Characterization of putative proteins encoded by variable ORFs in white spot syndrome virus genome

BackgroundWhite spot syndrome virus (WSSV) is the sole member of the novel Nimaviridae family, and the source of major economic problems in shrimp aquaculture. WSSV appears to have rapidly spread worldwide after the first reported outbreak in the early 1990s. Genomic deletions of various sizes occur at two loci in the WSSV genome, the ORF14/15 and ORF23/24 variable regions, and these have been used as molecular markers to study patterns of viral spread over space and time. We describe the dynamics underlying the process of WSSV genome shrinkage using empirical data and a simple mathematical model.Methodology/Principal FindingsWe genotyped new WSSV isolates from five Asian countries, and analyzed this information together with published data. Genome size appears to stabilize over time, and deletion size in the ORF23/24 variable region was significantly related to the time of the first WSSV outbreak in a particular country. Parameter estimates derived from fitting a simple mathematical model of genome shrinkage to the data support a geometric progression (k<1) of the genomic deletions, with k = 0.371±0.150.Conclusions/SignificanceThe data suggest that the rate of genome shrinkage decreases over time before attenuating. Bioassay data provided support for a link between genome size and WSSV fitness in an aquaculture setting. Differences in genomic deletions between geographic WSSV isolates suggest that WSSV spread did not follow a smooth pattern of geographic radiation, suggesting spread of WSSV over long distances by commercial activities. We discuss two hypotheses for genome shrinkage, an adaptive and a neutral one. We argue in favor of the adaptive hypothesis, given that there is support for a link between WSSV genome size and fitness.

We evaluated the prevalence of white spot syndrome virus(WSSV) in cultured crayfish(Procambarus clarkii) in Jiangsu Province,China.We collected 83 crayfish during the period between July 2008 and June 2010.Of these,42 were tested positive for WSSV using the loop-mediated isothermal amplification method(LAMP).Based on geographical and time considerations,we selected six positive samples for amplification of the variable and deletion regions of the WSSV genome.In addition,we used PCR amplification to clone and sequence these regions.We then conducted a pairwise and multiple alignment analysis to evaluate the degree of genetic diver-gence between different strains.The variable region was ~1 440 bp in length in most strains,except in isolate WSSV-XH(1 829 bp).The deletion region was 384 bp in length in six strains,and there was complete homol-ogy among the nucleotide sequences.We compared the isolate sequences to those in GenBank and found at least two distinct types of WSSV in Jiangsu Province.Given this,we hypothesize that the WSSV variable region and the deletion region may have evolved independently.

Recognition and removal of DNA damages is essential for cellular and organismal viability. Nucleotide excision repair (NER) is the sole mechanism in humans for the repair of carcinogenic UV irradiation-induced photoproducts in the DNA, such as cyclobutane pyrimidine dimers. The broad substrate versatility of NER further includes, among others, various bulky DNA adducts. It has been proposed that the 5'-3' helicase XPD (xeroderma pigmentosum group D) protein plays a decisive role in damage verification. However, despite recent advances such as the identification of a DNA-binding channel and central pore in the protein, through which the DNA is threaded, as well as a dedicated lesion recognition pocket near the pore, the exact process of target site recognition and verification in eukaryotic NER still remained elusive. Our single molecule analysis by atomic force microscopy reveals for the first time that XPD utilizes different recognition strategies to verify structurally diverse lesions. Bulky fluorescein damage is preferentially detected on the translocated strand, whereas the opposite strand preference is observed for a cyclobutane pyrimidine dimer lesion. Both states, however, lead to similar conformational changes in the resulting specific complexes, indicating a merge to a "final" verification state, which may then trigger the recruitment of further NER proteins.

