Evidence that an Unnamed Isometric Virus Associated with Potato Rugose Disease in Peru Is a New Species of Genus Torradovirus.

A previously uncharacterized torradovirus species infecting potatoes was detected by high-throughput sequencing from field samples from Peru and in customs intercepts in potato tubers that originated from South America in the United States of America and the Netherlands. This new potato torradovirus showed high nucleotide sequence identity to an unidentified isometric virus (SB26/29), which was associated with a disease named potato rugose stunting in southern Peru characterized over two decades ago. Thus, this virus is tentatively named potato rugose stunting virus (PotRSV). The genome of PotRSV isolates sequenced in this study were composed of two polyadenylated RNA segments. RNA1 ranges from 7,086 to 7,089 nt and RNA2 from 5,228 to 5,230 nt. RNA1 encodes a polyprotein containing the replication block (helicase-protease-polymerase), whereas RNA2 encodes a polyprotein cleaved into a movement protein and the three capsid proteins (CPs). Pairwise comparison among PotRSV isolates revealed amino acid identity values greater than 86% in the protease-polymerase (Pro-Pol) region and greater than 82% for the combined CPs. The closest torradovirus species, squash chlorotic leaf spot virus, shares amino acid identities of ∼58 and ∼41% in the Pro-Pol and the combined CPs, respectively. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Genomic characterization of a novel torradovirus infecting Arctium lappa L. in China.

Burdock (Arctium lappa L.) is not only a popular vegetable crop but also an important medicinal plant. In burdock plants with symptoms of leaf mosaic, a novel torradovirus tentatively named "burdock mosaic virus" (BdMV) was identified by high-throughput sequencing. The complete genomic sequence of BdMV was further determined using RT-PCR and the rapid amplification of cDNA ends (RACE) method. The genome is composed of two positive-sense single-stranded RNAs. RNA1 (6991 nt) encodes a polyprotein of 2186 aa, and RNA2 (4700 nt) encodes a protein of 201 aa and a polyprotein of 1212 aa that is predicted to be processed into one movement protein (MP) and three coat proteins (CPs). The Pro-Pol region of RNA1 and the CP region of RNA2 shared the highest amino acid sequence identity of 74.0% and 70.6%, respectively, with the corresponding sequences of lettuce necrotic leaf curl virus (LNLCV) isolate JG3. Phylogenetic analysis based on the amino acid sequences of the Pro-Pol and CP regions showed that BdMV clustered with other non-tomato-infecting torradoviruses. Taken together, these results suggest that BdMV is a new member of the genus Torradovirus.

Plant expression of NifD protein variants resistant to mitochondrial degradation.

To engineer Mo-dependent nitrogenase function in plants, expression of the structural proteins NifD and NifK will be an absolute requirement. Although mitochondria have been established as a suitable eukaryotic environment for biosynthesis of oxygen-sensitive enzymes such as NifH, expression of NifD in this organelle has proven difficult due to cryptic NifD degradation. Here, we describe a solution to this problem. Using molecular and proteomic methods, we found NifD degradation to be a consequence of mitochondrial endoprotease activity at a specific motif within NifD. Focusing on this functionally sensitive region, we designed NifD variants comprising between one and three amino acid substitutions and distinguished several that were resistant to degradation when expressed in both plant and yeast mitochondria. Nitrogenase activity assays of these resistant variants in Escherichia coli identified a subset that retained function, including a single amino acid variant (Y100Q). We found that other naturally occurring NifD proteins containing alternate amino acids at the Y100 position were also less susceptible to degradation. The Y100Q variant also enabled expression of a NifD(Y100Q)-linker-NifK translational polyprotein in plant mitochondria, confirmed by identification of the polyprotein in the soluble fraction of plant extracts. The NifD(Y100Q)-linker-NifK retained function in bacterial nitrogenase assays, demonstrating that this polyprotein permits expression of NifD and NifK in a defined stoichiometry supportive of activity. Our results exemplify how protein design can overcome impediments encountered when expressing synthetic proteins in novel environments. Specifically, these findings outline our progress toward the assembly of the catalytic unit of nitrogenase within mitochondria.

Expression of Cholesterol Hydroxylase/Lyase System Proteins in Yeast S. cerevisiae Cells as a Self-Processing Polyprotein.

