Difference Between Positive Sense And Negative Sense Rna

1CS Bond Life Sciences Center, University of Missouri, Columbia, MO 65201, USAude.iruossim@JiJ (J.J.); [email protected] (T.P.N.); [email protected] (R.R.)

2Department of Molecular Microbiology & Immunology, University of Missouri School of Medicine, Columbia, MO 65212, USAFind articles by

1CS Bond Life Sciences Center, University of Missouri, Columbia, MO 65201, USAude.iruossim@JiJ (J.J.); [email protected] (T.P.N.); [email protected] (R.R.)

2Department of Molecular Microbiology & Immunology, University of Missouri School of Medicine, Columbia, MO 65212, USA

3Center for AIDS Research, Laboratory of Biochemical Pharmacology, Department of Pediatrics, Emory University School of Medicine, Atlanta, GA 30322, USA; [email protected] (S.L.); ude.yrome@tissabl (L.C.B.); ude.yrome@anihcsr (R.F.S.)Find articles by

3Center for AIDS Research, Laboratory of Biochemical Pharmacology, Department of Pediatrics, Emory University School of Medicine, Atlanta, GA 30322, USA; [email protected] (S.L.); ude.yrome@tissabl (L.C.B.); ude.yrome@anihcsr (R.F.S.)Find articles by

1CS Bond Life Sciences Center, University of Missouri, Columbia, MO 65201, USAude.iruossim@JiJ (J.J.); [email protected] (T.P.N.); [email protected] (R.R.)

1CS Bond Life Sciences Center, University of Missouri, Columbia, MO 65201, USAude.iruossim@JiJ (J.J.); [email protected] (T.P.N.); [email protected] (R.R.)

2Department of Molecular Microbiology & Immunology, University of Missouri School of Medicine, Columbia, MO 65212, USAFind articles by

1CS Bond Life Sciences Center, University of Missouri, Columbia, MO 65201, USAude.iruossim@JiJ (J.J.); [email protected] (T.P.N.); [email protected] (R.R.)Find articles by

5Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, NY 10065, USA; ude.rellefekcor.liam@diliahcime (E.M.); ude.ub@1deeasm (M.S.); ude.rellefekcor@cecir (C.M.R.)Find articles by

5Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, NY 10065, USA; ude.rellefekcor.liam@diliahcime (E.M.); ude.ub@1deeasm (M.S.); ude.rellefekcor@cecir (C.M.R.)Find articles by

1CS Bond Life Sciences Center, University of Missouri, Columbia, MO 65201, USAude.iruossim@JiJ (J.J.); [email protected] (T.P.N.); [email protected] (R.R.)

2Department of Molecular Microbiology & Immunology, University of Missouri School of Medicine, Columbia, MO 65212, USAFind articles by

3Center for AIDS Research, Laboratory of Biochemical Pharmacology, Department of Pediatrics, Emory University School of Medicine, Atlanta, GA 30322, USA; [email protected] (S.L.); ude.yrome@tissabl (L.C.B.); ude.yrome@anihcsr (R.F.S.)Find articles by

3Center for AIDS Research, Laboratory of Biochemical Pharmacology, Department of Pediatrics, Emory University School of Medicine, Atlanta, GA 30322, USA; [email protected] (S.L.); ude.yrome@tissabl (L.C.B.); ude.yrome@anihcsr (R.F.S.)Find articles by

1CS Bond Life Sciences Center, University of Missouri, Columbia, MO 65201, USAude.iruossim@JiJ (J.J.); [email protected] (T.P.N.); [email protected] (R.R.)

2Department of Molecular Microbiology & Immunology, University of Missouri School of Medicine, Columbia, MO 65212, USAFind articles by

5Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, NY 10065, USA; ude.rellefekcor.liam@diliahcime (E.M.); ude.ub@1deeasm (M.S.); ude.rellefekcor@cecir (C.M.R.)Find articles by

1CS Bond Life Sciences Center, University of Missouri, Columbia, MO 65201, USAude.iruossim@JiJ (J.J.); [email protected] (T.P.N.); [email protected] (R.R.)

2Department of Molecular Microbiology & Immunology, University of Missouri School of Medicine, Columbia, MO 65212, USA

3Center for AIDS Research, Laboratory of Biochemical Pharmacology, Department of Pediatrics, Emory University School of Medicine, Atlanta, GA 30322, USA; [email protected] (S.L.); ude.yrome@tissabl (L.C.B.); ude.yrome@anihcsr (R.F.S.)