White spot syndrome virus (WSSV) replicates rapidly, can be extremely pathogenic and is a common cause of mass mortality in cultured shrimp. Variable number tandem repeat (VNTR) sequences present in the open reading frame (ORF)94, ORF125 and ORF75 regions of the WSSV genome have been used widely as genetic markers in epidemiological studies. However, reports that VNTRs might evolve rapidly following even a single transmission through penaeid shrimp or other crustacean hosts have created confusion as to how VNTR data is interpreted. To examine VNTR stability again, 2 WSSV strains (PmTN4RU and LvAP11RU) with differing ORF94 tandem repeat numbers and slight differences in apparent virulence were passaged sequentially 6 times through black tiger shrimp Penaeus monodon, Indian white shrimp Feneropenaeus indicus or Pacific white leg shrimp Litopenaeus vannamei. PCR analyses to genotype the ORF94, ORF125 and ORF75 VNTRs did not identify any differences from either of the 2 parental WSSV strains after multiple passages through any of the shrimp species. These data were confirmed by sequence analysis and indicate that the stability of the genome regions containing these VNTRs is quite high at least for the WSSV strains, hosts and number of passages examined and that the VNTR sequences thus represent useful genetic markers for studying WSSV epidemiology.

Pathogenicity of white spot syndrome virus (WSSV) after multiple passages in mud crab, Scylla olivacea

The transcriptional adaptor zinc (TAZ) fingers are a specialized class of zinc finger domains reported to exist only in eukaryotic transcriptional coactivator proteins. A putative protein within the shrimp white spot syndrome virus (WSSV) encodes for a TAZ domain, which is unique as no virus so far has been reported for the presence of this domain. Our study shows the viral TAZ domain to be similar to TAZ2 rather than TAZ1 domain of eukaryotic CREB-binding proteins and its paralog p300 proteins. Furthermore, as with eukaryotic TAZ2 domain which interacts and binds to several transcriptional factors including the p53 tumor suppressor protein, an in silico docking study of the WSSV-TAZ and the shrimp p53 transcriptional factor showed the two protein domains to be involved in a protein-protein interaction.

Identification of Two Major Virion Protein Genes of White Spot Syndrome Virus of Shrimp

The rbp9 gene of Drosophila melanogaster has been molecularly characterized and shown to be expressed solely in the CNS, where it encodes proteins with three RNA recognition motifs (RRMs). Sequencing of genomic and cDNA clones of rbp9 revealed a complex gene with three alternative promoters as well as alternative patterns of splicing. The deduced amino acid sequence of the RBP9 proteins is highly similar to those of three other nervous system-specific genes, human HuC and HuD and Drosophila elav, which also encode proteins with three RRMs. Developmental Northern analysis revealed that rbp9 is expressed from the late third instar larva through adult stages. The RBP9 protein was detected specifically in nuclei of the nervous system after morphogenesis of the adult CNS in the mid-pupal stage. Thus, the RBP9 protein does not appear until substantially later than rbp9 transcripts are detected. The adult nervous system nuclear-limited expression pattern, the presence of RRMs, and the high similarity to a group of nervous system-specific proteins in flies and humans suggest that rbp9 belongs to a nervous system-specific RRM protein gene subfamily that may participate in the processing of RNAs involved in the development of the CNS.

The SR proteins constitute a family of nuclear phosphoproteins, which are required for constitutive splicing and also influence alternative splicing regulation. Initially, it was suggested that SR proteins were functionally redundant in constitutive splicing. However, differences have been observed in alternative splicing regulation, suggesting unique functions for individual SR proteins. Homology searches of the Caenorhabditis elegans genome identified seven genes encoding putative orthologues of the human factors SF2/ASF, SRp20, SC35, SRp40, SRp75 and p54, and also several SR-related genes. To address the issue of functional redundancy, we used dsRNA interference (RNAi) to inhibit specific SR protein function during C.elegans development. RNAi with CeSF2/ASF caused late embryonic lethality, suggesting that this gene has an essential function during C.elegans development. RNAi with other SR genes resulted in no obvious phenotype, which is indicative of gene redundancy. Simultaneous interference of two or more SR proteins in certain combinations caused lethality or other developmental defects. RNAi with CeSRPK, an SR protein kinase, resulted in early embryonic lethality, suggesting an essential role for SR protein phosphorylation during development.

