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PubMed1980

Complete nucleotide sequence of alfalfa mosaic virus RNA 4.

Alfalfa mosaic virus RNA 4, the subgenomic messenger for viral coat protein, was partially digested with RNase T1 or RNase A and the sequence of a number of fragments was deduced by in vitro labeling with polynucleotide kinase and application of RNA sequencing techniques. From overlapping fragments, the complete primary sequence of the 881 nucleotides of RNA 4 was constructed: the coding region of 660 nucleotides (not including the initiation and termination codon) is flanked by a 5' noncoding region of 39 nucleotides and a 3' noncoding region of 182 nucleotides. The RNA sequencing data completely confirm the amino acid sequence of the coat protein as deduced by Van Beynum et al. (Fur.J. Biochem. 72, 63-78, 1977).

Base Sequence↗
PubMed1998

High prevalence of GB virus C/hepatitis G virus infection in different risk groups of HIV-infected patients.

OBJECTIVES: To investigate the prevalence of GB virus C (GBV-C)/hepatitis G virus (HGV) RNA and anti-E2 antibodies in different risk groups of HIV-infected patients compared to that in healthy blood donors, and to study the effects of possible interactions between HIV and GBV-C/HGV on the carrier state and hepatic changes. METHODS: Sera from 100 consecutive unselected HIV-infected outpatients and from 100 healthy blood donors were screened for GBV-C/HGV viremia and anti-E2 antibodies. Anti-E2 antibodies were detected using an immunoassay developed by Boehringer Mannheim according to the manufacturer's instructions. GBV-C/HGV RNA was extracted from sera and reverse transcribed. The resulting cDNA was amplified with a PCR developed in the laboratory with primers derived from the 5prime prime or minute noncoding region of the viral genome and detected with a specific capture probe. This procedure was validated by a French multicenter quality control group. RESULTS: Thirty-one of the 100 HIV-infected patients and 8% of the healthy blood donors displayed anti-E2 antibodies. Four HIV-infected patients and one healthy blood donor were found to be GBV-C/HGV viremic. When analyzed by risk factor for the acquisition of HIV, no differences in the prevalence of anti-E2 antibodies were found between intravenous drug users and homosexual and heterosexual patients. CONCLUSIONS: We found a high prevalence of GBV-C/HGV infection in the HIV-infected population, irrespective of the risk group factor for HIV infection, suggesting that the sexual route is as effective as the parenteral route for the acquisition of GBV-C/HGV. No biological alteration could be attributed to GBV-C/HGV, even in the viremic patients. HIV-infected patients were able to clear GBV-C/HGV viremia and to mount a humoral immune response.

Journal Article↗
PubMed2004

Direct role of a viroid RNA motif in mediating directional RNA trafficking across a specific cellular boundary.

The plasmodesmata and phloem form a symplasmic network that mediates direct cell-cell communication and transport throughout a plant. Selected endogenous RNAs, viral RNAs, and viroids traffic between specific cells or organs via this network. Whether an RNA itself has structural motifs to potentiate trafficking is not well understood. We have used mutational analysis to identify a motif that the noncoding Potato spindle tuber viroid RNA evolved to potentiate its efficient trafficking from the bundle sheath into mesophyll that is vital to establishing systemic infection in tobacco (Nicotiana tabacum). Surprisingly, this motif is not necessary for trafficking in the reverse direction (i.e., from the mesophyll to bundle sheath). It is not required for trafficking between other cell types either. We also found that the requirement for this motif to mediate bundle sheath-to-mesophyll trafficking is dependent on leaf developmental stages. Our results provide genetic evidence that (1) RNA structural motifs can play a direct role in mediating trafficking across a cellular boundary in a defined direction, (2) the bundle sheath-mesophyll boundary serves as a novel regulatory point for RNA trafficking between the phloem and nonvascular tissues, and (3) the symplasmic network remodels its capacity to traffic RNAs during plant development. These findings may help further studies to elucidate the interactions between RNA motifs and cellular factors that potentiate directional trafficking across specific cellular boundaries.

Base Sequence↗
PubMed2004

Tethering of eIF4G to adenoviral mRNAs by viral 100k protein drives ribosome shunting.

