The group of Negative-Stranded RNA Viruses (NSVs) includes many human pathogens, like the influenza, measles, mumps, respiratory syncytial or Ebola viruses, which produce frequent epidemics of disease and occasional, high mortality outbreaks by transmission from animal reservoirs. The genome of NSVs consists of one to several single-stranded, negative-polarity RNA molecules that are always assembled into mega Dalton-sized complexes by association to many nucleoprotein monomers. These RNA-protein complexes or ribonucleoproteins function as templates for transcription and replication by action of the viral RNA polymerase and accessory proteins. Here we review our knowledge on these large RNA-synthesis machines, including the structure of their components, the interactions among them and their enzymatic activities, and we discuss models showing how they perform the virus transcription and replication programmes. - Highlights: • Overall organisation of NSV RNA synthesis machines. • Structure and ...
TY - JOUR. T1 - Detection of negative-stranded hepatitis C virus RNA using a novel strand-specific reverse transcription-polymerase chain reaction. AU - Mizutani, Tetsuya. AU - Ikeda, Masanori. AU - Saito, Satoru. AU - Sugiyama, Kazuo. AU - Shimotohno, Kunitada. AU - Kato, Nobuyuki. PY - 1998/2/1. Y1 - 1998/2/1. N2 - We developed a novel single-tube reverse transcription-polymerase chain reaction (RT-PCR) for the specific detection of negative-stranded hepatitis C virus (HCV) RNA. By using in vitro synthesized positive- and negative-stranded HCV RNAs, it was demonstrated that as few as 50 copies of negative-stranded HCV RNA could be specifically detected with a set of primers that amplify a 232-base pair sequence unique to the 5-non-coding region of HCV RNA, while 108 copies of positive-stranded HCV RNA were not detected. In addition, we demonstrated that this method allows the detection as few as 100 copies of negative-stranded HCV RNA even with the coexistence of a 100-fold excess of ...
387440791 - EP 0832191 A4 2000-11-15 - RECOMBINANT VIRAL NUCLEIC ACIDS - [origin: WO9640867A1] The present invention relates to a recombinant viral nucleic acid selected from a (+) sense, single stranded RNA virus possessing a native subgenomic promoter encoding for a first viral subgenomic promoter, a nucleic acid sequence that codes for a viral coat protein whose transcription is regulated by the first viral subgenomic promoter, a second viral subgenomic promoter and a second nucleic acid sequence whose transcription is regulated by the second viral subgenomic promoter. The first and second viral subgenomic promoters of the recombinant viral nucleic acid do not have homologous sequences relative to each other. The recombinant viral nucleic acid provides the particular advantage that it systematically transcribes the second nucleic acid in the host. Host organisms encompassed by the present invention include procaryotes and eucaryotes, particularly animals and plants. The present invention also relates
Two copies of unspliced human immunodeficiency virus (HIV)-1 genomic RNA (gRNA) are preferentially selected for packaging by the group-specific antigen (Gag) polyprotein into progeny virions as a dimer during the late stages of the viral lifecycle. Elucidating the RNA features responsible for selective recognition of the full-length gRNA in the presence of an abundance of other cellular RNAs and spliced viral RNAs remains an area of intense research. The recent nuclear magnetic resonance (NMR) structure by Keane et al. [1] expands upon previous efforts to determine the conformation of the HIV-1 RNA packaging signal. The data support a secondary structure wherein sequences that constitute the major splice donor site are sequestered through base pairing, and a tertiary structure that adopts a tandem 3-way junction motif that exposes the dimerization initiation site and unpaired guanosines for specific recognition by Gag. While it remains to be established whether this structure is conserved in the context
Polivirüsün hücresel yaşam döngüsü (1) CD155 reseptörüne bağlanmasıyla başlar. Virüs endositozla alınır, ve viral RNA serbest kalır (2). Translation of the viral RNA occurs by an IRES-mediated mechanism (3). The polyprotein is cleaved, yielding mature viral proteins (4). The positive-sense RNA serves as template for complementary negative-strand synthesis, producing double-stranded replicative form (RF) RNA(5). Many positive strand RNA copies are produced from the single negative strand (6). The newly synthesized positive-sense RNA molecules can serve as templates for translation of more viral proteins (7) or can be enclosed in a capsid (8), which ultimately generates progeny virions. Lysis of the infected cell results in release of infectious progeny virions (9).[2] ...
The |i|Quick|/i|-RNA Viral Kit is a quick, purification system for viral RNA from plasma, serum, cell culture media, cellular suspensions, urine, blood, saliva and any other biological samples stored in DNA/RNA Shield™. DNA/RNA Shield™ ensures nucleic acid stability during sample storage/transport at ambient temperatures (4-25¬∞C). The reagent effectively lyses cells and inactivates nucleases and infectious agents (virus). The kit also features a specialized buffer system that facilitates complete viral particle lysis for efficient RNA isolation from samples containing enteroviruses, rhinoviruses, coronaviruses, HIV, HCV, influenza A virus, flaviviruses, measles virus, parainfluenza virus, parvovirus (a ssDNA virus), etc. Viral RNA is bound to the column, washed and eluted. The isolated high-quality viral RNA is ready for all downstream applications such as Next-Gen Sequencing, hybridization-based and RT/PCR detection.
This review is centered on the major strategies used by plant RNA viruses to produce the proteins required for virus multiplication. The strategies at the level of transcription presented here are synthesis of mRNA or subgenomic RNAs from viral RNA templates, and cap-snatching. At the level of translation, several strategies have been evolved by viruses at the steps of initiation, elongation and termination. At the initiation step, the classical scanning mode is the most frequent strategy employed by viruses; however in a vast number of cases, leaky scanning of the initiation complex allows expression of more than one protein from the same RNA sequence. During elongation, frameshift allows the formation of two proteins differing in their carboxy terminus. At the termination step, suppression of termination produces a protein with an elongated carboxy terminus. The last strategy that will be described is co- and/or post-translational cleavage of a polyprotein precursor by virally encoded ...
24. -T. and Nevins, J. R. Mol. Cell. BioI. Mol. Cell. BioI. 3: 2058-2065. 25. Feldman, L. , Imperiale, M. J. and Nevins, J. R. Sci. USA 79: 4952-4956. 1982. 28. Akusjarvi, G. and Persson, H. 29. Shaw, A. R. and Ziff, E. B. Nature 290: 113-118. Nature 292: 420-426. Cell 22: 905-916. 1983. Proc. Natl. Acad. 26. Fraser, N. , Nevins, J. , Ziff, E. B. and Darnell, J. E. BioI. 129: 643-656. 1979. 27. Nevins, J. R. and Wilson, M. C. 1: J. Mol. 1981. 1981. 1980. 30. Alt, F. , Bothwell, A. L. , Koshland, M. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. J. Initiation of protein synthesis: evidence for messenger RNA independent binding of methionyl-transfer RNA to the 40 S ribosomal subunit. J. Mol. BioI. 76:379-403, 1973. , Bose, K. K. Protein synthesis in rabbit reticulocytes: characteristics of a Met-tRNAfMet binding factor. Biochem. Biophys. Res. Commun. 48:1-9, 1972. , Kyner, D. and Acs,~. Protein initiation in eukaryotes: formation and function of a ternary complex composed of a ...
A study of sexual transmission of Zika virus among mice (link to paper) demonstrates beautifully that viral nucleic acid detected by polymerase chain reaction (PCR) is not the same as infectious virus.. Male mice were infected with Zika virus and then mated with female mice. Efficient sexual transmission of the virus from males to females was observed. This observation in itself is very interesting but is not the focus of my comments.. To understand the dynamics of sexual transmission, the authors measured Zika virus shedding in seminal fluid - by both PCR, to detect viral RNA, and by plaque assay, to detect infectious virus. The results are surprising (see figure - drawn in my hotel room).. Zika virus RNA persisted in semen for up to 60 days - far longer than did infectious virus, which could not be detected after about three weeks.. Many laboratories choose to assay the presence of viral genomes by PCR. This is an acceptable technique as long as the limitations are understood - it detects ...
Upon entry, the host cell senses an invasion. Recent evidence suggests that structured viral RNAs can act as Pathogen Associated Molecular Patterns (PAMPs) that are recognized by host Pattern Recognition Receptors (PRR) to activate cell signaling. An immediate goal of the laboratory is to understand how specific viral RNA-protein interactions influence the viral life cycle and the host immune response. The triphosphate group found at the 5 end of some viral RNA transcripts (5 3P) has been described recently as an important determinant of self versus non-self that allows cells to distinguish between viral and cellular RNAs. Our results suggest that the 5 3P cannot be the only determinant because some RNAs with a 5 3P are potent activators, while others cause no activation of innate immune signaling. We have identified a non-structured region in the hepatitis C virus (HCV) 3 untranslated region RNA that activates innate immune signaling by interacting with the RNA helicase RIG-I. We ...
The extra length that dimer RNA provides is critical in encouraging PKR to pair up and function properly. The length needed for one PKR to bind to RNA is fifteen base pairs, said Philip Bevilacqua, professor of chemistry, Penn State, one of the lead scientists on the project along with James Cole, associate professor, University of Connecticut. To get two PKRs to bind and dimerize, you need an RNA strand that is twice as long. Coles laboratory provided evidence of dimerization of RNA and PKR. In their experiments at Penn State, the scientists found the dimer RNA activated PKR from 9 to 118 times more than the single strand RNA, depending on the RNA type. TAR RNA dimerization activated the most PKR when the TAR did not exhibit structural defects. The researchers report their findings in a recent issue of the Journal of Molecular Biology. Adding these defects decreases the number of places where PKR can bind to the RNA, Heinicke explained. RNAs that showed the greatest degree of symmetry ...
The genomes of RNA viruses often contain RNA structures that are crucial for translation and RNA replication and may play additional, uncharacterized roles during the viral replication cycle. RNA structure with single-nucleotide resolution. In combination with orthogonal evolutionary analyses, we uncover several conserved RNA structures in the open reading frame of the viral genome. The…
NanoString has launched the nCounter RNA:Protein PanCancer Immune Profiling Panel.The entire nCounter line of instruments can analyze the panel, which measures both RNA and protein expression in samples containing as few as 150,000 cells. The new panel combines the measurement of 30 proteins with 770 RNA measurements specifically for immuno-oncology studies. The RNA measurements look at genes indicated in the hallmarks of cancer and the protein measurements focus on cell surface and immune checkpoint targets.
Id like to determine viral RNA level changes in virus producing cell culture transfected with as-DNA. Can anybody provide me with a protocol and anything to be noted in the operation? Thanks in advance.. ...
PubMed comprises more than 30 million citations for biomedical literature from MEDLINE, life science journals, and online books. Citations may include links to full-text content from PubMed Central and publisher web sites.
We have recently discovered that HIV-1 Integrase protein regulates particle maturation, a role completely independent of its catalytic activity. This involves the binding of integrase to viral RNA molecules, which ensures the encapsidation of viral RNA and IN in the viral core. Remarkably, integrase-RNA interactions can be targeted by a novel class of compounds that are currently in clinical trials. Our goal is to discover novel compounds that directly target integrase-RNA interactions and determine their mechanism of action, which will be of great value in the clinical setting.. ...