The Drosophila no-on-transient A (nonA) gene is involved in the visual behaviors and courtship song of the fly. The NONA polypeptide contains two copies of the RNA-recognition motif (RRM), a hallmark of proteins involved in RNA binding, and an adjacent conserved charged region. This 311-amino-acid region is found in four other proteins and largely overlaps the Drosophila-Behavior/Human Splicing (or DBHS) domain. The newest family member, Drosophila nAhomo, was discovered in a database search, and encodes a protein with 80% identity to NONA. In this study, three nonA mutations generated by chemical mutagenesis were sequenced and found to fall within the conserved region. Site-directed mutagenesis of the two RRMs, and within a (conserved) charged region located C-terminal to them, was performed to determine the significance of these domains with respect to whole-organismal phenotypes. Behavior and viability were assessed in transformed flies, the genetic background of which lacks the nonA locus. Point mutations of amino acid 548 in the charged region confirmed the etiology of the nonAdiss courtship-song mutation and showed that a milder substitution at this site produced intermediate singing behavior, although it failed to rescue visual defects. Mutagenesis of the RRM1 domain resulted in effects on viability, vision, and courtship song. However, amino acid substitutions in RNP-II of RRM2 led to near-normal phenotypes, and the in vivo nonA mutations located in or near RRM2 caused visual defects only. Thus, we suggest that the first RRM could be important for all functions influenced by nonA, whereas the second RRM may be required primarily for normal vision.

The white spot syndrome virus (WSSV) is a highly lethal and contagious pathogen to most cultivated shrimp species. The WSSV genome contains the ICP11 gene and its encoded protein acts as a multifunctinal DNA mimic protein that disrupts the nucleosome assembly by binding to the histone proteins H2A and H3. In addition, WSSV provokes severe nuclear hypertrophy and DNA damage. However, little is known about the influence of WSSV on the expression of the host's genes encoding for histones. Therefore, we investigated the effect of WSSV infection on the expression of the genes encoding histones in shrimp Penaeus vannamei. An RT-qPCR assay was performed to evaluate the temporal expression of H2A and H4 transcripts in the shrimp. Significant changes were observed in the expression of these genes, which coincided with the dynamics of replication of the virus. H2A reached its maximum expression levels at 12 hpi. Thus, it may be suggested that this is a viral strategy to evade the host's immune response in order to promote viral replication.

Mycobacterium tuberculosis is an intracellular bacterium that persists in phagosomes of myeloid cells. M. tuberculosis-encoded factors support pathogen survival and reduce fusion of phagosomes with bactericidal lysosomal compartments. It is, however, not entirely understood if host factors that mediate endosomal fusion affect M. tuberculosis intracellular localization and survival. Neither is it known if endosomal fusion influences induction of host immune reactivity by M. tuberculosis-infected cells. Lysosomal degradation of M. tuberculosis appears to be pivotal for making available lipid substrates for assembly into lipid-CD1d complexes to allow activation of CD1d-restricted invariant natural killer T (iNKT) cells. To clarify the role for endosomal fusion in M. tuberculosis survival and induction of host CD1d-mediated immune defense, we focused our studies on the invariant chain (Ii). Ii regulates endosome docking and fusion and thereby controls endosomal transport. Through direct binding, Ii also directs intracellular transport of the class II major histocompatibility complex and CD1d. Our findings demonstrate that upon infection of Ii-knockout (Ii(-/-)) macrophages, M. tuberculosis is initially retained in early endosomal antigen 1-positive lysosomal-associated membrane protein 1-negative phagosomes, which results in slightly impaired pathogen replication. The absence of Ii did not affect the ability of uninfected and infected macrophages to produce nitric oxide, tumor necrosis factor alpha, or interleukin-12. However, induction of cell surface CD1d was impaired in infected Ii(-/-) macrophages, and CD1d-restricted iNKT cells were unable to suppress bacterial replication when they were cocultured with M. tuberculosis-infected Ii(-/-) macrophages. Thus, while the host factor Ii is not essential for the formation of the M. tuberculosis-containing vacuole, its presence is crucial for iNKT cell recognition of infected macrophages.