2A peptide discovered in Picornaviridae is capable of self-cleavage providing an opportunity to carry out synthesis of several proteins using one transcript. Dissociation in the 2A sequence during translation leads to the individual proteins formation. We constructed cDNA including genes of the bovine cholesterol hydroxylase/lyase (CHL) system proteins-cytochrome P450scc (CYP11A1), adrenodoxin (Adx) and adrenodoxin reductase (AdR), that are fused into a single ORF using FMDV 2A nucleotide sequences. The constructed vectors direct the expression of cDNA encoding polyprotein P450scc-2A-Adx-2A-AdR (CHL-2A) in Escherichia coli and Saccharomyces cerevisiae. The induced bacterial cells exhibit a high level of CHL-2A expression, but polyprotein is not cleaved at the FMDV sites. In yeast S. cerevisiae, the discrete proteins P450scc-2A, Adx-2A and AdR are expressed. Moreover, a significant proportion of AdR and Adx is present in a fusion Adx-2A-AdR. Thus, the first 2A linker provides an efficient cleavage of the polyprotein, while the second 2A linker demonstrates lower efficiency. Cholesterol hydroxylase/lyase activity registered in the recombinant yeast cell homogenate indicates that the catalytically active CHL system is present in these cells. Consequently, for the first time the mammalian system of cytochrome P450 has been successfully reconstructed in yeast cells through expressing the self-processing polyprotein.

Branched-chain amino acids reduce hepatic iron accumulation and oxidative stress in hepatitis C virus polyprotein-expressing mice.

BACKGROUND & AIMS: Branched-chain amino acids (BCAA) reduce the incidence of hepatocellular carcinoma (HCC) in patients with cirrhosis. However, the mechanisms that underlie these effects remain unknown. Previously, we reported that oxidative stress in male transgenic mice that expressed hepatitis C virus polyprotein (HCVTgM) caused hepatic iron accumulation by reducing hepcidin transcription, thereby leading to HCC development. This study investigated whether long-term treatment with BCAA reduced hepatic iron accumulation and oxidative stress in iron-overloaded HCVTgM and in patients with HCV-related advanced fibrosis. METHODS: Male HCVTgM were fed an excess-iron diet that comprised either casein or 3.0% BCAA, or a control diet, for 6 months. RESULTS: For HCVTgM, BCAA supplementation increased the serum hepcidin-25 levels and antioxidant status [ratio of biological antioxidant potential (BAP) relative to derivatives of reactive oxygen metabolites (dROM)], decreased the hepatic iron contents, attenuated reactive oxygen species generation, and restored mitochondrial superoxide dismutase expression and mitochondrial complex I activity in the liver compared with mice fed the control diet. After 48 weeks of BCAA supplementation in patients with HCV-related advanced fibrosis, BAP/dROM and serum hepcidin-25 increased and serum ferritin decreased compared with the pretreatment levels. CONCLUSIONS: BCAA supplementation reduced oxidative stress by restoring mitochondrial function and improved iron metabolism by increasing hepcidin-25 in both iron-overloaded HCVTgM and patients with HCV-related advanced fibrosis. These activities of BCAA may partially account for their inhibitory effects on HCC development in cirrhosis patients.

Complete nucleotide sequence of a potato isolate of strain group C of Potato virus Y from 1938.

The complete genomic sequence of an isolate (PRI-509) of the C strain of Potato virus Y (PVY(C)), which was originally isolated from potato in 1938, was elucidated. The genomic RNA of PRI-509 consists of 9699 nucleotides, with the capacity to encode a polyprotein of 3061 amino acids with a molecular mass of 337 kDa.This is the first full-length sequence of a PVY (C) isolate from potato that belongs to the C1 phylogenetic subgroup, which was previously thought to exclusively contain non-potato isolates.

Foliar extracts from transgenic tomato plants expressing the structural polyprotein, P1-2A, and protease, 3C, from foot-and-mouth disease virus elicit a protective response in guinea pigs.

The expression of recombinant antigens in transgenic plants is increasingly used as an alternative method of producing experimental immunogens. In this report, we describe the production of transgenic tomato plants that express the structural polyprotein, P1-2A, and protease, 3C, from foot-and-mouth disease (FMDV). P1-2A3C was inserted into the plant binary vector, pBin438, and transformed into tomato plants using Agrobacterium tumefaciens strain, GV3101. The presence of P1-2A3C was confirmed by PCR, transcription was verified by RT-PCR, and recombinant protein expression was confirmed by sandwich-ELISA and Western blot analyses. Guinea pigs immunized intramuscularly with foliar extracts from P1-2A3C-transgenic tomato plants were found to develop a virus-specific antibody response against FMDV. Vaccinated guinea pigs were fully protected against a challenge infection, while guinea pigs injected with untransformed plant extracts failed to elicit an antibody response and were not protected against challenge. These results demonstrate that transgenic tomato plants expressing the FMDV structural polyprotein, P1-2A, and the protease, 3C, can be used as a source of recombinant antigen for vaccine production.