Although most mRNAs initiate translation by 5' ribosome scanning, some small fraction of mammalian and viral mRNAs utilize either of two alternate mechanisms, known as internal ribosome entry and ribosome shunting. Ribosome shunting is a poorly understood form of initiation in which 40S ribosome subunits are loaded onto mRNA through interactions with the m7GTP cap, but then bypass large segments of the mRNA as directed by cis-acting RNA shunting elements and trans-acting protein factors. Here, we describe the molecular mechanism by which ribosome shunting occurs with high efficiency on adenovirus late mRNAs. We show that the viral 100k protein possesses a selective binding element for the 5' noncoding region (5'NCR) of viral late mRNAs (known as the tripartite leader), forms a complex with initiation factor eIF4G and poly(A)-binding protein (PABP), and strongly and selectively enhances the level of both factors and 40S ribosome subunits on viral mRNAs in polysomes. Mutational and biochemical studies demonstrate that the ability of 100k protein to bind both the tripartite leader and eIF4G are critical to promote a high level of ribosome shunting. A molecular mechanism for ribosome shunting is described by which enhanced binding of eIF4G and possibly PABP with 100k protein, and simultaneous interaction with the tripartite leader 5'NCR, drives 40S ribosome recruitment and initiation on mRNAs.

Adenoviridae↗
PubMed1995

Genomic characterization of type 3 polioviruses isolated from vaccine-associated poliomyelitis cases in Brazil.

Eight strains of P3/Sabin-related polioviruses were analyzed; four from persistent paralytic poliomyelitis cases classified as vaccine associated, one from a transient paralysis case classified as transverse myelitis, one from a transient paralysis case classified as Guillain-Barré syndrome, one from a transient facial paralysis case, and one from a healthy vaccine. The serotypes of the viral isolates were identified by the neutralization test with hyperimmune equine sera and the relationship of the isolates with the P3/Sabin strain was demonstrated by molecular hybridization of the viral RNA of the isolates with a P3/Sabin-specific probe. The P3/Sabin relationship was confirmed by PCR, using a pair of specific primers for P3/Sabin-related isolates. The available data indicate that a U-->C mutation at nucleotide 472 in the 5' noncoding region of the genome of the type 3 Sabin strain increases the neurovirulence of this strain and this mutation was observed in all type 3 isolates from vaccine-associated cases. These eight P3/Sabin-related isolates were partially sequenced in the 5' noncoding region and seven presented a U-->C mutation at nucleotide 472, except the isolate from a transient paralysis case classified as transverse myelitis, that maintained a U at nucleotide 472. Although this virus maintaining U at nucleotide 472 may not be the etiological agent of the disease, the possibility that the virus was the causative agent of the disease could not be ruled out.

Base Sequence↗
PubMed1996

The variable 3' ends of a human cytomegalovirus oriLyt transcript (SRT) overlap an essential, conserved replicator element.

The genetically defined human cytomegalovirus (HCMV) lytic-phase replicator, oriLyt, comprises more than 2 kb in a structurally complex region that spans a variety of potential transcription control signals. Several transcripts originate within or cross oriLyt, and we are studying these oriLyt transcription units to determine whether they participate in initiating or regulating lytic-phase DNA synthesis. Results presented here establish the temporal accumulation and structure of the smallest replicator transcript, which we call SRT, and identify a single-sequence element essential to replicator function. SRT was detected as early as 2 h after HCMV infection of human fibroblast cells; transcript levels increased by 24 h and continued to increase thereafter. Consistent with its early appearance, treatment of HCMV-infected cells with the viral DNA polymerase inhibitor phosphonoformic acid had no effect on SRT accumulation; however, no SRT was detected in RNA preparations from cycloheximide-treated infected cells. Additional Northern (RNA) analysis localized the 0.2- to 0.25-kb SRT to an apparently noncoding segment near the center of the oriLyt core region. Reverse transcriptase PCR (rapid amplification of cDNA 5' ends [5'-RACE]) identified a single 5' end. In transient-transfection assays, the sequence immediately upstream of SRT functioned as a promoter responsive to HCMV infection when placed upstream of a reporter gene, suggesting that SRT is the product of a discrete transcription unit. RNA ligase-mediated 3'-RACE showed that SRT is not polyadenylated and has heterogeneous 3' ends within a roughly 45-nucleotide window overlapping an oligopyrimidine sequence having counterparts in the lytic-phase replicators of several herpesviruses. Mutation of the oligopyrimidine element showed that it is essential to oriLyt replicator function; it is the only essential single-sequence HCMV oriLyt replicator element described to date. Collectively, the location of SRT near the center of the oriLyt core region, its early expression, its overlapping relationship with a sequence element essential to replicator function, and its similarities to replicator transcripts in other systems suggest the possibility that SRT plays a role in initiating or regulating HCMV lytic-phase DNA synthesis.

