BSR 1.04 Virus Invasion of Host Cells - v6_09.06.24 PDF

BASIC SCIENCE RESEARCH Virus Invasion of Host Cells Block 1 Ronald Matias, PhD | August 16, 2024...

BASIC SCIENCE RESEARCH Virus Invasion of Host Cells Block 1 Ronald Matias, PhD | August 16, 2024 Trans 1.04 OVERVIEW ○ DNA is transcribed into messenger RNA (mRNA I. Viruses III. Replication Cycle of via cellular or viral RNA polymerase) and mRNA is A. 3 Broad Classes of Viruses translated into viral proteins Viruses A. Viral Attachment ○ DNA can also replicate using cellular or viral DNA II. Viral Infection B. SARS-CoV-2 polymerases A. Initiation of Infection: IV. Fusion Proteins Most eukaryotic DNA viruses replicate and assemble Collision of Virions A. Class I Fusion progeny viruses in the nucleus except for: and Cells B. Finding the “Right B. Class II Fusion C. Class III Fusion ○ ❗Poxvirus : replicate in cytoplasm Cell” V. