Infection with Strains of Citrus Tristeza Virus Does Not Exclude Superinfection by Other Strains of the Virus

Superinfection exclusion or homologous interference, a phenomenon in which a primary viral infection prevents a secondary infection with the same or closely related virus, has been observed commonly for viruses in various systems, including viruses of bacteria, plants, and animals. With plant viruses, homologous interference initially was used as a test of virus relatedness to define whether two virus isolates were “strains” of the same virus or represented different viruses, and subsequently purposeful infection with a mild isolate was implemented as a protective measure against isolates of the virus causing severe disease. In this study we examined superinfection exclusion of Citrus tristeza virus (CTV), a positive-sense RNA closterovirus. Thirteen naturally occurring isolates of CTV representing five different virus strains and a set of isolates originated from virus constructs engineered based on an infectious cDNA clone of T36 isolate of CTV, including hybrids containing sequences from different isolates, were examined for their ability to prevent superinfection by another isolate of the virus. We show that superinfection exclusion occurred only between isolates of the same strain and not between isolates of different strains. When isolates of the same strain were used for sequential plant inoculation, the primary infection provided complete exclusion of the challenge isolate, whereas isolates from heterologous strains appeared to have no effect on replication, movement or systemic infection by the challenge virus. Surprisingly, substitution of extended cognate sequences from isolates of the T68 or T30 strains into T36 did not confer the ability of resulting hybrid viruses to exclude superinfection by those donor strains. Overall, these results do not appear to be explained by mechanisms proposed previously for other viruses. Moreover, these observations bring an understanding of some previously unexplained fundamental features of CTV biology and, most importantly, build a foundation for the strategy of selecting mild isolates that would efficiently exclude severe virus isolates as a practical means to control CTV diseases.Superinfection exclusion or homologous interference is a phenomenon in which a preexisting viral infection prevents a secondary infection with the same or a closely related virus, whereas infection by unrelated viruses can be unaffected. The phenomenon was first observed by McKinney (57, 58) between two genotypes of Tobacco mosaic virus (TMV) and later with bacteriophages (21, 94). Since that time, the phenomenon has been observed often for viruses of animals (1, 13, 18, 34, 43, 47, 50, 85, 86-88, 102, 103) and plants (11, 30, 31, 32, 39, 40, 49, 77, 99, 100). In plant virology, homologous interference initially was used as a test of virus relatedness to define whether two virus isolates were “strains” of the same virus or represented different viruses (58, 77). Subsequently, it was developed into a management tool to reduce crop losses by purposely infecting plants with mild isolates of a virus to reduce infection and losses due to more severe isolates, which is referred to as “cross-protection” (reviewed in references 32 and 40).Homologous superinfection exclusion of animal viruses has been related to several mechanisms acting at various stages of the viral life cycle, including prevention of the incoming virus entry into cells (50, 86, 87), or inhibition of translation or interference with replication (1, 47, 50, 83). Several mechanisms have been postulated for homologous interference of plant viruses, including prevention of the disassembly of the challenge virus as it enters the cell resulting from the expression of the coat protein of the protector virus (67, 84; reviewed in reference 10) and induction of RNA silencing by the protector virus that leads to sequence-specific degradation of the challenge virus RNA (24, 69, 70). However, common mechanisms of superinfection exclusion, expected to be associated with the viruses of plants and animals, have not been elucidated.Citrus tristeza virus (CTV) is the largest and most complex member of the Closteroviridae family, which contains viruses with mono-, bi-, and tripartite genomes transmitted by a range of insect vectors, including aphids, whiteflies, and mealybugs (3, 6, 19, 20, 46). CTV has long flexuous virions (2,000 nm by 10 to 12 nm) encapsidated by two coat proteins and a single-stranded RNA genome of ∼19.3 kb. The major coat protein (CP) covers ca. 97% of the genomic RNA, and the minor coat protein (CPm) completes encapsidation of the genome at its 5′ end (25, 81). The RNA genome of CTV encodes 12 open reading frames (ORFs) (44, 64) (Fig. ​(Fig.1).1). ORFs 1a and 1b are expressed from the genomic RNA and encode polyproteins required for virus replication. ORF 1a encodes a 349-kDa polyprotein containing two papainlike protease domains plus methyltransferaselike and helicaselike domains. Translation of the polyprotein is thought to occasionally continue through the polymerase-like domain (ORF 1b) by a +1 frameshift. Ten 3′-end ORFs are expressed by 3′-coterminal subgenomic RNAs (sgRNAs) (37, 45) and encode the following proteins: major (CP) and minor (CPm) coat proteins, p65 (HSP70 homolog), and p61 that are involved in assembly of virions (79); a hydrophobic p6 protein with a proposed role in virus movement (20, 89); p20 and p23, which along with CP are suppressors of RNA silencing (54); and p33, p13, and p18, whose functions remain unknown. Remarkably, citrus trees can be infected with mutants with three genes deleted: p33, p18, and p13 (89).Open in a separate windowFIG. 1.(A) Schematic diagram of the genome organization of wild-type CTV (CTV9R) and its derivative CTV-BC5/GFP encoding GFP. The open boxes represent ORFs and their translation products. PRO, papainlike protease domain; MT, methyltransferase; HEL, helicase; RdRp, an RNA-dependent RNA polymerase; HSP70h, HSP70 homolog; CPm, minor coat protein; CP, major coat protein; GFP, green fluorescent protein. Bent arrows indicate positions of BYV (B_CP) or CTV CP (C_CP) sgRNA controller elements. Inserted elements are shown in gray. (B) Scheme of the “superinfection exclusion assay.” Young Madam Vinous sweet orange trees were initially inoculated with one of 13 tested CTV isolates. When primary infections were established, the trees were subsequently challenged with CTV-BC5/GFP. All inoculations were done by grafting of the infected tissue into the stem of a tree. The positions of primary (Pri) and challenge (Chl) graft inoculations are shown. The ability of the challenge virus to superinfect trees was determined by visual observation of GFP fluorescence in phloem-associated cells on the internal surface of bark from a young flash starting at about 2 months upon challenge inoculation. Scale bar, 0.4 mm.The host range of CTV is limited to citrus in which the virus infects only phloem-associated cells. CTV consists of numerous isolates that have distinctive biological and genetic characteristics (38, 48, 56, 72, 74, 75, 95). Recently, a classification strategy for CTV isolates was proposed based on sequence similarity. Analysis of nearly 400 isolates in an international collection revealed five major CTV genotype groups with some isolates undefined (38). For the purposes of the present study, strains are defined as phylogenetically distinct lineages of CTV based upon analysis of nucleotide sequences of the 1a ORF (38). This region of the genome shows high genetic diversity between CTV variants, with levels of sequence identity ranging between 72.3 to 90.3% (38, 48, 52, 74, 75; M. Hilf, unpublished data). Using this definition, T3, T30, T36, VT, and T68 are designated as strains. Individual virus samples are designated as isolates of one of these strains. The ORF 1a nucleotide sequences of isolates of the T36 and T68 strains are equally dissimilar to isolates of the T3, T30, and VT strains, with identities of 72.9, 73, and 72.4% and 77.6, 77.9, and 76.8%, respectively. Identities of ORF 1a range from 89.4 to 90.3% between isolates of the T3, T30, and VT strains. Sequences of ORF1a of isolates belonging to the T36 strain and those from the T68 strain show 72.3% identity. This compares to a range of 89 to 94.8% identity found in the more conserved 3′-half regions of the genomes of isolates from different CTV strains. Each strain is named after a “type isolate” and is composed of isolates with minor sequence divergence (generally less than 5% throughout genome) from the type member. However, isolates of a strain may have significant variations in symptoms and symptoms severity. Remarkably, field trees harbor complex populations of CTV, which are often composed of mixtures of different strains and recombinants between these strains (36, 48, 52, 68, 75, 96, 101). The genetic basis of such frequent coexistence of different strains within the same tree is unknown.CTV causes economically important diseases of citrus worldwide. One of the most effective management tools has been cross-protection when effective protecting isolates could be found. Preinfection with mild isolates allows commercial production of sweet oranges and limes in Brazil (16) and Peru (9) and grapefruit in South Africa (92). However, identification of protecting isolates has been empirical, difficult, and rare. Cross-protection usually has worked only in certain varieties, and the lack of effective protecting isolates has prevented its use in many varieties and citrus growing areas (15, 41, 61, 73). In general, there has been no understanding why some mild isolates were effective and others failed to protect. Because CTV diseases prevail in citrus growing areas worldwide, elucidation of the mechanisms of exclusion of one CTV variant by another one is an important goal.In the present study we examined relationships between different genotypes of CTV in terms of their ability to prevent superinfection by another isolate of the virus. We show that superinfection exclusion occurred only between minor genetic variants of the same strain (sequence group) and not between isolates of different strains. When isolates of the same strain were used for sequential plant inoculation, the primary infection provided full exclusion of the challenge isolate. In all combinations of virus isolates belonging to different strains, the primary infection of plants with one strain had no noticeable effect on the establishment of the secondary infection. The results obtained here help elucidate some previously unexplained fundamental features of CTV biology and pose the possibility of an existence of a novel mechanism for superinfection exclusion between virus variants.     