Molecular characterization of two Chinese isolates of Beet mosaic virus.

The complete genomic sequences of Beet mosaic virus Xinjiang (BtMV-XJ) and Inner Mongolia (BtMV-IM) isolates from China were determined and compared with US and German isolates, reported previously. Results showed that viral genome of the two isolates both comprise 9,591 nucleotides, and contain the large single open reading frame (ORF) encoding a single polyprotein of 3,085 amino acid residues, from which ten putative functional proteins may be produced by autolytic cleavage processing as the US (BtMV-Wa) and German (BtMV-G) isolates. Sequence comparisons showed that BtMV-XJ shared 89.8% and 98.3% overall nucleotide identity with BtMV-Wa and BtMV-G isolates, and BtMV-IM exhibited the overall identities of 91.6% and 93.8% with BtMV-Wa and BtMV-G, respectively. Further, analyses revealed that BtMV-XJ shared higher identities in almost every region to BtMV-G than to BtMV-Wa both at the nucleotide and the amino acid levels. While BtMV-IM in the regions (6,666-7,671 and 7,672-9,591) showed highest homology with BtMV-XJ and BtMV-G, especially, after nt 7,672 with similarity up to 99.2% with BtMV-G; the region (2,331-4,083) showed highest identity (98.0% nt identity) with BtMV-Wa. That suggested BtMV-XJ had a more close relationship to BtMV-G, while BtMV-IM was more likely to be a natural recombination virus. In addition, phylogenetic analysis of the available BtMV CP sequences showed that BtMV isolates fell into two distinct groups: Euroasia group (Europe and China) and America group (USA). To the best of our knowledge, this study reported the complete sequences of two BtMV isolates from Asia for the first time.

Strains of Peru tomato virus infecting cocona (Solanum sessiliflorum), tomato and pepper in Peru with reference to genome evolution in genus Potyvirus.

Two isolates (SL1 and SL6) of Peru tomato virus (PTV, genus Potyvirus) were obtained from cocona plants (Solanum sessiliflorum) growing in Tingo María, the jungle of the Amazon basin in Peru. One PTV isolate (TM) was isolated from a tomato plant (Lycopersicon esculentum) growing in Huaral at the Peruvian coast. The three PTV isolates were readily transmissible by Myzus persicae. Isolate SL1, but not SL6, caused chlorotic lesions in inoculated leaves of Chenopodium amaranticolor and C. quinoa. Isolate TM differed from SL1 and SL6 in causing more severe mosaic symptoms in tomato, and vein necrosis in the leaves of cocona. Pepper cv. Avelar (Capsicum annuum) showed resistance to the PTV isolates SL1 and SL6 but not TM. The 5'- and 3'-proximal sequences of the three PTV isolates were cloned, sequenced and compared to the corresponding sequences of four PTV isolates from pepper, the only host from which PTV isolates have been previously characterised at the molecular level. Phylogenetic analyses on the P1 protein and coat protein amino acid sequences indicated, in accordance with the phenotypic data from indicator hosts, that the PTV isolates from cocona represented a distinguishable strain. In contrast, the PTV isolates from tomato and pepper were not grouped according to the host. Inclusion of the sequence data from the three PTV isolates of this study in a phylogenetic analysis with other PTV isolates and other potyviruses strengthen the membership of PTV in the so-called "PVY subgroup" of Potyvirus. This subgroup of closely related potyvirus species was also distinguishable from other potyviruses by their more uniform sizes of the protein-encoding regions within the polyprotein.

The complete nucleotide sequence, genome organization, and specific detection of Beet mosaic virus.

Beet mosaic virus (BtMV) was identified almost five decades ago but has not been fully characterized at the molecular level. In this study, we have determined for the first time the complete nucleotide sequence of BtMV genomic RNA and have developed a specific molecular means for its diagnosis. The viral genome of BtMV comprises 9591 nucleotides, excluding the 3' terminal poly (A) sequence, and contains a single open reading frame (ORF) that begins at nt 166 and terminates at nt 9423, encoding a single polyprotein of 3086 amino acid residues. A 3' untranslated region of 168 nucleotides follows the ORF. The deduced genome organization is typical for a member of the family Potyviridae and includes 10 proteins: P1, HC-Pro, P3, 6K1, CI, 6K2, NIa-VPg, NIa-Pro, NIb and coat protein (CP). Nine putative protease cleavage sites were predicted computationally and by analogy with genome arrangements of other potyviruses. Conserved sequence motifs of homologous proteins of other potyviruses were found in corresponding positions of BtMV. BtMV is a distinct species of the genus Potyvirus with the most closely related species being Peanut mottle virus ( approximately 55% amino acid identity). Based on the nucleotide sequence obtained, we have developed a virus-specific RT-PCR assay for accurate diagnosis and differentiation of BtMV.