Patients are stratified by screening plasma viral RNA results (50,000 copies/ml or below vs above 50,000 copies/ml) and randomized to 1 of 2 treatment arms. Group 1 receives IDV 3 times daily plus d4T/3TC twice daily. Group 2 receives IDV/NFV/d4T/3TC twice daily. Patients remain on study medications for 24 weeks and are seen at the clinic once every 4 weeks after entering the study. At each clinic visit, blood samples are taken to evaluate CD4 cell count and plasma HIV RNA levels ...
Dear All, I am looking for a reliable method for the quantitation of in vitro transcribed RNA. If its quick and cheap alls the better. Also does anyone else have problems with the Ambion capped RNA making kit, Ithink they call it the mMESSAGE mMACHINE. We seem t get such variable results in term of yield its untrue, even when we make RNA from the same sample of linearised DNA (But on different days) Robert R. Woodward Email rw200 at cus.cam.ac.uk d ...
http://gateway.isiknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcAuth=Drexel&SrcApp=hagerty_opac&KeyRecord=0042-6822&DestApp=JCR&RQ=IF_CAT_ ...
The one-step RT-ddPCR kit for probes, introduced by Bio-Rad Laboratories, Inc., provides researchers with the ability to measure target RNA molecules with precision and sensitivity for applications such as gene expression analysis, miRNA analysis, and viral load quantitation.
BMV development was studied in well defmed physiological conditions, avoiding the use of antibiotics or excision of the roots. For an adequate labelling, to distinguish between host and viral RNAs, different periods of exposure to 32p were required, depending upon the post-inocu1ation time of infected plants and corresponding age of healthy controL Within the period of normal outlook of the plants, the rate of 32p· incorporation into BMV RNA was much higher than into barley nboso-mal RNA in infected tissue or in comparable healthy tissue.Analysis of the replicative structures isolated from infected plants revealed that only the three largest RNAs (A,K,B) had their own replicative intermediates (R I). This material was shown to con-tain a certain amount of intact viral RNA species, which were stable when treated with heat and for-mamide, excluding the possibility of hidden breaks. read more ...
Regarding HIV following statement not TRUE DNA retrovirus The genome of HIV is diploid, composed of 2 identical single stranded positive sense RNA copies. In association with viral RNA is the reverse transcriptase enzyme which is the characteristic
Regarding HIV following statement not TRUE DNA retrovirus The genome of HIV is diploid, composed of 2 identical single stranded positive sense RNA copies. In association with viral RNA is the reverse transcriptase enzyme which is the characteristic
[The episode starts with tons of virus organisms on Gumballs fur, then one of them stands on a hill-like lump of fur] Virus: Brothers, weve mutated many times over for this moment, but now we are ready. Today, we take this body; tomorrow, the REST OF THE WORLD! [The virus army starts cheering...
USP15 Participates in Hepatitis C Virus Propagation through Regulation of Viral RNA Translation and Lipid Droplet Formation. Kusakabe S, Suzuki T, Sugiyama Y, Haga S, Horike K, Tokunaga M, Hirano J, Zhang H, Chen DV, Ishiga H, Komoda Y, Ono C, Fukuhara T, Yamamoto M, Ikawa M, Satoh T, Akira S, Tanaka T, Moriishi K, Fukai M, Taketomi A, Yoshio S, Kanto T, Suzuki T, Okamoto T, Matsuura Y. J Virol. 2019 Mar 5;93(6). pii: JVI.01708-18. doi: 10.1128/JVI.01708-18. Print 2019 Mar 15. ...
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Consequently, despite the very simple reality that these RNA species are labeled as unstable, processing of many CUTs seems for being somewhat slow. domyhomeworkfor.me DNA can be a form of nucleic acid and it truly is composed of billions of nucleotides and a few certain amino acids. These organisms reside in
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Tusq X Plus-ன் பயன்பாடுகள், மருந்தளவு, பக்க விளைவுகள், நன்மைகள், தொடர்புகள் மற்றும் எச்சரிக்கைகள் ஆகியவற்றை கண்டுபிடியுங்கள்.
Tusq X Plus-ன் பயன்பாடுகள், மருந்தளவு, பக்க விளைவுகள், நன்மைகள், தொடர்புகள் மற்றும் எச்சரிக்கைகள் ஆகியவற்றை கண்டுபிடியுங்கள்.
TY - JOUR. T1 - Mizoribine inhibits hepatitis C virus RNA replication. T2 - Effect of combination with interferon-α. AU - Naka, Kazuhito. AU - Ikeda, Masanori. AU - Abe, Ken Ichi. AU - Dansako, Hiromichi. AU - Kato, Nobuyuki. N1 - Funding Information: We thank T. Nakamura, A. Morishita, and T. Maeta for their helpful experimental assistance. This work was supported by Grants-in-Aid for the third-term comprehensive 10-year strategy for cancer control, and for research on hepatitis from the Ministry of Health, Labor, and Welfare of Japan, and by Grants-in-Aid for scientific research from the Organization for Pharmaceutical Safety and Research (OPSR).. PY - 2005/5/13. Y1 - 2005/5/13. N2 - Interferon (IFN)-α monotherapy, as well as the more effective combination therapy of IFN-α and ribavirin, are currently used for patients with chronic hepatitis C caused by hepatitis C virus (HCV) infection, although the mechanisms of the antiviral effects of these reagents on HCV remain ambiguous, and side ...
Summary of Facts and Submissions. I. European patent No. 0 846 181 with the title cDNA corresponding to the antigenome of nonsegmented negative strand RNA viruses, and process for the production of such viruses encoding additional antigenically active proteins was granted on European patent application No. 96928446.2 (published as WO 97/06270). The patent was granted with 21 claims.. II. Claim 1 of the patent as granted read as follows:. 1. A method for the production of an infectious non-segmented negative-strand RNA virus of the family Paramyxoviridae comprising. (a) introducing a cDNA molecule contained in a plasmid, wherein said cDNA molecule comprises the entire (+)-strand sequence of said negative- strand RNA virus operatively linked to an expression control sequence, which allows the synthesis of anti-genomic RNA transcripts bearing the authentic 3 -termini, and wherein said cDNA molecule consists of an integral multiple of six nucleotides, into a helper cell expressing an ...