Base Sequence↗
PubMed2005

Identification and characterization of a novel, psoriasis susceptibility-related noncoding RNA gene, PRINS.

To identify genetic factors contributing to psoriasis susceptibility, gene expression profiles of uninvolved epidermis from psoriatic patients and epidermis from healthy individuals were compared. Besides already characterized genes, we identified a cDNA with yet unknown functions, which we further characterized and named PRINS (Psoriasis susceptibility-related RNA Gene Induced by Stress). In silico structural and homology studies suggested that PRINS may function as a noncoding RNA. PRINS harbors two Alu elements, it is transcribed by RNA polymerase II, and it is expressed at different levels in various human tissues. Real time reverse transcription-PCR analysis showed that PRINS was expressed higher in the uninvolved epidermis of psoriatic patients compared with both psoriatic lesional and healthy epidermis, suggesting a role for PRINS in psoriasis susceptibility. PRINS is regulated by the proliferation and differentiation state of keratinocytes. Treatment with T-lymphokines, known to precipitate psoriatic symptoms, decreased PRINS expression in the uninvolved psoriatic but not in healthy epidermis. Real time reverse transcription-PCR analysis showed that stress signals such as ultraviolet-B irradiation, viral infection (herpes simplex virus), and translational inhibition increased the RNA level of PRINS. Gene-specific silencing of PRINS by RNA interference revealed that down-regulation of PRINS impairs cell viability after serum starvation but not under normal serum conditions. Our findings suggest that PRINS functions as a noncoding regulatory RNA, playing a protective role in cells exposed to stress. Furthermore, elevated PRINS expression in the epidermis may contribute to psoriasis susceptibility.

Base Sequence↗
PubMed1997

[Establishment of semi-nested polymerase chain reaction for detecting Coxsackie B virus RNA and the application in clinical diagnosis of viral myocarditis].

The aim of this study was to determine the applicability of the polymerase chain reaction (PCR) technique for the detection of CBV RNA and for routine diagnostic use in clinics. To this end, three oligomeric regions of great homology among the CBVs were selected from the conserved 5'-noncoding region of the CBV genome, and were designated as a set of primers and probe. Semi-nested CBV PCR assay was established. The sensitivity of the method is at 0.1 TCID50 in diluted virus stocks, and this method doesn't react with other related viruses. After the optimum reaction system was determined, the technique was then used in clinical samples. In patients with myocarditis, 62 of 111 serum samples were positive by PCR. The CSF samples from patients with meningitis were also tested, and the positive result was found in 7 out of 20 (35%) specimens. Using the same 111 samples, the PCR technique was compared with the ELISA. The detection rates of the two methods for CBV were 56% and 45%, respectively, with a coincident rate of 77%.

Enterovirus B, Human↗
PubMed2026

NAT10 is critical to block RNA sensing-induced IFN-β transactivation in viral infection.

UNLABELLED: Cells detect invading viruses and produce type I interferons (IFNs) to stimulate an innate antiviral effector response. However, IFN levels must be fine-tuned to achieve antiviral efficacy while limiting hyperinflammatory and tissue-damaging effects. Here, we report that NAT10, a histone and cytidine acetyltransferase, regulates the production of type I IFNs and RNA virus infections. Depletion of NAT10 increased the expression of IFN-β and IFN-stimulated genes, and correspondingly impaired viral replication. Mechanistically, NAT10 dynamically associated with the IFN-β promoter and also negatively regulated IRF3's chromatin associations through modulation of long noncoding RNAs that inhibit IRF3. Treatment of cells with Remodelin, a NAT10 inhibitor, similarly increased IFN-β expression and inhibited viral infections. Overall, our findings reveal NAT10 is a potential host-directed target for antiviral treatment via regulation of type I IFN. IMPORTANCE: Type I interferons (IFNs) signaling pathway is critical to cellular defense and innate immunity against evading pathogens, including viruses. However, induction of type I IFNs is fine-tuned to achieve the antiviral consequence while maintaining host cellular homeostasis. This paper presents a novel mechanism for the NAT10 protein to silence IFN-β induction through modulation of IRF3 activity at the promoter of IFN-β, and further demonstrates the therapeutic potential of the NAT10 inhibitor Remodelin to restrict viral infection while inducing IFN-β.

Interferon-beta↗
PubMed1996

Mapping of host range restriction of the rakkyo strain of tobacco mosaic virus in Nicotiana tabacum cv. bright yellow.