RNA viruses are highly successful pathogens and are the causative agents for many important diseases. To fully understand the replication of these viruses it is necessary to address the roles of both positive-strand RNA ((+)RNA) and negative-strand RNA ((−)RNA), and their interplay with viral and host proteins. Here we used branched DNA (bDNA) fluorescence in situ hybridization (FISH) to stain both the abundant (+)RNA and the far less abundant (−)RNA in both hepatitis C virus (HCV)- and Zika virus-infected cells, and combined these analyses with visualization of viral proteins through confocal imaging. We were able to phenotypically examine HCV-infected cells in the presence of uninfected cells and revealed the effect of direct-acting antivirals on HCV (+)RNA, (−)RNA, and protein, within hours of commencing treatment. Herein, we demonstrate that bDNA FISH is a powerful tool for the study of RNA viruses that can provide insights into drug efficacy and mechanism of action.

Positive-strand RNA, (+)RNA, viruses include many human pathogens, such as the families picornaviridae (e.g., poliovirus [1]), togaviridae (e.g., rubella virus [2]), and flaviviridae (e.g., Dengue virus [3], Zika virus [4], and hepatitis C virus [5]). While these viruses pursue a wide range of replication strategies in diverse hosts, they also share key features and are defined by their use of a (+)RNA genome. This genomic RNA fulfils three distinct functions: (1) it is the mRNA from which proteins are produced; (2) it is the template from which the negative-strand RNA ((−)RNA) is transcribed, to serve as the replicative intermediate; and (3) it codes the genetic information that must be packaged into assembling particles and transferred to target cells. At any given time, an infected cell will contain many (+)RNA molecules performing these roles, although any single (+)RNA molecule may only function in one capacity at one time. As the (+)RNA molecules are identical to one another, they cannot be differentiated by sequence and their separate roles can only be determined through simultaneous analysis of the interacting cofactors.

Following infection of a target cell, the viral RNA first serves as mRNA, exploiting the host-cell translational machinery to direct synthesis of the viral proteins; these proteins include enzymes that are responsible for synthesis of first the negative, then the positive strand of the genome, and proteins that modify the environment of the cell to support viral replication. Positive-strand RNA viruses replicate their genomes in the cytoplasm of infected cells, in association with virus-induced membrane structures, often termed the “membranous web” [6]. These membranes provide a foundation on which to anchor the viral replication complex (RC), and in combination with viral proteins, may provide protection against surveillance by the innate immune system. In the RC, the virus synthesizes new (+)RNA. At early times post-infection, the new (+)RNA will be used to generate more viral proteins and (−)RNA; at later time points, (+)RNA is packaged into particles made up of the viral structural proteins, and released from the cell. To better understand the replication of these viruses, their interactions with the host cell, and ultimately, how to combat them, it is necessary to consider both (+) and (−)RNA, and their interaction with proteins, viral and cellular.

While there are thousands of antibodies available to specifically identify viral and cellular proteins, and sufficient fluorescent tags to allow co-visualization of multiple proteins in a single sample, these imaging approaches are often incompatible with conventional methods for visualization of nucleic acids (e.g., hybridization of fluorescently labeled oligonucleotide probes). Branched DNA (bDNA) in situ hybridization is a technique that exploits sequence specific probes, and branching preamplifier and amplifier DNAs, to produce an intense localized signal [7]. Unlike conventional FISH methods, bDNA FISH is readily compatible with immunofluorescence, allowing simultaneous analysis of nucleic acids and proteins ( ). Various bDNA approaches have been developed for commercial use, including RNAscope [8], PrimeFlow [9], and ViewRNA [10]. These techniques have been applied to quantify and localize specific nucleic acids and the cells that harbor them. The compatibility of PrimeFlow with flow cytometry has proven particularly useful for analysis of human immunodeficiency virus (HIV)-1 latency and reservoirs [11,12,13], while RNAscope and ViewRNA have been employed diagnostically for histological staining [14,15,16,17,18,19], and in cell biology to visualize cellular and viral RNAs [20,21,22]. These techniques have frequently been employed in low resolution imaging approaches, such as histology and flow cytometry, that exploit the robust signal to clearly identify rare infected cells. A variation on the robust detection of an abundant RNA in rare cells is the detection of less abundant targets; these FISH methods have sufficient sensitivity to identify individual nucleic acid molecules, sometimes referred to as single molecule FISH. One such application of this sensitivity has been to modify the experimental conditions to achieve labelling of the viral nucleic acids in HIV-infected cells [22,23,24,25]. Most infected cells in the clinical context only contain a small number of integrated proviruses, typically just one [26]; thus, the signal amplification of bDNA FISH renders it ideally suited to the visualization of such a low abundance target.