A positive-sense single-stranded RNA virus (or (+)ssRNA virus) is a virus that uses positive sense, single-stranded RNA as its genetic material. Single stranded RNA viruses are classified as positive or negative depending on the sense or polarity of the RNA. The positive-sense viral RNA genome can also serve as messenger RNA and can be translated into protein in the host cell. Positive-sense ssRNA viruses belong to Group IV in the Baltimore classification. Positive-sense RNA viruses account for a large fraction of known viruses, including many pathogens such as the hepatitis C virus, West Nile virus, dengue virus, and SARS and MERS coronaviruses, as well as less clinically serious pathogens such as the rhinoviruses that cause the common cold. Positive-sense ssRNA viruses have genetic material that can function both as a genome and as messenger RNA; it can be directly translated into protein in the host cell by host ribosomes. The first proteins to be expressed after infection serve genome ...
In this study we developed a novel highly adapted HCV replicon that harbors two synergistic mutations in NS3 and one in NS5A. The ECF of this RNA was ∼5 × 105 CFU per μg of RNA, which is ∼20-fold higher than that of the best-adapted replicon we described recently (29). By analyzing this and several other HCV RNAs that differed with respect to their levels of cell culture adaptation, we found a clear correlation between the ECF and RNA replication as determined by two different transient-transfection assays. These results demonstrate that cell culture-adaptive mutations increase RNA replication. They also provide an explanation of how adaptive replicons are generated. As shown with the parental replicon carrying the luciferase gene, this nonadapted RNA replicated only transiently and at a low level in transfected cells. During this time, mutations must have been generated by the viral RNA polymerase that in a few instances were adaptive. By increasing the level of RNA replication, cells ...
A negative-sense single-stranded RNA virus (or (-)ssRNA virus) is a virus that uses negative sense, single-stranded RNA as its genetic material. Single stranded RNA viruses are classified as positive or negative depending on the sense or polarity of the RNA. The negative viral RNA is complementary to the mRNA and must be converted to a positive RNA by RNA polymerase before translation. Therefore, the purified RNA of a negative sense virus is not infectious by itself, as it needs to be converted to a positive sense RNA for replication. These viruses belong to Group V on the Baltimore classification. In addition, negative-sense single-stranded RNA viruses have complex genomic sequences, cell cycles, and replication habits that use various protein complexes to arrange in specific conformations and carry out necessary processes for survival and reproduction of their genomic sequences. The complexity of negative-sense single-stranded RNA viruses carries into its ability to suppress the innate immune ...
TY - JOUR. T1 - Importance of the positive-strand RNA secondary structure of a murine coronavirus defective interfering RNA internal replication signal in positive-strand RNA synthesis. AU - Repass, John F.. AU - Makino, Shinji. PY - 1998/10. Y1 - 1998/10. N2 - The RNA elements that are required for replication of defective interfering (DI) RNA of the JHM strain of mouse hepatitis virus (MHV) consist of three discontinuous genomic regions: about 0.46 to 0.47 kb from both terminal sequences and an internal 58-nucleotide (nt)-long sequence (58-nt region) present at about 0.9 kb from the 5 end of the DI genome. The internal region is important for positive-strand DI RNA synthesis (Y. N. Kim and S. Makino, J. Virol. 69:4963-4971, 1995). We further characterized the 58-nt region in the present study and obtained the following results. (i) The positive-strand RNA structure in solution was comparable with that predicted by computer modeling. (ii) Positive-strand RNA secondary structure, but not ...
Both genomic and subgenomic replicative intermediates (RIs) and replicative-form (RF) structures were found in 17CL1 mouse cells that had been infected with the A59 strain of mouse hepatitis virus (MHV), a prototypic coronavirus. Seven species of RNase-resistant RF RNAs, whose sizes were consistent with the fact that each was derived from an RI that was engaged in the synthesis of one of the seven MHV positive-strand RNAs, were produced by treatment with RNase A. Because the radiolabeling of the seven RF RNAs was proportional to that of the corresponding seven positive-strand RNAs, the relative rate of synthesis of each of the MHV positive-strand RNAs may be controlled by the relative number of each of the size classes of RIs that are produced. In contrast to alphavirus, which produced its subgenome-length RF RNAs from genome-length RIs, MHV RF RNAs were derived from genome- and subgenome-length RIs. Only the three largest MHV RF RNAs (RFI, RFII, and RFIII) were derived from the RIs that ...
TY - JOUR. T1 - NMR structure of stem-loop SL2 of the HIV-1 Ψ RNA packaging signal reveals a novel A-U-A base-triple platform. AU - Amarasinghe, Gaya K.. AU - De Guzman, Roberto N.. AU - Turner, Ryan B.. AU - Summers, Michael F.. PY - 2000/5/26. Y1 - 2000/5/26. N2 - The genome of the human immunodeficiency virus type-1 (HIV-1) contains a stretch of ~120 nucleotides known as the ψ-site that is essential for RNA packaging during virus assembly. These nucleotides have been proposed to form four stem-loops (SL1-SL4) that have both independent and overlapping functions. Stem-loop SL2 is important for efficient recognition and packaging of the full-length, unspliced viral genome, and also contains the major splice-donor site (SD) for mRNA splicing. We have determined the structure of the 19-residue SL2 oligoribonucleotide by heteronuclear NMR methods. The structure is generally consistent with the most recent of two earlier secondary structure predictions, with residues G1-G2-C3-G4 and C6-U7 forming ...