Transgenic expression in Arabidopsis of a polyprotein construct leading to production of two different antimicrobial proteins.

We developed a method for expression in Arabidopsis of a transgene encoding a cleavable chimeric polyprotein. The polyprotein precursor consists of a leader peptide and two different antimicrobial proteins (AMPs), DmAMP1 originating from Dahlia merckii seeds and RsAFP2 originating from Raphanus sativus seeds, which are linked by an intervening sequence ("linker peptide") originating from a natural polyprotein occurring in seed of Impatiens balsamina. The chimeric polyprotein was found to be cleaved in transgenic Arabidopsis plants and the individual AMPs were secreted into the extracellular space. Both AMPs were found to exert antifungal activity in vitro. It is surprising that the amount of AMPs produced in plants transformed with some of the polyprotein transgene constructs was significantly higher compared with the amount in plants transformed with a transgene encoding a single AMP, indicating that the polyprotein expression strategy may be a way to boost expression levels of small proteins.

Comprehensive screening for human immunodeficiency virus type 1 subtype-specific CD8 cytotoxic T lymphocytes and definition of degenerate epitopes restricted by HLA-A0207 and -C(W)0304 alleles.

For this report, the rapid identification and characterization of human immunodeficiency virus type 1 (HIV-1)-derived broadly cross-subtype-reactive CD8 cytotoxic T lymphocyte (CTL) epitopes were performed. Using a gamma interferon (IFN-gamma) Elispot assay-based approach and a panel of recombinant vaccinia viruses expressing gag, env, pol, and nef genes representing the seven most predominant subtypes and one circulating recombinant form of HIV-1, the subtype specificity and cross-subtype reactivity of a CD8 response were directly measured from circulating peripheral blood mononuclear cells (PBMC). Enhanced sensitivity of detection of CD8 responses from cryopreserved PBMC was achieved using autologous vaccinia virus-infected B-lymphoblastoid cell lines as supplemental antigen-presenting cells. Of eleven subjects studied, six exhibited broadly cross-subtype-reactive CD8-mediated IFN-gamma production (at least seven of eight subtypes recognized) to at least one major gene product from HIV-1. Screening of subjects showing broadly cross-subtype-specific responses in the vaccinia virus-based enzyme-linked immunospot (Elispot) assay using a panel of overlapping peptides resulted in the identification of cross-subtype responses down to the 20-mer peptide level in less than 3 days. Three subjects showed broad cross-subtype reactivity in both the IFN-gamma Elispot assay and the standard chromium release cytotoxicity assay. Fine mapping and HLA restriction analysis of the response from three subjects demonstrated that this technique can be used to define epitopes restricted by HLA-A, -B, and -C alleles. In addition, the ability of all three epitopes to be processed from multiple subtypes of their parent proteins and presented in the context of HLA class I molecules following de novo synthesis is shown. While all three minimal epitopes mapped here had previously been defined as HIV-1 epitopes, two are shown to have novel HLA restriction alleles and therefore exhibit degenerate HLA binding capacity. These findings provide biological validation of HLA supertypes in HIV-1 CTL recognition and support earlier studies of cross-subtype CTL responses during HIV-1 infection.

Comparison of the complete sequences of five different isolates of Potato virus A (PVA), genus Potyvirus.

The complete nucleotide (nt) and deduced amino acid (aa) sequences of isolates Ali, U, Her (from potato, Solanum tuberosum) and TamMV (from tamarillo, Solanum betacea) of Potato virus A (PVA, genus Potyvirus) were determined and compared with the previously reported sequence of PVA isolate B11. Most parts (proteins) of the polyprotein showed over 95% aa sequence similarity. The cylindrical inclusion (CI) protein and the 6K 1 protein were the most conserved proteins among the five isolates. TamMV was the most different isolate. Sequence similarity between TamMV and the other isolates was the lowest in regions close to the 5'-end [5'-non-translated region (NTR) and P1 region] and 3'-end (N-terminus of coat protein) of the genome. However, the termini of the genome (the first 60 nt of the 5'-NTR and the entire 3'-NTR) were highly similar in all five isolates. A frameshift region in the replicase (NIb) was identified the PVA isolates Ali, B11, Her and U, as compared to TamMV and other potyviruses.