The rakkyo strain (TMV-R) and the common strain (TMV-U1) of tobacco mosaic virus exhibit distinct host range differences. TMV-R infects rakkyo plants, a monocot host that TMV-U1 is unable to infect. However, TMV-R causes only latent infection in Nicotiana tabacum cv. Bright Yellow (BY) in inoculated leaves, whereas TMV-U1 infects BY systemically and induces mosaic symptoms. Complete nucleotide sequencing of the TMV-R genomic RNA revealed amino acid changes in the 130K/ 180K replicase proteins, the 30K protein, and the coat protein and nucleotide changes in the 5' and 3' noncoding regions compared to TMV-U1. To identify viral components involved in determination of differences in host range, we have mapped determinants for the differential infection phenotype in BY plants by constructing chimeric viruses between the two strains in the present study. Examination of the infection phenotypes of the 14 constructed chimeric viruses in BY showed that determinants defining the differential infection phenotype in BY reside in the 130K/180K replicase proteins and the 3' noncoding region. Cognate combination of the 130K/180K replicase proteins and the 3' noncoding region of TMV-U1 origin is required to produce systemic infection in BY plants.

DNA-Directed DNA Polymerase↗
PubMed2006

RNA world - the dark matter of evolutionary genomics.

For a long time, molecular evolutionary biologists have been focused on DNA and proteins, whereas RNA has lived in the shadow of its famous chemical cousins as a mere intermediary. Although this perspective has begun to change since genome-wide transcriptional profiling was successfully extended to evolutionary biology, it still echoes in evolutionary literature. In this mini-review, new developments of RNA biochemistry and transcriptomics are brought to the attention of evolutionary biologists. In particular, the unexpected abundance and functional significance of noncoding RNAs is briefly reviewed. Noncoding RNAs control a remarkable range of biological pathways and processes, all with obvious fitness consequences, such as initiation of translation, mRNA abundance, transposon jumping, chromosome architecture, stem cell maintenance, development of brain and muscles, insulin secretion, cancerogenesis and plant resistance to viral infections.

Animals↗
PubMed1981

Cellular homologue (c-src) of the transforming gene of Rous sarcoma virus: isolation, mapping, and transcriptional analysis of c-src and flanking regions.

The tumorigenic properties of Rous sarcoma virus are attributable to a 60,000-dalton protein, pp60v--src, encoded by a single viral gene, v-src. A homologous gene, c-src, that contains the information for a 60,000-dalton protein, pp60c--src, has been identified in all tested vertebrate cells. By screening a recombinant DNA library representative of the chicken genome, we isolated two overlapping DNA fragments that contain more than 30 kilobases (kb) of DNA spanning the coding sequences for pp60c--src. This 30-kb region is devoid of moderately or highly repeated sequences and shares homology with the entire viral gene and noncoding sequences 5' of v-src. Although v-src has an uninterrupted coding sequence, c-src is interrupted by a minimum of seven intervening sequences. At least two polyadenylylated RNAs seem to be encoded within the DNA we have isolated. The larger RNA, approximately 3.9 kb, is the presumptive c-src mRNA; the smaller transcript of about 2 kb hybridizes to DNA sequences several kb from the 3' end of the v-src/c-src homology area.

Animals↗
PubMed1988

The nucleocapsid gene of infectious hematopoietic necrosis virus, a fish rhabdovirus.

The complete nucleotide sequence of the infectious hematopoietic necrosis virus (IHNV) nucleocapsid gene has been determined using cDNA clones of genomic and messenger RNAs. Genomic clones were generated by using random DNA oligomers to prime cDNA synthesis and were mapped to their respective locations on the genome by the use of cDNA probes derived from viral mRNAs. Interesting features of the IHNV nucleocapsid gene sequence elucidated by the sequencing of these clones include short homologies with N genes of other rhabdoviruses at the 5' and 3' nontranslated termini of the mRNA, as well as an exceptionally long 5' noncoding region of the mRNA, suggesting a leader RNA may be coupled to the N mRNA. A comparison of the IHNV N protein coding sequence with other rhabdoviral N genes shows some homologies at the amino acid level which indicates the possible evolutionary relationship of these N proteins. The determination of the nucleotide sequences of IHNV genes and intergenic regions will be useful for studying the mechanisms of rhabdoviral transcription and replication.

Amino Acid Sequence↗
PubMed1999

Characterization of the Hantaan nucleocapsid protein-ribonucleic acid interaction.

