Medical Pharmacology Chapter 36:  Antiviral Drugs

• Antiviral Drugs

  • More about Influenza Viruses.

    • Orthomyxoviruses and paramyxoviruses are the two families originally described by "myxoviruses".⁴ 

      • The origination of the myxoviruses name was based on the finding that virions associated with influenza and some other enveloped, negative-strand RNA viruses bind to sialic acid residues in mucoproteins. (Here "ortho = correct and para = alternative).

      • The Orthomyxoviridae family not only includes influenza viruses A, and but also two tick-borne mammalian viruses in the Thogotovirus genus (from the Kenyan Thogoto forest where the virus was first isolated) and a virus (Isarvirus genus) which infects Atlantic salmon.

        • Thogotoviral Virion⁶

          Thogotovirus virion:  enveloped and spherical6 Attribution:  ViralZone http://viralzone.expasy.org/all_by_species/79.html

    • Influenza type A viruses cause most flu epidemics and are commonly found in both avian and mammalian species.⁴ 

      • By contrast, influenza type B is found only in humans; whereas, type C infects both humans and pigs.

    • Orthomyxoviruses exhibit both common structure and replication process.⁴ 

      • Their negative-strand RNA is comprised of 6-8 gene segments, individually associated with helical nucleocapsids.

      • These nucleocapsids are packaged in a host cell plasma membrane-derived lipid envelope forming the virion.⁴ 

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          Thogotoviral Genome⁶

          Thogotoviral genome, illustrating segmented ssRNA(-) linear form, containing 6-7 segments coding for 7-9 proteins.6 Viral RNA polymerase (PB1, PB2 and PA) transcribes one mRNA from each segment.6 Attribution:  ViralZone http://viralzone.expasy.org/all_by_species/79.html

    • Influenza viral genome segments:

      • 11 viral proteins are produced by influenza type A viruses.⁴ 

        • 9 of these 11 are packaged in virions.⁴  

          • Genome segments 1 though 6 each code for one protein as follows:

            • 3 RNA polymerase subunits (PB2, PB1, and PA)

            • Gycoprotein hemagglutinin (HA) envelope glycoprotein

            • Nucleocapsid protein NP and an additional envelope glycoprotein, neuraminidase (NA).⁴  

              • For some influenza A isolates, an additional small protein may also be encoded, PB1-F2.

          • mRNAs transcribed from genome 7 and 8 may be spliced which results in 2 distinct mRNAs, coding for different viral proteins.⁴  

            • Genome segment 7 encodes the sequence for matrix proteins (M1) as well as a third envelope protein, M2.

            • Genome segment 8 encodes for NS1 and for NS2, both nonstructural proteins.

            • All of these proteins have important roles in viral replication.⁴ 

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          Influenza A Virion⁷ 

          Ribonucleoprotein comples:  a viral RNA segment linked with the nucleoprotein (NP) and 3 polymerase proteins, PA, PB1 and PB2.7 Matrix protein (M1) is interacts with both the ribonucleoprein and viral envelope.7 Figure Attribution:  Modified slightly from "An influenza A virion" from reference 7 (An influenza A virion: Horimoto T Kawaoka Influenza:  Lessons from Past Pandemics, Warnings from Current Incidents; Nature Reviews|Microbiology 3:  591-600, August 2005 and Scitable by nature education:  An influenza A virion http://www.nature.com/scitable/content/an-influenza-a-virion-26874

      • Role of hemagglutinin in influenza viral replication:

        • Influenza virus has an ability to agglutinate red blood cells.⁴

          • This action appears mediated by influenza hemagglutinin (so named) protein (HA), a protein which binds to sialic acid-containing receptors.

          • These receptors are not only localized on red blood cells but also on most other cells.

          • HA forms a "trimer" in the virus enzyme and is key for virion binding to the cell surface and also for viral genomic entry into the cell.

            • Entry of the virus into the cell occurs by a fusion pathway.⁴

        • HA, a type I transmembrane protein, after synthesis, is first localized in endoplasmic reticulum membrane and later transported by the Golgi apparatus to the plasma membrane where it can be incorporated into virions.⁴ 

          • The role of influenza hemagglutinin protein, HA, in membrane fusion is dependent on cellular proteinases that mediate cleavage of HA into two subunits.

            • This cleavage is necessary for membrane fusion activity.

              • The first subunits, HA₁, is the surface subunit which contains the sialic acid binding region.

              • HA₂ is localized in the virus envelope and contains the fusion peptide.

            • Initially, the fusion peptide resides in an inaccessible hydrophobic region of HA, close to the viral envelope lipid membrane.⁴ 

              • The fusion activation process is initiated subsequent to virion binding to cell receptors.

