The Single-stranded Viral RNA

Actiially, the first demonstration that the infectivity of an animal virus resided exclusively in its ENA was provided by Colter al in 1957 (l6)s Experimental evidence was then produced that the MA extracted from mengovirus-infected Ehrlich ascites cells was infectious. This observation was soon extended to poliovirus RNA (17). Retrospectively considered, it was a very happy circumstance indeed that this pioneer work was carried out in the picornavirus system, for we now know that similar experiments performed with other RRA-containing animal viruses (e.g. influenza) would never have produced such a clearcut result. 

The infectivity of the viral s s RRA (be it from virions or extracted from infected cells) is abolished by nucleases, but is not affected by treatment of the host-cell with Actinomycin R, a-amanitin, or cordycepin (I8), (see below). 

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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... 

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.

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... 

Antibodies to mono-, di-, and trinucleotides of the usual nucleic acid bases react with denatured DNA but not with native DNA, in which the bases are not accessible. It has been difficult in many cases to measure their reactions with ribosomal RNA or single-stranded viral RNA, partly because of the extensive secondary and tertiary folding that may mask many of the bases and partly because of the difficulty in removing all ri-bonuclease from serum. Some reaction with RNA was measurable with an antiadenosine serum after careful efforts were made to remove ribonu-clease. Antibodies to anticodon sequences reacted with tRNA, as did antibodies to modified bases of tRNA and antibodies occurring spontaneously in sera of NZB/NZW mice or human patients with systemic lupus erythematosus. ... 

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. 

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. 

Flaviviruses are small, enveloped viruses, with cubic symmetry, approximately 45 nm in diameter, which replicate in vertebrate and invertebrate cells. The nucleocapsid contains the single-stranded plus RNA associated with a nucleocapsid protein, C, which has a molecular weight of 13K. The viral envelope consists of one large glycoprotein, E, of molecular weight approximately 55K, and a small 8K membrane-associated protein, M, which is not glycosylated (Wes-taway, 1980 Matthews, 1982). The structural components of flaviviruses have been reviewed by Russell et al. (1980) and the replication strategy of these viruses reviewed by Westaway (1980). 

While in physiological salt solutions up to 80% of single-stranded viral RNA is adsorbed to HeLa cells within 5-10 minutes at 37° C (Borriss and Koch, 1964a), the figure is only 20% for RF-RNA and RI-RNA (Wentzky and Koch, 1971 Wiegers and Koch, 1972). 

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. 

Hepatitis E is a non-enveloped single-stranded messenger RNA virus of unclassified genus.18 The HEV is similar to HAV in that the virus is harvested in contaminated feces, thus infecting people via the fecal-oral route. High HEV levels in the bile often prompt viral shedding in the feces. The severity of hepatic damage is dependent on the HEV strain Mex 14, Sar 55, or the US 2 strain.19 No cases of chronic hepatitis E have yet been documented. 

This enzyme is associated with the virions of RNA tumor viruses such as the Rous sarcoma virus (RSV). The enzyme has remarkable enzymatic activity in that it can catalyze several seemingly diverse steps in the synthesis of double-stranded DNA from the single-stranded RNA viral genome. The enzyme uses a tRNA for tryp-tophan as a primer to make a copy of DNA that is complementary to the viral RNA. The resulting RNA-DNA hybrid is converted to a double-stranded DNA molecule by ribon-uclease (RNase)H and DNA-dependent DNA polymerase activities that are intrinsic to reverse transcriptase. 

Some E. coli bacteriophages, including f2, MS2, R17, and Qj8, as well as some eukaryotic viruses (including influenza and Sindbis viruses, the latter associated with a form of encephalitis) have RNA genomes. The single-stranded RNA chromosomes of these viruses, which also function as mRNAs for the synthesis of viral proteins, are replicated in the host cell by an RNA-dependent RNA polymerase (RNA replicase). All RNA viruses—with the exception of retroviruses—must encode a protein with RNA-dependent RNA polymerase activity because the host cells do not possess this enzyme. 

Genes in all cellular organisms are made of DNA. The same is true for some viruses, but for others the genetic material is RNA. Viruses are genetic elements enclosed in protein coats that can move from one cell to another but are not capable of independent growth. One well-studied example of an RNA virus is the tobacco mosaic virus, which infects the leaves of tobacco plants. This virus consists of a single strand of RNA (6930 nucleotides) surrounded by a protein coat of 2130 identical subunits. An RNA-directed RNA polymerase catalyzes the replication of this viral RNA. 

Fig. 7.5. Some typical molecular weiglit-mobility (or sedimentation coefficient) relationstiips for electrophoresis of RNA in polyacrylamide gels, showing conformational effects a) set of single-stranded viral and ribosomal RNAs in 2.4% gel (Bishop et al. 1967). b) shows a typical separation of different species on a single gel, displayed as a densitometric trace c) native single-stranded RNAs ( ), formaldehyde treated RNAs (O). polyriboadenylic acid fractions with (A) and without (A) formaldehyde treatment, and polyribouridylic acid fractions ( ) treated with formaldehyde. These data show that the mobilities are little affected by single-stranded stacking, but substantially by partial base pairing, and that formaldehyde treatment is inadequate to reduce RNA to the conformation of a single-stranded unpaired polynucleotide (from Finder et al., 1974).

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