Similar to the integrated provirus of retroviruses, the (−)RNA of (+)RNA viruses is present in low amounts relative to the (+)RNA [27,28,29,30]. It is, nevertheless, essential for the synthesis of (+)RNA and, as it lacks the multiple functions of (+)RNA, it is a more reliable marker of the RC. Here we use hepatitis C virus (HCV)-infected human hepatoma cells and Zika virus (ZIKV)-infected Vero cells to demonstrate the specific labelling of (+)RNA and (−)RNA, allowing analysis of viral activity at an individual cell level. To validate the approach, we applied bDNA FISH to a phenomenon we had previously examined [31], that of the rapid response of HCV-infected cells to a variety of anti-HCV direct-acting antiviral agents (DAAs). Using this approach we visualized the rapid decline in HCV RNA associated with the use of NS5A inhibitors [31,32,33]. This new assay represents a novel approach to evaluate other RNA viruses in future studies.

Huh-7.5.1 cells are derived from the human hepatoma 7 cell line, and have been previously described [34]. Cells were propagated in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS). Vero-E6 cells were obtained from ATCC and cultured in DMEM supplemented with 10% FBS. Vero cells are derived from the kidney of an African green monkey and lack the genes coding type I interferon, making them suitable for the growth of many viruses [35,36,37]. Jc1-FLAG2(p7-nsGluc2A), hereafter Jc1/Gluc2A, was described previously [38]. Zika virus (isolate MR766) [39,40] was obtained from Alexander Franz (University of Missouri), and propagated and titered in Vero cells.

Daclatasvir (DCV, BMS-790052) and Danoprevir (DNV, RG7227) were purchased from Selleckchem. Ledipasvir (LDV, GS-5885) was purchased from MedChem Express. Sofosbuvir (SOF, GS-7977) was purchased from Acme Bioscience. Mouse monoclonal primary antibody 9E10 specific for NS5A was used as previously described [41]. Mouse monoclonal antibodies specific for HCV core (C7-50) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; G-9) were purchased from Abcam and Santa Cruz Biotechnology, respectively. Alexa Fluor 647 conjugated secondary antibody and Hoechst 33258 for nuclear staining were purchased from Invitrogen.

The initial reverse transcription step of the HCV 5′ UTR was carried out as previously described [42]. Briefly, total RNA was extracted using an RNeasy kit (Qiagen, Hilden, Germany), and quantified by absorbance at 260 nm. A quantity of 50 ng of RNA was denatured at 70 °C for 8 min with dNTPs and either the RC21 primer 5′-CTCCCGGGGCACTCGCAAGC-3′ (for the positive strand) or the tag-RC1 primer 5′-ggccgtcatggtggcgaataaGCCTAGCCATGGCGTTAGTA-3′ (for the negative strand), followed by incubation at 4 °C for 5 min. ThermoscriptTM reverse transcriptase (Invitrogen) was added to the denatured RNA template and incubated at 60 °C for 1 h, followed by RNase H treatment for 20 min at 37 °C. Reverse transcribed cDNA was mixed with RC1 (5′-GCCTAGCCATGGCGTTAGTA-3′) and RC21 primers for positive strand amplification and tag (5′-ggccgtcatggtggcgaataa-3′) and RC21 primers for negative strand amplification. Amplification was conducted by denaturation at 95 °C for 10 min, followed by 40 cycles of denaturation at 95 °C for 15 s and annealing/extension at 60 °C for 1 min using PerfeCTa SYBR Green FastMix (Quanta Biosciences, Beverly, MA, USA). Amplification was carried out in an Applied Biosystems®® 7500 Fast Real-Time PCR Instrument (ABI). In vitro transcribed RNA from the HCV infectious clone was used to generate a standard curve.

