Medical Pharmacology Chapter 36:  Antiviral Drugs

• Viral Genome and Virus Classification

  • The viral genome regulates expression of proteins enabling genome replication.¹ 

    • The characteristics of those pathways responsible for synthesis of early viral proteins are intimately associated with both chemical nature and strandedness of genomic DNA or RNA.

    • Viruses that utilize single-strand RNA can be subcategorized into two groups.

      • Some viruses contain the "sense" RNA strands containing coding structures that may be directly translated into viral proteins.

        • In this case the strand is described as the positive (or plus) strand.

        • Other viruses package the complementary base sequence or "anti-sense" strand.

          • This latter type describes the negative (or minus) strand. Initiation of replication cycles are different between these two groups.¹ 

      • Strandedness as well as other virus genome characteristics represents one of several bases of viral classification.² 

      • Other virus genomic properties in addition to strandedness include:  

        • Genomic size in kilobases (kb) or kilobase pairs (kpb)

        • Whether the genome is arranged linearly or circularly

        • Linear vs. circular (ring) DNA topologies³ 

        • Genomic segments (number and size)

        • Nucleotide sequence

        • G + C (guanine + cytosine) content

       

      • DNA A-T and G-C bonding

        Wikiality123:  "Image showing A-T and G-C bonding that occurs in DNA (12/2008) http://www.commons.wikimedia.org/wiki/File:AT-GC.jpg Blue arrows point to hydrogen bonding site GC content percentage4: ((G+C)/(A+T+G+C)) x 100

      • Presence of special aspects such as:

        • Repetitive elements

          • Tandem repeats are described by successive identical (or nearly identical) nucleotide (DNA) repeat units.⁷ 

            • A tandem repeat is defined as a pattern (e.g. in DNA) of one or more nucleotides repeated with repetitions adjacent.

              • For example:   ATTCG ATTCG ATTCG where the sequence ATTCG is found repeated three times.

          •  

            Short Tandem Repeats⁹

            Rosalind:  Learning bioinformatics, Glosssary--Short Tandem Repeats

          • However, there may be variation in both length of repeat units as well as length of the whole repeats making absolute classification challenging.

            • Large repeat units (largest) are described as "satellites."

            • The term satellite is derived from centrifugation of DNA in density gradients.

            • In this case major bands form; however, smaller bands which differ in CG/AT content are also described.

              • The smaller bands likely occur due to large "monotonous" repeats.

              • The term "minisatellites" described shorter tandem repeats 10 to 60 nucleotide repeats); whereas, "microsatellites" exhibit very short repeat units of the order of 1-5 bp.⁷

               

          • The Epstein-Barr virus (EBV) genome exhibits an unusually high content of repetitive sequences.⁵  

            • Taylor B "Simplified diagram of the structure of EVB. http://commons.wikimedia.org/wiki/File:Viral_Tegument.svg#mediaviewer/File:Viral_Tegument.svg

            • This virus is characterized by linear genomic DNA on the order of 170 kilobases (kb) with a high content of these repetitive sequences.

            • Tandem repeats are common among latent EBV genes, i.e. those genes expressed in the latent viral cycle.⁵

            The EBV genome has been used even as a model for repetitive sequences within eukaryotic cell chromosomes.

            • In the case of EBV repeat sequences have been described  at several internal as well as terminal regions.⁶

        • 5'-terminal cap

          • Directionality and molecular biology refers to the end-to-end chemical orientation of a single nucleic acid strand.

            • This is based on a chemical convention of naming nucleotide sugar-ring carbon atoms using numbers and giving rise to a 5' end (five prime end) and a 3' end (three prime end). The term upstream as it relates to genes and protein regulatory binding sites refers to "towards the 5'-end  or downstream i.e. towards the 3'-end. See figures below.

              • Directionality in Molecular Biology¹⁰

                A sugar-ring (furanose) molecule with carbon atoms labeled using the notation described above. The 5's is upstream; with the 3' downstream. DNA and RNA are synthesized going from the 5' to the 3' direction.

              •  

                DNA Chemical Structure¹⁰

                "In the DNA segment shown, the 5' to 3' directions are: DOWN the left strand and UP the right strand.  Chemical structure of DNA, with colored label identifying the four bases as well as the phosphate and deoxyribose components of the backbone."