A recent genome-wide bioinformatic analysis indicated that 54% of human genes undergo alternative polyadenylation. Although it is clear that differential selection of poly(A) sites can alter gene expression, resulting in significant biological consequences, the mechanisms that regulate polyadenylation are poorly understood. Here we report that the neuron-specific members of a family of RNA-binding proteins, Hu proteins, known to regulate mRNA stability and translation in the cytoplasm, play an important role in polyadenylation regulation. Hu proteins are homologs of the Drosophila embryonic lethal abnormal visual protein and contain three RNA recognition motifs. Using an in vitro polyadenylation assay with HeLa cell nuclear extract and recombinant Hu proteins, we have shown that Hu proteins selectively block both cleavage and poly(A) addition at sites containing U-rich sequences. Hu proteins have no effect on poly(A) sites that do not contain U-rich sequences or sites in which the U-rich sequences are mutated. All three RNA recognition motifs of Hu proteins are required for this activity. Overexpression of HuR in HeLa cells also blocks polyadenylation at a poly(A) signal that contains U-rich sequences. Hu proteins block the interaction between the polyadenylation cleavage stimulation factor 64-kDa subunit and RNA most likely through direct interaction with poly(A) cleavage stimulation factor 64-kDa subunit and cleavage and polyadenylation specificity factor 160-kDa subunit. These studies identify a novel group of mammalian polyadenylation regulators. Furthermore, they define a previously unknown nuclear function of Hu proteins.

The involvement of Ral and its downstream molecules in receptor-mediated endocytosis was examined. Expression of either RalG23V or RalS28N, which are known to be constitutively active and dominantnegative forms, respectively, in A431 cells blocked internalization of epidermal growth factor (EGF). Stable expression of RalG23V or RalS28N in CHO-IR cells also inhibited internalization of insulin. Internalization of EGF and insulin was not affected by full-length RalBP1 which is an effector protein of Ral, but was inhibited by its C-terminal region which binds directly to Ral and POB1. POB1 is a binding protein of RalBP1 and has the Eps15 homology (EH) domain. Deletion mutants of POB1 inhibited internalization of EGF and insulin. However, internalization of transferrin was unaffected by Ral, RalBP1, POB1 and their mutants. Epsin and Eps15 have been reported to be involved in the regulation of endocytosis of the receptors for EGF and transferrin. The EH domain of POB1 bound directly to Epsin and Eps15. Taken together with the observation that EGF and insulin activate Ral, these results suggest that Ral, RalBP1 and POB1 transmit the signal from the receptors to Epsin and Eps15, thereby regulating ligand-dependent receptor-mediated endocytosis.

Gbp1p is a putative telomere-binding protein from Chlamydomonas reinhardtii that contains two RNA recognition motifs (RRMs) which are commonly found in heterogeneous nuclear ribonucleoproteins (hnRNPs). Previously we demonstrated that Gbp1p binds single-stranded DNA (ssDNA) containing the Chlamydomonas telomeric sequence but not the RNA containing the cognate sequence. Here we show that at lower protein concentrations Gbp1 can also bind an RNA containing the cognate sequence. We found that mutation of the two RRM motifs of Gbp1p to match the highly conserved region of hnRNP RRMs did not alter the affinity of Gbp1p for either RNA or DNA. The ability of Gbp1p to associate with either of these two nucleic acids is governed by the dimerization state of the protein. Monomeric Gbp1p associates with either ssDNA or RNA, showing a small binding preference for RNA. Dimeric Gbp1p has a strong preference for binding ssDNA and shows little affinity for RNA. To the best of our knowledge, this is the first example of a protein that qualitatively shifts its nucleic acid binding preference upon dimerization. The biological implications of a telomere-binding protein that is regulated by dimerization are discussed.