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Inhibiting the recognition
of the cis-acting sequence

Inhibition of the recognition of the cis-acting sequence clearly involves two questions:

1. What to target?
2. How to target it?

Attack the cis-acting sequence itself

Evidence that altering the cis-acting sequence inhibits virus growth

• The cis-acting sequence is required for initiating viral RNA synthesis, and mutations in the cis-acting sequence are deleterious for virus growth. The following examples are by no means comprehensive:
  • Togaviruses: Conserved 5' and 3' terminus of the Sindbis virus genome [Kuhn et al. 1990; Niesters and Strauss, 1990; Kuhn, et al., 1992; Hill et al., 1997]
    
  • Rhabdoviruses: Conserved promoter of vesicular stomatitis virus [Barr, et al., 1997a; Barr, et al., 1997b; Schnell, et al., 1996; Stillman and Whitt, 1997], and the terminal sequences [Wertz, et al., 1994; Pattnaik, et al., 1995]
    
  • Bunyaviruses: Conserved 3' terminus of the S segment of Rift Valley fever virus [Prehaud, et al., 1997]
    
  • Reoviruses: Conserved terminal sequences of the rotavirus genome [Chen et al., 1994; Gorziglia and Collins, 1992; Wentz, et al., 1996].

• In fact, even a small inhibition of the activity of the cis-acting sequence can result in dramatic inhibition of virus growth. This is in part because a small decrease in efficiency is amplified during the multiple rounds of viral RNA synthesis in infected cells.
  • Togaviruses: Most if not all possible mutations in a 5 nt region (positions -13 to -9) of the Sindbis virus promoter result in defective growth of the virus, even though some are only 2-fold less active [Hertz and Huang, 1995 Hertz and Huang, 1995]
    
  • Orthomyxoviruses: Similarly, mutations in the 3' 15 nt of the influenza A viral RNA promoter have a spectrum of effects on its template activity in vitro, from minimal effect to as much as >10-fold inhibition. In comparison, the same mutations dramatically inhibited promoter activity in vivo [Piccone, et al., 1993].
    

Potential methods: sequester and / or destroy the cis-acting sequence

using:
• RNAses or ribozymes specific for the cis-acting sequence
• Conventional nucleic acids (DNA or RNA) as antisense inhibitors
• A combination of RNAse and antisense, e.g., 2-5A-antisense chimeras [Player et al., 1998]
• Nucleic acid analogs as antisense inhibtors (e.g., peptide nucleic acids [Hanvey, et al., 1992])

Challenges

• How to deliver the inhibitor to the interior of the infected cell?

  Nucleic acids tend to be unstable, and inefficient at crossing cell membranes.

  • Use nucleic acid analogs designed to be nuclease-resistant, and better at crossing cell membranes?
  • Make transgenic animals / vectors that express the ribozyme/antisense inhibitor, to render them virus-resistant?

Attack the cognate proteins

Evidence that inhibiting the cognate protein inhibits virus growth

• Excess cis-acting sequence competitors inhibit virus growth

  Successive passaging of animal viruses at high multiplicities of infection frequently results in the preferential accumulation of defective interfering (DI) genomes [Perrault, 1981]. The DI genomes typically have extensive genetic rearrangements and lack some or most of the sequences of the normal virus [O'Hara, et al., 1984; Monroe and Schlesinger, 1984]. Indeed, in most cases the DI genomes are neither transcribed nor translated. They are consequently defective, unable to complete the lifecycle alone. However, they do retain the cis-acting sequences, that are recognized for replicating and packaging the DI genomes. If a source of the requisite replicase and structural proteins is available, e.g., provided in trans by coinfecting, normal helper virus, the DI genomes will be replicated and packaged into infectious particles. Continued passaging at high multiplicities of infection selects for DI genomes that are replicated and packaged efficiently, resulting in the enrichment of the DI genomes at the expense of the helper virus (e.g., Holland, 1980; Horodyski, et al., 1983; Schlesinger and Weiss, 1986].

  DI genomes may therefore be viewed as collections of cis-acting sequences, that act as decoys or competitive inhibitors to inhibit virus growth. They demonstrate that the viral proteins responsible for replication, transcription and packaging can be overwhelmed by competing cis-acting sequences, resulting in the inhibition of normal virus growth. Furthermore, this inhibition is not peculiar to replicating DI molecules, nor is it dependent on the presence of multiple cis-acting sequences (the replication and packaging signals) on a single DI molecule. For example:

  • over-expression of a single cis-acting sequence (TAR) resulted in growth inhibition of the human immunodeficiency virus [Sullenger, et al., 1991]
  • Sense and antisense RNAs corresponding to the 3' terminus, as well as antisense RNA to the 5' noncoding region of the foot-and-mouth virus genome were able to inhibit virus growth in culture. The inhibition is probably by hybridizing to the target viral sequences [Gutierrez, et al., 1994]. However, it is possible that the RNAs also act as 'decoys' or competitive inhibitors.
  • Sense RNAs, decoys, containing the 3' terminal regions of the turnip yellow mosaic virus were able to inhibit virus replication [Morch, et al., 1987].

Potential methods:

• Nucleic acid decoys (DNA or RNA).
• Synthetic competitive inhibitors (e.g., from screening compound libraries or combinatorial chemistry libraries?) that have better pharmacological properties: solubility, stability, ability to cross cell membranes efficiently.

Challenges

• Is the cognate protein(s) of viral or host origin?
  This is discussed in more detail on a separate page.

• Attack of the cognate viral protein is much prefered, to minimize effects on host processes.
  • How to identify which of the viral protein(s) is the cognate protein for each cis-acing sequence?
    Brute force screening might not be unreasonable, as there are no more than a few candidate viral proteins to examine
  .
• How to identify potential inhibitors?
  • Screening of libraries?
  • In vitro selection methods?

• Need inhibitors that interfere with the binding site for the cis-acting sequence.
  • Sites elsewhere on the cognate protein might be quite mutable in comparison.
  • Therefor will need assays for interference of binding as a screening tool.

• How to deliver the inhibitor to the interior of the infected cell.

Those that are not available through PubMed of the National Library of Medicine, USA.

• Holland, J. J., S. I. T. Kennedy, B. L. Semler, C. L. Jones, L. Roux, and E. A. Grabau. 1980. Defective interfering RNA viruses and the host-cell response, p. 137-192. In H. Fraenkel-Conrat and R. R. Wagner (ed.), Comprehensive Virology, vol. 16. Plenum Press, New York.
  
• Schlesinger, S., and B. G. Weiss. 1986. Defective RNAs of Alphaviruses, p. 149-166. In S. Schlesinger and M. J. Schlesinger (ed.), The Togaviridae and Flaviviridae. Plenum Press, New York.