                 

          • Most eukaryotic mRNAs contain a special blocked nucleotide structure at their 5' end, named the cap  structure.¹² 

            • Analysis of cellular and viral mRNAs resulted in a generalized cap structure, which in the case of human respiratory syncytial virus, consists of a 7-methyl guanosine links to an  unmethylated guanosine via a 5'-5' pyrophosphate linkage formed utilizing the α  and β phosphates of  GTP.

            • The complete cap structure  is ^m7G(5')ppp(5')Gp,  devoid of ribose 2'-O-methylation. The capping process, including methylation, is associated with transcription.¹² 

          •  

            Viral RNA 5'end structure and maturation¹³

            " Nearly all mammalian viruses modify their RNA 5'-end through the covalent addition of a cap structure (majority) or a protein (minority). Although the widely acquired RNA cap structure is chemically identical (m7GpppN), viruses have evolved a large variety of mechanisms to synthesize or acquire this crucial structure. The mechanisms of 5'-end maturation vary among order and families as presented in the above schematic (for clarity, only a few viral families are presented). Within the Flaviviridae family, the Flavivirus synthesize a typical cap structure while the Hepacivirus and Pestivirus are the only mammalian viruses representative to harbor an unmodified 5'-triphosphate end. Their RNA 5' UTR instead folds into a highly structured three-dimensional confirmation termed IRES. Of notice, the Retroviridae RNA harbor both, a cap structure and an IRES, as well as the Picornaviridae RNA that harbors both an IRES and a 5'-VPg-linked protein. (Adapted from Decroly et al (2012))."  RNA 5′-end Maturation: A Crucial Step in the Replication of Viral Genomes

          •  

            "Canonical 5' RNA Cap Synthesis Pathway"¹³

            "RNA cap-1 structures are conventionally synthesized by the sequential γ-phosphate hydrolysis by: an RTPase GMP transfer by a GTase N7-methylation by an N7MTase, and 2'O-methylation by a 2'OMTase.  The contribution of each substrate to the formation of the final 5' RNA cap structure is highlighted by a color code: pppRNA (black), GTP (blue) and SAM (green and red)."13

            The reason that viruses have adopted a similar cap structure to that observed in eukaryotic cells is likely evolutionary pressure. 13 Suggested earlier, synthesis of a cap structure at the 5'-end of RNA requires several steps.13   In the general case, three sequential enzymatic steps are required to form the 7-methylguanosine 5'-5'-triphosphate bridged cap structure.13 The directional 5' to 3' polymerization of nucleotide triphosphates (NTP) during RNA synthesis results in nascent RNA exhibiting at their 5'-end a triphosphate group which came from the initial nucleotide triphosphate (NTP). Initially, this 5'-triphosphate RNA end is converted into a 5'-diphosphate end by hydrolysis of the terminal phosphate (or γ-phosphate) by an RNA triphosphatase (RTPase). A two-step reaction that ensues, catalyzed by an RNA guanylyltransferase (GTase). The first step involves enzymatic binding and GTP hydrolysis forming a covalent enzyme-GMP intermediate. In the second step, this complex catalyzes GMP transfer to the 5'-end of the diphosphorylated acceptor RNA (ppRNA). Finally, an RNA (guanine-N-7)-methyltransferase (N7MTase) using the methyl group donor S-Adenosyl methionine (SAM), allowing methylation of the guanosine residue of the cap structure at the N7 position. These enzyme catalyzed steps result in a basic and minimal RNA-0 structure (m7GpppN) allowing for subsequent methylation steps using S-Adenosyl methionine as the methyl donor.13

        • 5'-terminal covalently linked protein²

          • One of the early examples of a virus containing a protein attached to the 5' terminal of his DNA is the hepatitis B virus.¹⁴ 

            • The hepatitis B virus DNA is a small circular molecule, partially double-stranded.

              There is, moreover, a single-stranded portion of the length 15-50% of the circle links in different molecules.

            • Purification studies suggested that the HBV DNA contained a tightly attached protein since DNA could not be removed from aqueous solution without prior protease digestion.