The nucleocapsid (N) protein functions in hantavirus replication through its interactions with the viral genomic and antigenomic RNAs. To address the biological functions of the N protein, it was critical to first define this binding interaction. The dissociation constant, K(d), for the interaction of the Hantaan virus (HTNV) N protein and its genomic S segment (vRNA) was measured under several solution conditions. Overall, increasing the NaCl and Mg(2+) in these binding reactions had little impact on the K(d). However, the HTNV N protein showed an enhanced specificity for HTNV vRNA as compared with the S segment open reading frame RNA or a nonviral RNA with increasing ionic strength and the presence of Mg(2+). In contrast, the assembly of Sin Nombre virus N protein-HTNV vRNA complexes was inhibited by the presence of Mg(2+) or an increase in the ionic strength. The K(d) values for HTNV and Sin Nombre virus N proteins were nearly identical for the S segment open reading frame RNA, showing weak affinity over several binding reaction conditions. Our data suggest a model in which specific recognition of the HTNV vRNA by the HTNV N protein resides in the noncoding regions of the HTNV vRNA.

Capsid↗
PubMedDate not supplied

Effective resistance to potyvirus infection conferred by expression of antisense RNA in transgenic plants.

Approximately 660 nt including the carboxy-terminal portion of the bean yellow mosaic potyvirus (BYMV) coat protein gene, complete 3' noncoding sequence and a short poly(A) tract were introduced to produce antisense RNA in transgenic Nicotiana benthamiana. Original (R0) transformants were selfed, and homozygous second generation (R2) populations challenged with infected sap, purified virus, or viral RNA. One transgenic line of 10 examined was extremely resistant to infection by mechanical inoculation of 100 micrograms/ml BYMV or 50 micrograms/ml BYMV RNA (the highest concentrations tested); no virus could be detected in inoculated leaves of this line. Nine other lines were systemically infected when inoculated with BYMV or BYMV RNA, with initial symptoms indistinguishable from those in nontransgenic plants. One of these lines subsequently developed reduced symptoms and then symptomless, virus-free leaves (complete recovery from BYMV infection), while other lines produced leaves with reduced symptoms and reduced virus titer compared to the controls (partial recovery). No transgenic lines had resistance to infection, nor recovery from symptom expression, following inoculation with pepper mottle or turnip mosaic potyviruses. Antisense RNA from 3' regulatory regions can confer multiple degrees of resistance to potyviruses, including extreme resistanc to infection, presumably by interference in virus replication.

Base Sequence↗
PubMed1998

Diversity among isolates of squash mosaic virus.

cDNA clones of RNA-2 of two isolates of squash mosaic virus (SqMV) were constructed and sequenced, revealing 87% sequence similarity. In Northern blot hybridization analyses, DNA probes made from these clones defined two SqMV hybridization subgroups. This grouping was verified by reciprocal hybridizations of purified RNA from five SqMV isolates, as probed with cDNA made from a member of each subgroup. Comparison of the RNA-2 sequence among the two SqMV isolates, and the reported sequence of other comoviruses, showed that SqMV constitutes one of four major branches in a phylogenetic tree of the genus. Analysis of the terminal noncoding sequences showed that although potentially similar folding patterns may form, neither nucleotide sequence nor secondary structural elements are highly conserved among comoviruses. In vitro translation products from purified RNA-1 of each subgroup (encoding the viral proteases) were found to process the polyprotein generated by in vitro translation of purified RNA-2 from either subgroup.

Amino Acid Sequence↗
PubMed1997

Genotypic distribution of hepatitis C virus in different regions of Thailand.

The genotypic distribution of hepatitis C virus (HCV) isolated from blood donors from four major regions of Thailand was studied by reverse hybridization assays. PCR-amplified products from the 5' noncoding and core regions of the viral genome were hybridized to genotype- and subtype-specific probes which were immobilized on the nitrocellulose membrane. Of 332 anti-HCV-positive plasma samples studied, 71% contained HCV RNA. HCV genotype 3a was the most prevalent genotype (39%), followed by genotype 1b (20%) and genotype 6 group variants (18%). HCV genotype 1a was identified among 9% of all isolates. Other genotypes (genotype 1 which was neither 1a nor 1b, genotype 3b, and an unclassified genotype) were uncommon. There was no difference in the mean age of the donors infected with different HCV genotypes. The genotypic distribution pattern of HCV was similar among HCV isolates from different regions of Thailand.

Genes, Viral↗