Medical Pharmacology Chapter 36:  Antiviral Drugs

• Antiviral Drugs

  • More about Influenza Viruses.

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

      • Nucleocapsid release from the virion: the role of the M2 protein

        • Influenza A Virion ⁶ 

          Ribonucleoprotein comples:  a viral RNA segment linked with the nucleoprotein (NP) and 3 polymerase proteins, PA, PB1 and PB2.6 Matrix protein (M1) is interacts with both the ribonucleoprein and viral envelope.6 Figure Attribution:  Modified slightly from "An influenza A virion" from reference 6 (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 natureeducation:  An influenza A virion http://www.nature.com/scitable/content/an-influenza-a-virion-26874

        •  

          Cut-away View of an Influenza A Virion ⁷ 

          Herstellung E, Eickmann M Structure of swine influenza virus showing different types of antigens present on and inside the capsid. http://commons.wikimedia.org/wiki/File:Flu_und_legende_color_c.jpg

      • The M2 protein is small with an N-terminal external domain, along with a single transmembrane domain with a larger internal domain.⁴ 

        • The essential feature of the M2 protein resides in its ability to form a highly selective transmembrane ion channel that allows protons (H⁺) to partition into the membrane.

          • Structurally, the M2 protein creates a pore utilizing 4 parallel transmembrane α-helices (tetrameric) which structurally defines the viral envelope pore.

            •  

              Influenza Matrix Protein M2⁸

              Thatsongic Strain sequence info:  matrix protein 2, Influenza A virus http://commons.wikimedia.org/wiki/File:M2struct.png Note the tetrameric α-heliceal structure

            • An important step in virus replication is release of the viral nucleocapsid from the virion and it is this process that is made easier by the M2 H⁺ channel.

              • The consequence of this channel facilitating proton translocation to the interior of the virion causes endosomal acidification.⁴

              •  As a result of this acidification (higher [H⁺]), the affinity of the matrix protein M1 for the nucleocapsid is reduced, thus statistically increasing the likelihood of nucleocapsid release into the cytoplasm, following membrane fusion.

                • The anti-influenza viral drug amantadine is a specific inhibitor at the M2 membrane H⁺ channel.

                  • Therefore, with amantadine-mediated channel blockade, viral nucleocapsid release is diminished or incomplete, thus blocking infection.⁴

                    • The antiinfluenza Drug Amantadine (Symmetrel):  M2

                      H⁺ Membrane Channel Blocker

      • Viral RNA Replication

        • Orthomyxoviruses (influenza virus) replicate in the host cell nucleus, by contrast to most other RNA viruses.⁴ 

          1. 1. Virion genomes are initially transported to the nucleus for transcription.

          2. 2. Viral mRNAs then move to the cytoplasm for translation into protein.

          3. 3. Viral proteins then move back to the nucleus for the purpose of regulating genomic replication.

          4. 4. Nucleocapsids are formed using the newly replicated viral genome with the nucleocapsids then moving back to the cytoplasm, allowing virion assembly at the host cell plasma membrane.

          5. 5. Nucleocapsids move from the cytoplasm to the nucleus through a nuclear membrane pore.

             1. The NP protein, nucleocapsid protein, as well as polymerase proteins containing nuclear localization recognition capabilities, mediating interaction with cellular importin-α.

                1. The resulting complex then interacts with importin-β.

                   1. This assembly docks with nuclear pore complexes and facilitates translocation of proteins across the nuclear membrane important for replication .

                   2. Copying of viral negative-sense RNA genome segments, described earlier, is then initiated utilizing cellular RNAs as transcription primers.⁴ 

          6.  

             "Classical Nuclear Import Cycle"⁹

             "In the cytoplasm, cargo containing a classic nuclear localization signal (cNLS) is bound by the heterodimeric import receptor, importin α/importin β.9,10 Importin α recognizes the cNLS and importin β mediates interaction with the nuclear pore during translocation."9 Once inside the nucleus, RanGTP binding results in a conformational change in importin β, which releases the IBB region (Importin Beta Binding) of importin α.9  This autoinhibitory domain, together with Nup2 (Nup2, a nucleoporin = karyopherin release factor) and Cse1 (Cse1 = the export receptor for importin α) facilitates cNLS association and delivery of the cNLS cargo in the nucleus.9,11,12  Lastly, importin α is recycled back to the cytoplasm by the export receptor, Cse1, in complex with RanGTP.9  Energy for nuclear transport is dependent upon the small Ras family GTPase, Ran which cycles between a GTP- and GDP-bound state.9,13,14 *Carriers that are involved in active transport of macromolecular material between cytoplasmic and nuclear compartments are referred to generally as "karyopherins."9

      • Precursor Viral mRNA Capping:

        • "RNA processing:  5' cap, 3'polyA tail and Splicing"¹⁶

          Attribution:  Cavnar P RNA processing:  5'cap, 3'polyA tail, and Splicing https://www.youtube.com/watch?v=m2lOf3ker9M (3/2013) Peter Cavnar YouTube Channel https://www.youtube.com/user/PCB2131/videos

        • Primers for synthesis of viral mRNAs are "capped" 5' ends of cellular pre-messenger RNAs.⁴ 

          • Capping represents an an altered nucleotide on the 5'-end of, in this case precursor messenger RNA.

            • The capping process, which occurs only in the nucleus, is important in the creation of both stable and mature mRNA.

              • The 5' cap (on the 5' mRNA end) is composed of a guanine nucleotide linked to the mRNA by means of a 5' to 5' triphosphate bridge.

                •  

                  "5' Cap Structure"¹⁵

          • Capped viral mRNAs are derived from these host-cell cellular pre-mRNAs that had been used to prime transcription.

            • This is the mechanism by which orthomyxoviruses (e.g. influenza virus) makes capped messenger RNAs even without its own coding for capping enzyme.

              • Normally, cellular pre-messenger RNAs are exported to the cytoplasm where they provide a pool of cellular pre-messenger RNA, thus supporting influenza virus mRNA production.

                • If this pool is not actively replenished by an intact cellular RNA synthesis system, the cellular pre-messenger RNA pool needed by the virus for its mRNA synthesis would become rapidly depleted.⁴  

                  • Thus, for influenza virus, given the need for cellular RNA synthesis, agents that interfere with cellular RNA synthesis inhibit influenza viral replication.

                    • Examples of such agents include:

                      • actinomycin-D

                        • Actinomycin-D (Dactinomycin)

                      • α-amanitin.⁴

                        • α-Amanitin

        • The reader is directed to reference 4 for detailed discussion of additional, detailed steps in influenza A viral replication and regulation.⁴