Poliovirus RNA replicates in membrane-associated replication complexes in the cytoplasm of infected cells. By using a reversible inhibitor of poliovirus RNA replication, it is possible to synchronize viral RNA replication. The processing of the viral polyprotein results in the formation of the individual viral proteins along with stable intermediates in the processing pathway. To expand the utility of the in vitro complementation assay, experiments were designed to determine if all of the viral replication proteins could be provided in trans to support the replication of mutant RNA templates. The authors engineered two transcript RNAs (DJB2 and DJB15) that contained large out-of-frame deletions in the polyprotein coding sequence. The results to date using the in vitro complementation assay indicate that the 5 cloverleaf, the 3 nontranslated region (NTR), and the poly(A) tail are the minimum sequences required for negative-strand synthesis. Previous studies have shown that the 5 cloverleaf plays an
Figure 2 shows the genome organization of GRV; those of other umbraviruses are very similar. For each RNA, there is at the 5′ end a very short non-coding region preceding ORF1, which encodes a putative product of 31-37 kDa. ORF2, which slightly overlaps the end of ORF1, could encode a product of 63-65 kDa but lacks an AUG initiation codon near its 5′ end. However, immediately before the stop codon of ORF1 there is a 7 nt sequence that is associated with frameshifting in several plant and animal viruses, and it seems probable that ORF1 and ORF2 are translated as a single polypeptide of 94-98 kDa by a mechanism involving a −1 frameshift. The predicted product contains, in the ORF2 region, sequence motifs characteristic of viral RdRp. A short untranslated region separates ORF2 from ORF3 and ORF4, which overlap each other almost completely in different reading frames and each yield a putative product of 26-29 kDa. The ORF4 product contains sequences characteristic of plant virus MPs. The ORF3 ...
TY - JOUR. T1 - Molecular cloning of full-length HIV-1 genomes directly from plasma viral RNA. AU - Fang, Guowei. AU - Weiser, Barbara. AU - Visosky, Aloise A.. AU - Townsend, Laura. AU - Burger, Harold. PY - 1996. Y1 - 1996. N2 - Human immunodeficiency virus type 1 (HIV-1) in plasma reflects the replicating virus population at any point in time in vivo. Studies of the relationship of the complete HIV-1 genome to pathogenesis therefore need to focus on plasma virions. Since dual infections and recombination can occur in vivo, cloning an intact plasma virus genome as a single full-length molecule is desirable. For these reasons, we developed an efficient method to clone full-length HIV-1 genomes directly from plasma viral RNA. This method used reverse transcription and long polymerase chain reaction (PCR) amplification. Virion-associated RNA was isolated from plasma samples and then reverse- transcribed to make cDNA for PCR amplification. Two different strategies were employed to amplify the ...
Viral RNA load in the nasopharyngeal swabs peaked early at median 7.56 (range 6.19-10.56) log10 copies/mL and decreased over time (p,0.001 for trend) (Figure, panel A). The positivity of the specimens was 75% during week 2 and 55% during week 3 (Appendix Table 2). In comparison, the median initial fecal RNA load was 7.68 (range ,4.10-10.27) log10 copies/mL and remained steadily high (p = 0.148 for trend) for ,3 weeks (Figure, panel B). Fecal positivity remained ,80%. The median RNA load in fecal samples was significantly higher than that for nasopharyngeal swab specimens during week 2 (7.26 vs. 6.19 log10 copies/mL; p = 0.006) and week 3 (7.61 versus 5.49 log10 copies/mL; p = 0.006). Except for 1 case, the RNA load in saliva declined rapidly with time (p = 0.003 for trend) (Figure, panel C). Positivity in saliva samples was 80% in week 1 but dropped sharply to 33% in week 2 and 11% in week 3.. We collected urine specimens from the 12 patients after a median of 3 (range 0-8) days and plasma ...
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Hepatitis C trojan (HCV) is an enveloped, positive strand RNA computer virus of about 9. attributable to inherently different properties of low density particles, to association of these particles with factors stimulating fusion, or to co-floatation of factors enhancing fusion activity in genus of the Flaviviridae family (1). Based on sequence comparison, patient isolates are classified into seven genotypes, differing in their nucleotide sequence by 30C35% (2C5). The two viral surface proteins, E1 (residues 192C383) and E2 (residues 384C746), are processed by transmission peptidases of the endoplasmic reticulum from a 3,000-amino acid-long polyprotein encoded by the HCV genome (examined in Ref. 2). The E1 (31 kDa) and E2 (70 kDa) proteins are glycosylated in their large amino-terminal ectodomains (6) and are anchored in the viral membrane by their carboxyl-terminal transmembrane domains. E1 and E2 form a heterodimer stabilized by noncovalent interactions. This oligomer is usually thought to be ...
Early biochemical experiments established that the minimal RNA synthesis machinery of NNS RNA viruses comprises the N encased genomic RNA associated with the viral polymerase, an L-P complex (Emerson and Yu, 1975; Mellon and Emerson, 1978). The atomic structure of N‐RNA complexes from VSV and rabies virus provided evidence that the RNA must somehow be dissociated from N for copying by the polymerase (Albertini et al, 2006; Green et al, 2006). The co‐crystal structure of the PCTD of VSV with the N‐RNA complex led to a model in which P brings L to the RNA template by binding directly between N molecules, and this interaction is perhaps also required to keep L associated with the N‐RNA during copying (Green and Luo, 2009). By now providing the first direct evidence that L can actually use RNA in the absence of the N and P, we have defined the minimal RNA synthesis components as L and RNA. We conclude that while N and P play important roles in viral RNA synthesis they are not essential for ...