MeSHer: identifying biological concepts in microarray assays based on PubMed references and MeSH terms

SUMMARY: MeSHer uses a simple statistical approach to identify biological concepts in the form of Medical Subject Headings (MeSH terms) obtained from the PubMed database that are significantly overrepresented within the identified gene set relative to those associated with the overall collection of genes on the underlying DNA microarray platform. As a demonstration, we apply this approach to gene lists acquired from a published study of the effects of angiotensin II (Ang II) treatment on cardiac gene expression and demonstrate that this approach can aid in the interpretation of the resulting 'significant' gene set. AVAILABILITY: The software is available at http://www.tm4.org. SUPPLEMENTARY INFORMATION: Results from the analysis of significant genes from the published Ang II study.     

Structure of the O-Specific polysaccharide from Shewanella japonica KMM 3601 containing 5,7-Diacetamido-3,5,7,9-tetradeoxy-D-glycero-D-talo-non-2-ulosonic acid

Structure of the O-specific polysaccharide chain of the lipopolysaccharide (LPS) of Shewanella japonica KMM 3601 was elucidated. The initial and O-deacylated LPS as well as a trisaccharide representing the O-deacetylated repeating unit of the O-specific polysaccharide were studied by sugar analysis along with ¹H and ¹³C NMR spectroscopy. The polysaccharide was found to contain a rare higher sugar, 5,7-diacetamido-3,5,7,9-tetradeoxy-d-glycero-d-talo-non-2-ulosonic acid (a derivative of 4-epilegionaminic acid, 4eLeg). The following structure of the trisaccharide repeating unit was established: →4)-α-4eLegp5Ac7Ac-(2→4)-β-d-GlcpA3Ac-(1→3)-β-d-GalpNAc-(1→.     

Myofibrillar troponin exists in three states and there is signal transduction along skeletal myofibrillar thin filaments