            • These observations from 1980 followed earlier findings of proteins associated with the 5' terminal of linear nucleic acids tightly bound to adenoviruses, enteroviruses, and B. subtilis phage ϕ29.¹⁴ 

          • In the case of adenovirus, a 5' prime terminal precursor protein serves as a primer for the start of viral DNA replication.¹⁵ 

            • The mechanism involves covalent binding of the first nucleotide in the DNA chain with this precursor protein remaining covalently bound to the 5' terminal of viral DNA.

            • Upon virion maturation the precursor protein is cleaved to the terminal 5' protein.

              • The 5' virion terminal protein is thus derived from the carboxyl (COOH) terminus of this precursor protein.

              • The mature virion terminal 5' protein enables viral DNA to circularize, process which is both protease-sensitive and dependent on noncovalent interactions.

              • In this case, the covalent linkage between DNA and the 5' terminal protein is mediated by a phosphodiester bond between the β-hydroxyl group of the terminal protein' s serine residue and the 5'-hydroxyl of the terminal deoxycytosine residue.¹⁵ 

          • VPg:  The presence of genome-linked viral proteins was initially identified approximately 40 years ago in studies of polioviral RNA.^16,17 

            •  

              Poliovirus type 1:  genomic structure showing VPg, Cloverleaf, and IRES locations

              De Jesus NH Epidemics to eradication Virol J 4:  70, 2007 http://www.ncbi.nlm.nih.gov/pubmed/17623069 and http://en.wikipedia.org/wiki/Poliovirus

            • Detailed chemical analysis of the poliovirion indicated that the 5'-terminal end of the virion RNA exhibited the following structure: protein-pU-U-A-A-A-A-C-A-G. ¹⁶

            • The authors suggested that the protein-nucleotide structure function as a primer for initiation of poliovirus RNA synthesis.

            • This primer activity depends on the protein first becoming uridylylated which provides a free hydroxyl, allowing initial interaction with viral encoded RNA-dependent RNA polymerase.

              VPg uridylylation is catalyzed by viral RNA-dependent RNA polymerase. 

              Uridylylation of VPg is thus a required step in poliovirus genome replication. ^{16, 17,18}

        • Isomerization

          • The genome of herpes simplex virus (HSV) exhibits isomerization. HSV genomes are described as linear molecules composed of both long (L) and short (S) segments.¹⁹ 

            • Each segment is associated with unique sequences designated U_L and U_S. (U for "unique")

            • Genomic replication occurs at three origins, one localized in U_L (Ori_L) and two (diploid) localized in the short repeating region (Ori_S).

              • Characteristics of replication intermediates suggested a "rolling circle mechanism" responsible for HSV DNA synthesis.

              • Furthermore, the L and S segments invert relative to each other, resulting in four equimolar genomic isomers.

                • Isomers (two) may occur by cleavage of concantemers at alternative L- S junctions with remaining isomers occur due to homologous recombination between sequences in the large repeated regions of DNA that flank L-S junctions.

                  • Isomerization might be associated with formation of circular replication templates derived from linear virion DNA.¹⁹

                   

          • Herpes Simplex Viral Genome²⁰

            "Top:  Organization of unique and repeated sequence elements.  Repeated sequence elements are shown in small letters (a,b,c) and unique sequence elements are shown in capital letters (UL, Us). Elements shown with a prime are inverted. Below:  four isomeric forms of viral DNA with different orientations of the long (L) and short (S) components due to frequent recombination at the repeated sequence elements."20

            Attribution:  Figure 24-1 (Roizman B Campadelli-Fiume G Longnecker R Herpesviruses Chapter 24 (in Acheson NH,  Fundamentals of molecular virology, 2nd edition) Figure 24-1, p 287, 2011.)

             

        • 3'-terminal poly(A) tract²

          • Almost all fully processed eukaryotic mRNA exhibit a poly(A) tail located at the 3' end.²¹ 

            • This poly(A) appears important in nearly all aspects of mRNA metabolism.

            • Proposed functions include:

              • Enhancement of mRNA stability

              • Increased mRNA translational efficiency and

              • Participation in mRNA transport from the nucleus to the cytoplasm.

          • The underlying polyadenylation involves activities beyond addition of poly(A) tails to mRNAs.²¹