In these studies, we found that paralogous viral RNA structures originating from duplications in the 3′ UTR of flavivirus genomes are under different selective pressures in different hosts. The data support the hypothesis that duplicated DB elements, present in most MBFV genomes, conserve redundant functions, but also have evolved divergent host-specific activities that modulate viral RNA replication. Our work provides mechanistic details by which DB structures regulate viral genome conformation by either enhancing or competing with long-range RNA-RNA interactions that promote viral RNA synthesis. We propose a model in which different requirements for viral replication in mosquito and human cells may explain the conservation of duplicated RNA structures in flavivirus 3 UTRs.. Formation of two DB structures with PK interactions in the DENV genome was originally predicted (15-17) and then supported by chemical and enzymatic probing (5, 24, 51). Interestingly, predictions of RNA folding ...
Hepatitis C virus (HCV) is a single-stranded plus-sense RNA virus that is transmitted by blood-to-blood contact, and infects the human liver. HCV has a unique dependence on the liver-specific microRNA miR-122, where miR-122 binds the 5´ un-translated region of the viral RNA at two tandem sites and increases viral RNA abundance. The mechanisms of augmentation are not yet fully understood, but the interaction is known to stabilize the viral RNA, increase translation from the viral internal ribosomal entry site (IRES), and result in increased viral yield. In an attempt to create a small animal model for HCV, we added miR-122 to mouse cell lines previously thought non-permissive to HCV, which rendered these cells permissive to the virus, additionally showing that miR-122 is one of the major determinants of HCV hepatotropism. We found that some wild-type and knockout mouse cell lines - NCoA6 and PKR knockout embryonic fibroblasts - could be rendered permissive to transient HCV sub-genomic, but not ...
Nonsegmented negative-strand (NNS) RNA viruses initiate infection by delivering into the host cell an extremely specific RNA synthesis machine comprising the genomic RNA completely encapsidated from the viral nucleocapsid protein and from the viral polymerase. cap-forming actions. The capping enzyme maps to a globular site, which can be juxtaposed towards the band, as well as the cap methyltransferase maps to a far more distal and connected globule flexibly. Upon P binding, L goes through a substantial rearrangement that may reveal an optimal placing of its practical domains for transcription. The structural map of L provides fresh insights in to the interrelationship of its different domains, and their rearrangement on P binding thats likely very important to RNA synthesis. As the set up of conserved areas involved with catalysis can be homologous, the structural insights acquired for VSV L most likely extend to all or any NNS RNA infections. rows in Fig. 4with averages 8C10 in Fig. 1row in ...
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The HCV replication complex. After clathrin-mediated endocytosis, fusion of HCV with cellular membranes, and uncoating the viral nucleocapsid, the single-stranded positive-sense RNA genome of the virus of approximately 9600 nucleotides is released into the cytoplasm to serve as a messenger RNA for the HCV polyprotein precursor. The HCV genome contains a single large open reading frame encoding for a polyprotein of approximately 3100 amino acids. The translated section of the HCV genome is flanked by the strongly conserved HCV 3′ and 5′ untranslated regions (UTR). The 5′ UTR is comprised of four highly structured domains forming the internal ribosome entry site (IRES), which is a virus-specific structure to initiate HCV mRNA translation. From the initially translated polyprotein, the structural HCV protein core (C) and envelope 1 and 2 (E1, E2); p7; and the six nonstructural HCV proteins NS2, NS3, NS4A, NS4B, NS5A and NS5B, are processed by both viral and host proteases. The core protein ...
The overall goal of our research is to understand the structure and function of RNA molecules. Most of our early work focused on ribosomal RNA (rRNA), characterizing the role of the RNA in protein synthesis (Vila et al 1994, Thompson et al 2001). More recently, we have turned our attention to understanding the structure and function of viral RNA molecules, particularly enteroviral genomic RNA. We have conducted studies to learn the structure of the internal ribosome entry site (IRES) RNA found in picornaviruses (Kim et al., 2005, Bailey and Tapprich 2007) and we have determined virulence determinants in picornal genomic RNA (Prusa et al. 2014). In our initial studies we have used chemical modification and primer extension to deduce the structure of the IRES elements in coxsackievirus B3. This analysis has been completed for virulent wild type viruses, attenuated mutant viruses and avirulent viruses. We have shown localized structural changes in the IRES RNA that correlate with virulence. These ...
