon.

In sheep, susceptibility to scrapie is mainly influenced by polymorphisms of the PrP gene. In goats, there are to date few data related to scrapie susceptibility association with PrP gene polymorphisms. In this study, we first investigated PrP gene polymorphisms of the French Alpine and Saanen breeds. Based on PrP gene open reading frame sequencing of artificial insemination bucks (n=404), six encoding mutations were identified at codons 127, 142, 154, 211, 222 and 240. However, only seven haplotypes could be detected: four (GIH154RQS, GIRQ211QS, GIRRK222S and GIRRQP240) derived from the wild-type allele (G127I142R154R211Q222S240) by a single-codon mutation, and two (S127IRRQP240 and GM142RRQP240) by a double-codon mutation. A case–control study was then implemented in a highly affected Alpine and Saanen breed herd (90 cases/164 controls). Mutations at codon 142 (I/M), 154 (R/H), 211 (R/Q) and 222 (Q/K) were found to induce a significant degree of protection towards natural scrapie infection. Compared with the baseline homozygote wild-type genotype I142R154R211Q222/IRRQ goats, the odds of scrapie cases in IRQ211Q/IRRQ and IRRK222/IRRQ heterozygous animals were significantly lower [odds ratio (OR)=0.133, P<0.0001; and OR=0.048, P<0.0001, respectively]. The heterozygote M142RRQ/IRRQ genotype was only protective (OR=0.243, P=0.0186) in goats also PP240 homozygous at codon 240. However, mutated allele frequencies in French Alpine and Saanen breeds were low (0.5–18.5 %), which prevent us from assessing the influence of all the possible genotypes in natural exposure conditions.

Several studies have failed to demonstrate the presence of immune responses to infectious prions during the course of prion disease, reflecting the identical primary structure of normal and disease-associated isoforms and the widespread expression of the normal cellular form of prion protein, PrPC, leading to B- and/or T-cell tolerance of disease-associated isoforms and also possibly because antigen-presenting cells are unable to process the highly aggregated, detergent-insoluble, protease-resistant form, PrPSc. Under certain circumstances, PrPSc can be revealed to the immune system in immunogenic form, and it has been shown previously that anti-PrP antibodies can be induced to prions immunoadsorbed to Dynabeads using specific anti-PrP monoclonal antibodies, even in PrP-sufficient mice. This study demonstrated in a murine scrapie model that PrP–Dynabeads effectively stimulated the immune system to produce anti-PrP IgM antibodies over prolonged periods after repeated immunization. It was also shown that these immune responses prolonged incubation times in murine scrapie.

The interferon (IFN) system is a major effector of the innate immunity that allows time for the subsequent establishment of an adaptive immune response against a wide-range of pathogens. Their diverse biological actions are thought to be mediated by the products of specific but usually overlapping sets of cellular genes induced in the target cells. Ubiquitin ligase members of the tripartite motif (TRIM) protein family have emerged as IFN-induced proteins involved in both innate and adaptive immunity. In this report, we provide evidence that TRIM22 is a functional E3 ubiquitin ligase that is also ubiquitinated itself. We demonstrate that TRIM22 expression leads to a viral protection of HeLa cells against encephalomyocarditis virus infections. This effect is dependent upon its E3 ubiquitinating activity, since no antiviral effect was observed in cells expressing a TRIM22-deletion mutant defective in ubiquitinating activity. Consistent with this, TRIM22 interacts with the viral 3C protease (3C PRO ) and mediates its ubiquitination. Altogether, our findings demonstrate that TRIM22 E3 ubiquitin ligase activity represents a new antiviral pathway induced by IFN against picornaviruses.

Approximately 15 % of human prion disease is associated with autosomal-dominant pathogenic mutations in the prion protein (PrP) gene. Previous attempts to model these diseases in mice have expressed human PrP mutations in murine PrP, but this may have different structural consequences. Here, we describe transgenic mice expressing human PrP with P102L or E200K mutations and methionine (M) at the polymorphic residue 129. Although no spontaneous disease developed in aged animals, these mice were readily susceptible to prion infection from patients with the homotypic pathogenic mutation. However, while variant Creutzfeldt–Jakob disease (CJD) prions transmitted infection efficiently to both lines of mice, markedly different susceptibilities to classical (sporadic and iatrogenic) CJD prions were observed. Prions from E200K and classical CJD M129 homozygous patients, transmitted disease with equivalent efficiencies and short incubation periods in human PrP 200K, 129M transgenic mice. However, mismatch at residue 129 between inoculum and host dramatically increased the incubation period. In human PrP 102L, 129M transgenic mice, short disease incubation periods were only observed with transmissions of prions from P102L patients, whereas classical CJD prions showed prolonged and variable incubation periods irrespective of the codon 129 genotype. Analysis of disease-related PrP (PrPSc) showed marked alteration in the PrPSc glycoform ratio propagated after transmission of classical CJD prions, consistent with the PrP point mutations directly influencing PrPSc assembly. These data indicate that P102L or E200K mutations of human PrP have differing effects on prion propagation that depend upon prion strain type and can be significantly influenced by mismatch at the polymorphic residue 129.