RNA, double-stranded viral

Induced by Double-stranded Viral RNA Receptors identical... 

The feature common to the cytotoxic effects brought on by nonreplicating influenza virus, poxvirus, and defective-interfering vesicular stomatitis virus is the high multiplicity of infection required. This has led to the assumption that the toxic effect is caused by one or more components of the parental input virion, most likely protein in origin. However, Cordell-Stewart and Taylor (1971, 1973) have provided evidence that the double-stranded viral RNA isolated from cells infected with bovine enterovirus causes a rapid cytopathic effect as determined by trypan-blue uptake or Cr release from affected Ehrlich ascites tumor cells or L1210 cells toxic effects are reduced or do not occur in cells exposed to single-stranded or heat-denatured double-stranded viral RNA and the toxic effect of bovine enteroviral double-stranded RNA is not abolished by inhibitors of protein synthesis such as puromycin or cycloheximide. 

Cordell-Stewart, B., and Taylor, M. W., 1971, Effect of double-stranded viral RNA on mammalian cells in culture, Proc. Natl. Acad. Sci. USA 68 1326. 

Differences in the capacity of inhibition by polynucleotides not involved in complementary hydrogen bonds and by double-helical complexes of synthetic polyribonucleotides, or double-stranded viral RNA allow the conclusion that it is above all the regions of associated base pairs which are recognized in the RNA by anti-poly I poly C antibodies. Such complementary double-stranded helical regions have been described especially in tRNA but they have also been shown to exist in ribosomal RNA. These two kinds of RNA were therefore isolated and studied separately. Although both fractions precipitate anti-poly I poly C antibodies, their reactivity is nevertheless very different and rRNA precipitates eight times as much antibody as tRNA. Since tRNA possesses an important tertiary structure, this low reactivity could be explained by the non-accessibility of antigenic sites. 

Immune sera against poly A poly U, anti-poly I poly C and anti-poly G were used as controls. Anti-poly G does not precipitate any of the RNAs tested, whereas antibodies to the two complementary complexes precipitate all the RNAs, regardless of origin or fraction (Table 7 to 9). Thus antipoly G poly C antibodies show a specificity for ribosomal RNA from animal cells, and for certain viral RNAs furthermore they distinguish between rRNA and tRNA. In contrast, antibodies obtained by immunization with other polynucleotide complexes such as poly A poly U and poly I poly C show no specificity for any RNA whatever its type or origin, with the exception however of double-stranded viral RNA (Stollar, 1970). 

After the virus has attached to CD4 and chemokine receptors, another viral glycoprotein (gp41) assists with viral fusion to the cell and internalization of the viral contents. The viral contents include single-stranded RNA, an RNA-dependent DNA polymerase (also known as reverse transcriptase), and other enzymes. Using the single-stranded viral RNA as a template, reverse transcriptase synthesizes a complementary strand of DNA. The single-stranded viral RNA is removed from the newly formed DNA strand by ribonuclease H, and reverse transcriptase completes the synthesis of double-stranded DNA. The... 

FIGURE 10.10 RNAi sequence. First the vims approaches and attaches itself to the cell wall (A). The virus injects double-stranded RNA (B) into the cell cytoplasm. The dicer attacks the dsRNA, breaking it into smaller units (C and D). These smaller units are then acted on by RISC, forming single-stranded viral RNA (E), which are rendered incapable of forming their own protein by attachment to a complement contained within the cell s own single-stranded RNA (F). 

Like all other retroviruses, human immunodeficiency virus type 1 (HIV-1) contains the multifunctional enzyme reverse transcriptase (RT). Retroviral RTs have a DNA polymerase activity that can use either an RNA or a DNA template and an RNase H activity. HIV-1 RT is essential for the conversion of single-stranded viral RNA into a linear double-stranded DNA that is subsequently integrated into the host cell chromosomes [1-4]. In this conversion process HIV-1 RT catalyzes the incorporation of approximately... 

HIV is an RNA virus, meaning the viral genome is encoded as RNA instead of DNA. One key step in the virus cycle is to synthesize double-stranded viral DNA using viral RNA as a template. A viral enzyme called reverse transcriptase (RT) accomplishes this task. The reverse transcription process may be blocked by nucleoside reverse transcriptase inhibitors (NRTIs). NRTIs have already been discussed somewhat in Chapter 6. 

Antisense nucleotides production of an RNA sequence complementary to single-stranded viral RNA, which triggers the formation of double-stranded duplex, inhibiting viral RNA replication. 

Fig. 12.23. The life cycle of a retrovirus. The virus contains two identical RNA strands, only one of which is shown for clarity. After penetrating the plasma membrane, the single-stranded viral RNA genome is reverse-transcribed to a double-stranded DNA form. The viral DNA migrates to the nucleus and integrates into the chromosomal DNA, where it is transcribed to form a viral RNA transcript. The viral transcript can form the viral RNA genome for progeny viruses, or can be translated to generate viral structural proteins.