Coronaviruses are positive-strand RNA viruses that are important infectious agents of both animals and humans. A common feature among positive-strand RNA viruses is their assembly of replication-transcription complexes in association with cytoplasmic membranes. Upon infection, coronaviruses extensively rearrange cellular membranes into organelle-like replicative structures that consist of double-membrane vesicles and convoluted membranes to which the nonstructural proteins involved in RNA synthesis localize. Double-stranded RNA, presumably functioning as replicative intermediate during viral RNA synthesis, has been detected at the double-membrane vesicle interior. Recent studies have provided new insights into the assembly and functioning of the coronavirus replicative structures. This review will summarize the current knowledge on the biogenesis of the replicative structures, the membrane anchoring of the replication-transcription complexes, and the location of viral RNA synthesis, with particular
RNA viruses infecting vertebrates differ fundamentally in their ability to establish persistent infections with markedly different patterns of transmission, disease mechanisms and evolutionary relationships with their hosts. Although interactions with host innate and adaptive responses are complex and persistence mechanisms likely multi-factorial, we previously observed associations between bioinformatically predicted RNA secondary formation in genomes of positive-stranded RNA viruses with their in vivo fitness and persistence. To analyse this interactions functionally, we transfected fibroblasts with non-replicating, non-translated RNA transcripts from RNA viral genomes with differing degrees of genome-scale ordered RNA structure (GORS). Single-stranded RNA transcripts induced interferon-β mediated though RIG-I and PKR activation, the latter associated with rapid induction of antiviral stress granules. A striking inverse correlation was observed between induction of both cellular responses with
Once transcribed, the nascent full-length RNA of HIV-1 must travel to the appropriate host cell sites to be translated or to find a partner RNA for copackaging to form newly generated viruses. In this report, we sought to delineate the location where HIV-1 RNA initiates dimerization and the influence of the RNA transport pathway used by the virus on downstream events essential to viral replication. Using a cell-fusion-dependent recombination assay, we demonstrate that the two RNAs destined for copackaging into the same virion select each other mostly within the cytoplasm. Moreover, by manipulating the RNA export element in the viral genome, we show that the export pathway taken is important for the ability of RNA molecules derived from two viruses to interact and be copackaged. These results further illustrate that at the point of dimerization the two main cellular export pathways are partially distinct. Lastly, by providing Gag in trans, we have demonstrated that Gag is able to package RNA from either
This observation argues that RNPs are not randomly incorporated into virions, and is consistent with the presence of specific signals in each RNA segment that enable the RNPs to be packaged as a complete set. The mechanisms by which these signals are recognized, and how they ensure incorporation of one copy of each RNA segment into the particle, are not known.. There is clear evidence for a selective mechanism during the packaging of the bacteriophage ψ6 genome. Viral particles contain one copy each of a S, M, and L dsRNA segment. All particles contain a complete complement of genome segments, as indicated by the fact that every virus particle is infectious. Only the S RNA segment can enter newly formed particles; once that segment is packaged, then the M RNA can enter. The L RNA can only enter particles that contain both the S and M segments. Precise packaging is therefore the result of a serial dependence of packaging of the RNA segments.. Muramoto, Y., Takada, A., Fujii, K., Noda, T., ...
Viral RNA-dependent RNA Polymerase Assay. The Viral RNA-dependent RNA Polymerase Assay is developed using a RNA polymerase in the Flaviviridae,. a family of positive, single-stranded, enveloped RNA viruses. The assay is based on measurement of the. RNA molecules synthesized by the RNA polymerase using RNA as a template in the presence of NTPs.. The assay can be performed in a 384-well or 96-well plate format for tests of theenzyme activities of RNA. polymerases in the Flaviviridae family and high throughput screening of inhibitors. ...
Protein and RNA synthesis are inhibited when VSV infects certain cells. UV-inactivation analysis of the virus indicates that transcription of two regions of the viral genome are required for efficient inhibition. The larger of the two viral products represents transcription of approximately 1500 nucleotides and may represent the N protein gene, while the smaller product is approximately 40 nucleotides long. The latter product is thought to be encoded at the 3-proximal end of the genome.^ Two viral mutants have been shown to be deficient in the expression of the smaller transcription product and result in less efficient inhibition of both protein and RNA synthesis. Analysis of these mutants and the UV-inactivated wild-type virus have allowed for the establishment of conditions where the effects of either the large or the small transcription product can be observed independent of the other. This will allow for the correlation of a viral product with inhibition, and thereby establish the causative
My group uses X-ray crystallography as a central technique to study the structure-function relationships of complexes involving RNA of various kinds in eukaryotic cells. This includes the transcription/replication machinery of segmented negative strand RNA viruses (e.g. influenza), complexes involved in sorting of Pol II transcripts into their appropriate processing pathways and innate immune system pattern recognition receptors, notably the response to viral RNA via Rig-I like helicases.. Keywords: Protein-RNA recognition / aminoacyl tRNA synthetases / RNA metabolism / virus structure / influenza virus polymerase / innate immunity / Rig-I like helicases / X-ray crystallography. Subject area(s): Microbiology, Virology & Pathogens , RNA , Structural Biology & Biophysics. ...
role of rna-protein interactions in the internal initiation of translation of plus-strand rna viruses : a novel target for antiviral therapeutics
Although changing therapies is a common strategy in the treatment of HIV disease, guidelines are needed to help clinicians and patients decide when a change in antiretroviral therapy is indicated. The technology of measuring HIV RNA in plasma has been suggested as a tool for monitoring clinical drug efficacy. However, uncertainty remains about whether aggressive antiretroviral treatment to lower HIV RNA and maintain low levels for as long as possible will confer clinical benefit in comparison with management based on monitoring CD4 counts and HIV-related symptoms.. Patients are randomized to a decision making strategy for initiating or changing therapy based on current clinical practice alone vs. decision making based on plasma HIV RNA quantitation in addition to current clinical practice in patients with ,= 300 CD4+ cells/mm3. All patients in the RNA arm as well as a subset (n = 183) of those in the CCP arm will have a plasma HIV RNA quantitation drawn every 4 months. The results of these ...
The armored L-RNA (2,248 bp) expressed by our two-plasmid coexpression system differs in several respects from the virus-like particles previously described by Pickett and Peabody (17). These authors also used a two-plasmid expression system; their goal was to determine whether the 21-nt Operator (pac site) would confer MS2-specific packageability on nonbacteriophage RNA in vivo. The E. coli was induced such that the Operator-lacZ hybrid RNA was coexpressed with the MS2 coat protein. The specificity of the Pickett and Peabody bacteriophage packaging system, however, was poor since the host E. coli RNA was packaged in preference to the Operator-lacZ RNA. In other studies, Pickett and Peabody modified the packaging of the Operator-lacZ RNA by changing the ratios of coat protein to Operator-lacZ RNA produced in E. coli. By increasing the concentration of the Operator-lacZ RNA and decreasing the concentration of the coat protein, these researchers were able to encapsulate mainly the Operator-lacZ ...