ZIKV RT-qPCR was performed using the same general approach, with the following differences. RC21 and RC1 were replaced by Tag-ZK21 primer (5′-ggccgtcatggtggcgaataaCCTGACAACACTAAaATTGGTGC-3′) and Tag-ZK1 primer (5′-ggccgtcatggtggcgaataaAGGATCATAGGTGATGAAGAAAAGT-3′). cDNA synthesis was performed using SuperScript III First-Strand Synthesis System (Invitrogen), following the manufacturers’ instructions. qPCR amplification was conducted using PowerUp SYBR Green Master Mix (Applied Biosystems, Foster City, CA, USA), in a PikoReal 96 Real-Time PCR system (ThermoFisher Scientific). The cycle conditions were uracil-DNA glycosylase (UDG) activation at 50 °C for 2 min, dual-lock DNA polymerase at 95 °C for 2 min, followed by 40 cycles of denaturation at 95 °C for 15 sec, annealing at 55 °C for 15 sec, and extension at 72 °C for 1 min. An MR766 infectious clone [43] was used to generate a standard curve, and was subject to the same strand specific RT-qPCR protocol.

The key difference between the negative and positive sense RNA virus is that the negative sense RNA virus comprises viral RNA, which is complementary to the viral mRNA, while positive sense RNA virus comprises viral mRNA, which can be translated into proteins directly.

Main Difference – Positive vs Negative Sense RNA Virus

Viruses are the smallest form of obligate parasites that require a host cell for their replication. They consist of a DNA or RNA genome covered by a protein capsid. The viruses that consist of a genome made up of DNA are called DNA viruses while viruses made up of RNA are called RNA viruses. Positive and negative sense RNA viruses are the two types of single-stranded RNA viruses classified based on the type of genome. Positive sense RNA is also known as plus-strand or sense strand while negative sense RNA is also known as minus-strand or antisense strand. The main difference between positive and negative sense RNA virus is that positive sense RNA virus consists of viral mRNA that can be directly translated into proteins whereas negative sense RNA virus consists of viral RNA that is complementary to the viral mRNA.

Key Terms: mRNA, Negative Sense, Positive Sense, RNA-Dependent RNA Polymerase (RdRp), Single-Stranded RNA Virus, Transcription, Translation

What is a Positive Sense RNA virus?

A positive sense RNA virus is a type of virus that contains a positive sense single-stranded RNA as its genetic material. These viruses have the ability to function as messenger RNA and have the potential to be translated directly into protein inside the host. According to the Baltimore classification system, positive sense single-stranded RNA virus belongs to the group IV. These RNA viruses are responsible for a larger fraction of RNA viruses including Hepatitis C virus, West Nile virus and dengue virus and the viruses responsible for SARS and MERS. They also include in the category that causes mild disease conditions such as common cold.

Since positive sense RNA virus genomes have the ability to act as messenger RNA, their genomes directly translated into proteins by host ribosomes. Once viral proteins are produced inside the host, they recruit the RNA to produce viral replication complexes. Viral replication continues through double-stranded RNA intermediates.

Difference Between Positive Sense And Negative Sense Rna

The involvement of double-stranded RNA provides the opportunity for the virus to invade immune responses. All these virus genomes encode the synthesis of a type of RNA protein known as RNA dependent polymerase. In these phenomena, RNA is synthesized from an RNA template. There are few types of host cell proteins that are recruited by these positive sense single-stranded RNA virus. These include RNA binding proteins, membrane remodelling proteins, chaperone proteins. All these proteins involve in the exploitation of host cells secretary pathways needed for viral replication.

4. Branched DNA In Situ Hybridization (bDNA FISH) for Strand-Specific Nucleic Acid Visualization

bDNA FISH for cultured adherent cells was used for HCV RNA detection using the RNAscope method, with some modifications [8]. Cells were fixed in 4% paraformaldehyde for 30 min at room temperature (RT), washed three time in phosphate buffered saline (PBS), then incubated in PBS supplemented with 0.1% Tween-20 (PBS-T) for 10 min at RT, followed by two wash steps with PBS. Coverslips were immobilized on Superfrost glass slides using a small drop of nail polish. A circle was drawn around the coverslip using an ImmEdge hydrophobic barrier pen (Vector Laboratories). Protease treatment (Protease 3) was diluted 1:15 in PBS and incubated on the sample in a humidified HybEZ oven at 40 °C for 15 min and washed twice in PBS. Specific V-HCV-GT2a probe for (+)RNA (Catalogue number, 441361; Advanced Cell Diagnostics, Newark, CA, USA) was added to the coverslip and incubated in humidified HybEZ oven at 40 °C for 2 h, followed by HCV-GT2a-sense-C2 probe for (−)RNA (Catalogue number, 441371) diluted 1:50 in probe dilution buffer for an additional 2 h. Probes were used sequentially rather than simultaneously because they target the same region of the viral genome ( ) and would likely anneal to one another if applied together. Two consecutive wash steps were performed in 1× wash buffer (Catalog number, 310091; Advanced Cell Diagnostics) with agitation at RT for 2 min in every wash step after this point, and all incubations were performed in a humidified HybEZ oven at 40 °C. bDNA amplification was performed using a series of amplifiers (RNAscope; Advanced Cell Diagnostics). Amplifier hybridization 1-Fluorescent (Amp 1-FL) was added to the coverslip for 30 min, followed by Amp 2-FL hybridization for 15 min. Amp 3-FL hybridization was then added for 30 min, followed by Amp 4-FL hybridization for 15 min. If samples were to be stained by immunofluorescence, this was performed after the RNAscope staining. Anti-NS5A antibody and anti-HCV core antibody were diluted 1:1000 in PBS-T, and incubated on coverslips for 1 h at RT. Secondary anti-mouse Alexa Fluor 647 was diluted 1:2000 in PBS-T and incubated on coverslips for 1 h at RT. Nuclei were stained with DAPI for 1 min or Hoechst 33258 at 0.5 µg/mL for 10 min at room temperature in PBS-T. Coverslips were washed 3 times in PBS-T after each incubation. Finally, coverslips were detached and mounted on fresh slides using ProLong Gold Antifade reagent (Thermo Fisher Scientific, Waltham, MA, USA). Most s were obtained using a Leica TCP SP8 MP confocal fluorescence equipped with a 63× HC PL APO CS2 oil-immersion objective (numerical aperture 1.4), and a tunable supercontinuum white light laser. The excitation/emission bandpass wavelengths used to detect DAPI, Alexa 488, ATTO 550, and Alexa 647 were set to 405/420–480, 488/505–550, 550/560–610, 568/580–630, and 647/655–705 nm, respectively. Pixel size under these conditions was 0.18 μm. Within any given dataset, conditions such as the laser intensity, exposure time and pinhole were kept constant, to allow comparison of the s. s of the HCV-infected cells for were captured using a Nikon C2 confocal microscope, with a 60× APO oil-immersion objective (numerical aperture 1.4). Excitation lasers were 405 nm, 488 nm, and 561 nm.

Nucleic Acid Target Probe Name Catalog# Target Region of Genome (Coded Protein)
HCV (+)RNA V-HCV-GT2a 441361 4268–5505 (NS3-NS4B)
HCV (−)RNA HCV-GT2a-sense-C2 441371 4268–5505 (NS3-NS4B)
ZIKV (+)RNA V-ZIKA-pp-O2 464531 866–1763 (M-E)
ZIKV (−)RNA V-ZIKA-pp-O2-sense-C2 478731-C2 866–1763 (M-E)

ZIKV-infected cells were stained by the same protocol as HCV, with the following changes: the protease pre-treatment used a 1:2 dilution, rather than 1:15; and the ZIKV-specific probes were V-ZIKA-pp-O2 for the (+)RNA (Catalogue number, 464531) and V-ZIKA-pp-O2-sense-C2 for the (−)RNA (Catalogue number, 478731-C2).

In order to quantify the differential drug effects on (+) and (−) strands of HCV RNA, we manually acquired 30 s of each biological replicate drug treatment experiment and performed cellular analysis and analysis using BioTek Gen5 software. Abundance of (+) and (−)RNA was quantified and plotted in two ways: number of fluorescent foci per infected cell, and fluorescence intensity per infected cell. ZIKV staining was performed similarly, with an additional calculation of the area of (−)RNA foci.

Graphs were plotted using Microsoft Excel. For significance testing, GraphPad Prism 6 was used to perform one-way ANOVA with Dunnett’s post-test for multiple comparisons, comparing each test condition to a control sample, or Tukey’s post-test for multiple comparisons comparing all conditions to one another.

FAQ

What is positive sense and negative sense DNA?

The main difference between positive and negative sense RNA virus is that positive sense RNA virus consists of viral mRNA that can be directly translated into proteins whereas negative sense RNA virus consists of viral RNA that is complementary to the viral mRNA.

Is human RNA positive or negative sense?

Depending on the context, sense may have slightly different meanings. For example, negative-sense strand of DNA is equivalent to the template strand, whereas the positive-sense strand is the non-template strand whose nucleotide sequence is equivalent to the sequence of the mRNA transcript.

What is the meaning of positive-sense RNA?

The genome is between 7,212 (human rhinovirus B; type 14) and 8,450 (FMD virus) nucleotides in length (Table 14.2). The RNA has a positive sense. Accordingly, viral proteins can be directly translated from the RNA without requiring an intermediate transcription step.

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