Double-stranded (ds) RNA-dependent protein kinase (PKR) was the first PRR shown to rect ise a product of viral replication, namely dsRNA. PKR is induced by IFN and in a critical host antiviral defence mechanism and as such it is both a PRR and an IFN eflFeaor protein. The discovery of PKR explained the observation that in cells pretreated with IFN, the translation ofviral mRNAs were blocked. - PKR is activated in response to viral, cellular or synthetic dsRNA of 30... 

One way of searching for the presence of inhibitors of polypeptide initiation in infected cells was to add cytoplasmic fractions from virus infected cells to a cell-free system from rabbit reticulocytes. This system initiates the synthesis of new polypeptide chains at a very high rate. Cytoplasm from poliovirus infected HeLa cells, but not from uninfected cells, inhibited protein synthesis in the reticulocyte lysate (59) The inhibitor was isolated and identified as double-stranded (ds) RNA (60). To study the effect of ds RNA on host and viral protein synthesis, a cell-free system from HeLa cells was developed which initiated translation on endogenous cellular or viral mRNA. When added to this system, the ds RNA was found to inhibit the translation of both cellular and viral mRNAs (61). Furthermore, measurement of the amount of ds RNA present in cells early in infection (61, 62) revealed that an insufficient quantity was present to act as a direct agent of protein synthesis inhibition. 

Koch, G. Bishop,J.M.(1968).The effect of polycations on the interaction of viral RNA with mammalian cells studies on the infectivity of single- and double-stranded poliovirus RNA, F/ra/ogy.35,9-17. 

One approach was to prepare extracts of poliovirus-infected HeLa cells and to assay for an inhibitor of initiation by addition to a reticulocyte lysate translation reaction. The reticulocyte lysate had been well-characterized as a system capable of efficient initiation in vitro. A potent inhibitor of initiation was indeed found in the cytoplasm of poliovirus-infected cells, which was not present in similar preparations from uninfected HeLa cells (Hunt and Ehrenfeld, 1971), and the inhibitor was subsequently identified as double-stranded poliovirus RNA (Ehrenfeld and Hunt, 1971). In order to qualify as a specific inhibitor of host cell protein synthesis, however, it was necessary to demonstrate that viral protein synthesis was immune to the inhibitory effects of the putative shut-off factor. When double-stranded RNA was put to this test, no specificity between cellular and viral protein synthesis could be demonstrated (Celma and Ehrenfeld, 1974) rather, all protein synthesis was inhibited equally. In addition, concentrations of viral double-stranded RNA required to... 

A number of methods are in use to titrate the infectivity of viral nucleic acids in mammalian virus-cell systems in vitro. The first method to be applied to tissue culture cells was based on treatment with hypertonic salt or sucrose solutions and a subsequent influx of RNA when the cells were returned to an isotonic environment (Alexander et al., 1958 Koch et al., i960). Later it was shown that the infectivity of poliovirus-specific RNAs, including double-stranded (RF-RNA) and multistranded (RI-RNA) molecules, can be determined with an agar cell suspension plaque assay, provided the cells are sensitized for RNA infection by polycation treatment (for review see Pagano, 1970). Several other methods have been used. These include exposure of cells... 

HLA virus (5 [xg/ml) and their sensitivity to infection by isolated RNA was analyzed. We found (see Table 5) the cells exposed to HLA virus for one minute at 37° C prior to addition of single- or double-stranded poliovirus RNA yielded 100 times more infective centers after 15 min incubation at 37° C than untreated cells (Breindl and Koch, 1972). This result reveals that the poliovirus coat proteins are able to sensitize HeLa cells to infection by viral RNA. 

The RNA isolated from E, coli after interaction with single-stranded viral RNA in the absence of inhibitors of protein synthesis shows a time-dependent increase in infectivity and a conversion of input label from single-stranded to double-stranded forms (RF-RNA and RI-RNA). Newly synthesized RNA hybridizes efficiently with melted poliovirus RF-RNA (Koch and Vollertsen, 1972b). The events following infection of E, coli by viral RNA are essentially the same as the ones described above following infection by RF-RNA. 

Interferons [alFN, piFN and ylFN]. Interferons are a family of glycosylated proteins and are cytokines which are produced a few hours after cells have been infected with a virus. Interferons protect cells from viral infections and have antiviral activities at very low concentrations ( 3 x 10 M, less than 50 molecules are apparently sufficient to protect a single cell). Double stranded RNA are very efficient inducers of IFNs. There are three main types of IFNs. The aIFNs are synthesised in lymphocytes and the piFNs are formed in infected fibroblasts. The a and P families are fairly similar consisting of ca 166 to 169 amino acids. Although ylFNs are also small glycosylated proteins (ca 146 amino acids), they are different because they are not synthesised after viral infections but are produced by lymphocytes when stimulated by mitogens (agents that induced cell division). 

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