The 3 ends of the S and M messenger RNAs isolated from BHK21 cells infected with Germiston virus were analyzed by mapping with RNase T2 or nuclease S1. The transcription termination signal was found to be located approximately 115 and 80 nucleotides upstream from the 3 end of the S and M genomic RNA templates, respectively. Both mRNAs were found to possess several adenosine residues at their 3 ends, but were not polyadenylated. They have acquired at their 5 end a heterologous 12- to 18-nucleotide-long sequence, which is not coded for by the virus. Sequencing of the 5 terminal region from single molecules cloned into pBR327 revealed that these primers are rich in C and G residues and possess a U or a C adjacent to the viral sequence.
The bunyavirus genome is monomeric and consists of three segments of linear negative-sense (or ambisense, depending on the genus) RNA. The terminal sequences of each segment are base-paired. Because of this, the RNAs form non-covalently closed circles. The nucleotide sequences at the 3-terminus and the 5-terminus are complimentary, forming panhandle structures. The 5-terminus is not capped. The complete genome is 10500-22700 nucleotides long. The three segments of the genome are labeled L, M, and S. The L segment is 6300-12000 nucleotides long and encodes the viral RNA polymerase. The M segment is 3500-6000 nucleotides long and encodes two glycoproteins as a single gene product that is usually co-translationally cleaved. The S segments is 1000-2200 nucleotides long and encodes the coat protein. (sources: Descriptions of Plant Viruses, ICTVdB) ...
Genome RNA replication of all (+)RNA viruses takes place in close association with rearranged intracellular membranes. We are only beginning to understand the biogenesis and ultrastructure of these virus-induced membrane structures. In collaboration with the virology groups of LUMC (Prof. Dr. Eric Snijder) and the University of Utrecht (Prof. Dr. Frank van Kuppeveld), EM and tomography approaches will be used to gain more insight into the architecture of the rearranged membranes, the localization of the viral replication enzymes, and the localization of host factors that are hijacked by picornavirus to facilitate replication of their RNA genome.. Host institute ...
The multiscale model of hepatitis C virus (HCV) dynamics, which includes intracellular viral RNA (vRNA) replication, has been formulated in recent years in order to provide a new conceptual framework for understanding the mechanism of action of a variety of agents for the treatment of HCV. We present a robust and efficient numerical method that belongs to the family of adaptive stepsize methods and is implicit, a Rosenbrock type method that is highly suited to solve this problem. We provide a Graphical User Interface that applies this method and is useful for simulating viral dynamics during treatment with anti-HCV agents that act against HCV on the molecular level.
Recent studies have shown that replication of hepatitis C virus (HCV) is dependent on miR-122 expression.[20] miR-122 regulates HCV by binding directly to two adjacent sites close to the 5 end of HCV RNA.[21] Although these experiments were conducted using genotype 1a and 1b HCV RNA, the miR-122 binding sites are highly conserved across different genotypes, and miR-122 is also required for replication of infectious type 2a HCV.[22] As miRNAs generally function to repress gene expression by binding to 3UTR sites, this positive regulation of viral replication via a 5UTR represents a novel function for miR-122. The mechanism of regulation is not yet clear. miR-122 stimulates translation of HCV RNA, but not to a sufficient extent to explain its effects on viral replication, indicating that a second stage of the viral replication cycle must also be regulated.[23][24] HCV RNA synthesis is not affected by miR-122, suggesting that regulation of other processes such as RNA stability may occur.[25][26] ...
Hiv rna pcr test - What is the window period for p42 antigen/ antibody dual 4th gen HIV test? And the window period for HIV RNA PCR test? When can it be conclusive? See below. Please consult this site for a detailed answer. Nucleic acid tests are conclusive about a month after acquiring infection. Http://www. Sfaf. Org/hiv-info/testing/hiv-test-window-periods. Html
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Plays an essential role in viral RNA transcription and replication by forming the heterotrimeric polymerase complex together with PB1 and PB2 subunits. The complex transcribes viral mRNAs by using a unique mechanism called cap-snatching. It consists in the hijacking and cleavage of host capped pre-mRNAs. These short capped RNAs are then used as primers for viral mRNAs. The PB2 subunit is responsible for the binding of the 5 cap of cellular pre-mRNAs which are subsequently cleaved after 10-13 nucleotides by the PA subunit that carries the endonuclease activity.
Single-stranded RNA viruses have evolved to survive extremely high mutation rates.The ubiquity and effect of ssRNA viral diseases makes an understanding of the theoretical and mechanical underpinnings of rapid viral evolution vital to our ability to control them. In this body of work, we explore some of the ways in which ssRNA viruses can uncouple the rate at which variation is generated (mutation rate) from the rate at which variation is observed (measured rate of molecular evolution).. ...
Hepatitis A virus (HAV) is a RNA hepatovirus (in the picornavirus family) - It is a naked capsid (unenveloped), linear single-stranded, positive-sense RNA virus with a cubic (icosahedral) symmetry -it replicates in the cytoplasm of the intestinal mucosa by using viral RNA polymerase. -The virus is transmitted by the fecal-oral route -More than 90% of…
Description: An enzyme that catalyses RNA-template-directed extension of the 3- end of an RNA strand by one nucleotide at a time, and can initiate a chain de novo. (Enzyme Nomenclature, 1992, p293 ...
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Nucleic acids are polymers of nucleotides, including DNA and RNA. DNA/RNA synthesis is an anabolic mechanism generally involving the chemical reaction of phosphate, pentose sugar, and a nitrogenous base. Certina enzymes are required to facilitate the event. Defects or deficiencies in these enzymes can lead to a variety of diseases, and a lot of chemicals that affect DNA/RNA synthesis are used in diseases relating to cell proliferation, such as cancers and microbial infections.
Ilkka Julkunen, Department of Virology, University of Turku and Virology Unit, National Institute for Health and Welfare (THL), Helsinki, Finland, will give a seminar on Mechanisms of activation of innate immune responses in viral RNA and virus infection stimulated human cells Host: Malin Flodström Tullberg