                • In this step, virions into the cell by means of an endosomal vesicle and with a decrease in endosomal pH (increased acidity), a change in HA confirmation is induced.

                  • One consequence of this change in conformation is the transition of the fusion peptide out of the inaccessible, hydrophobic region towards the endosomal membrane.

                    • There, this new HA confirmation allows insertion of the protein into the membrane.

                    • Concurrent with that step is a change in location of HA2, anchored in the viral envelope, and now moved closer to the extended N-terminal region.

                      • "Spring-loaded mechanism for viral membrane fusion"^10,13

                        "Spring-loaded mechanism for viral membrane fusion. In the native conformation of influenza hemagglutinin (HA) (left), the HA1 subunits (yellow balls) occupy the distal end of the structure, atop a trimeric coiled coil region of HA2 (blue). The fusion peptides (green) are buried in the core of the HA2 structure. On induction of low pH, fusion-activating conformational changes occur (right). The noncovalent interactions between HA1 and HA2 weaken and the loop regions of HA2 (red) “spring” into helical conformations, extending the central trimeric coiled coil and propelling the fusion peptides to the top of the structure to interact with the target membrane."10 Attribution:  This figure corresponds to Figure 2 in reference 10; Figure 2 is cited as an adaptation from reference 13 (Eckert DM Kim PS Mechanisms of Viral membrane Fusion and Its Inhibition. Annu. Rev. Biochem. 70:  777-810 2001.;Carr CM Kim PS A Spring-loaded mechanism for the Conformational Change of Influenza Hemagglutinin. Cell 73(4):  823-832, May 21, 1993. (second sourced from reference 10)

                    • In this stage, one end of HA1 is associated with the endosomal membrane with the other end associated with viral envelope.

                      • The described conformational change allows the approximation of these two membranes and membrane fusion.⁴

            •  

              X-Ray Crystal Structure of Trimeric Influenza Hemagglutinin (bromelain-released form)^10,11

              "High-resolution structure of native influenza hemagglutinin (HA).10,11 The X-ray crystal structure of the trimeric HA, including all of HA1 and the first 175 residues of HA2 .10  Three HA1 monomers (yellow) sit atop a stalk-region composed of three HA2 monomers (two gray, one mostly blue). The first 20 residues of the predicted fusion peptide (green) are indicated. Residues 55–76 (red), which are in a loop conformation, become helical during the HA-mediated fusion process. Residues 106–112 (orange), helical here, change to a loop conformation to form the trimer-of-hairpins during the fusion process."10 Attribution: This figure corresponds to Fig. 1b of reference 10 (Eckert DM Kim PS Mechanisms of Viral membrane Fusion and Its Inhibition. Annu. Rev. Biochem. 70:  777-810 2001.)

            •  

              Conformational changes of viral fusion proteins leading to membrane fusion¹²

              "Schematic representation of a working model for viral membrane fusion mediated by class I fusion proteins. Influenza virus, which is internalized into an endosome, is shown as an example. In the native state of the fusion protein — which is a trimer — most of the surface subunit (green) is exposed. Part of the transmembrane subunit, including the fusion peptide, is not exposed. Following fusion-activating conditions,conformational changes occur to ‘free’ the fusion peptide (red) from its previously unexposed location. In the case of influenza HA, this occurs by a ‘spring-loaded’ mechanism. The ‘pre-hairpin’ intermediate spans two membranes — with the transmembrane domain positioned in the viral membrane and the fusion peptide inserted into the host-cell membrane. The prehairpin intermediate forms a trimer of hairpins, and membrane fusion occurs, which leads to pore formation and release of the viral genome into the cytoplasm." Attribution:  Figure here corresponds to Figure 5(a) in reference 12 (Dimitrov DS Virus Entry:  Molecular Mechanisms and Biomedical Applications. Nature Reviews|Microbiology 2:  109-122 February 2004.)

        •  

          Influenza Hemagglutinin⁹

          Attribution:  Harrison S (Harvard/HHMI)  Influenza Hemagglutinin https://www.youtube.com/watch?v=64wF8ZOSVGU (11/2013) iBiology Techiques YouTube Channel:  https://www.youtube.com/channel/UCimxROEIlNsiRqT9X5tg2XQ

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          Viral Membrane Fusion⁸

          Attribution: Harrison S Viral membrane fusion (Part 2) https://www.youtube.com/watch?v=qcepGvFUM38 (3/2012) iBiology Techiques YouTube Channel:  https://www.youtube.com/channel/UCimxROEIlNsiRqT9X5tg2XQ