Activation of striated muscle contraction is a highly cooperative signal transduction process converting calcium binding by troponin C (TnC) into interactions between thin and thick filaments. Once calcium is bound, transduction involves changes in protein interactions along the thin filament. The process is thought to involve three different states of actin-tropomyosin (Tm) resulting from changes in troponin's (Tn) interaction with actin-Tm: a blocked (B) state preventing myosin interaction, a closed (C) state allowing weak myosin interactions and favored by calcium binding to Tn, and an open or M state allowing strong myosin interactions. This was tested by measuring the apparent rate of Tn dissociation from rigor skeletal myofibrils using labeled Tn exchange. The location and rate of exchange of Tn or its subunits were measured by high-resolution fluorescence microscopy and image analysis. Three different rates of Tn exchange were observed that were dependent on calcium concentration and strong cross-bridge binding that strongly support the three-state model. The rate of Tn dissociation in the non-overlap region was 200-fold faster at pCa 4 (C-state region) than at pCa 9 (B-state region). When Tn contained engineered TnC mutants with weakened regulatory TnI interactions, the apparent exchange rate at pCa 4 in the non-overlap region increased proportionately with TnI-TnC regulatory affinity. This suggests that the mechanism of calcium enhancement of the rate of Tn dissociation is by favoring a TnI-TnC interaction over a TnI-actin-Tm interaction. At pCa 9, the rate of Tn dissociation in the overlap region (M-state region) was 100-fold faster than the non-overlap region (B-state region) suggesting that strong cross-bridges increase the rate of Tn dissociation. At pCa 4, the rate of Tn dissociation was twofold faster in the non-overlap region (C-state region) than the overlap region (M-state region) that likely involved a strong cross-bridge influence on TnT's interaction with actin-Tm. At sub-maximal calcium (pCa 6.2-5.8), there was a long-range influence of the strong cross-bridge on Tn to enhance its dissociation rate, tens of nanometers from the strong cross-bridge. These observations suggest that the three different states of actin-Tm are associated with three different states of Tn. They also support a model in which strong cross-bridges shift the regulatory equilibrium from a TnI-actin-Tm interaction to a TnC-TnI interaction that likely enhances calcium binding by TnC.     

Poly(amidoamine) dendrimer peripherally modified with 4-N,N-dimethylaminoethyleneamino-1,8-naphthalimide as a sensor of metal cations and protons.

A new poly(amidoamine) dendrimer from second generation whose periphery comprises sixteen fluorescent 4-N,N-dimethylaminoethylamino-1,8-naphthalimide units has been synthesized and characterized. In DMF, the dendrimer shows sensitivity to the presence of Cu(2+), Fe(3+) and protons. The changes in the fluorescence intensity of the material are in opposite directions if acids or metals are present. Fluorescence enhancements (FE from 5 to 9 depending on solvent) are recorded when the photoinduced electron transfer (PET) originating from the donating amine to the electron accepting naphthalimide is inhibited by the protonation of the N,N-dimethylamino groups. In the case of Cu(2+) cations, a fluorescence quenching (FQ of 6) is first observed, followed by fluorescence partial restoration. In the Fe(3+) case, the same behaviour is observed with a final FE of 2. The successive complexations of these cations by the dendrimer core and by the external rim of the dendrimer may explain the results.     

Silicatein Genes in Spicule-Forming and Nonspicule-forming Pacific Demosponges

Silicatein genes are known to be involved in siliceous spicule formation in marine sponges. Proteins encoded by these genes, silicateins, were recently proposed for nanobiotechnological applications. We studied silicatein genes of marine sponges Latrunculia oparinae collected in the west Pacific region, shelf of Kuril Islands. Five silicatein genes, LoSilA1, LoSilA1a, LoSilA2, and LoSilA3 (silicatein-α group), LoSilB (silicatein-β group), and one cathepsin gene, LoCath, were isolated from the sponge L. oparinae for the first time. The deduced amino acid sequence of L. oparinae silicateins showed high-sequence identity with silicateins described previously. LoCath contains the catalytic triad of amino acid residues Cys-His-Asn characteristic for cathepsins as well as motifs typical for silicateins. A phylogenetic analysis places LoCath between sponge silicateins-β and L-cathepsins suggesting that the LoCath gene represents an intermediate form between silicatein and cathepsin genes. Additionally, we identified, for the first time, silicatein genes (AcSilA and AcSilB) in nonspicule-forming marine sponge, Acаnthodendrilla sp. The results suggest that silicateins could participate also in the function(s) unrelated to spiculogenesis.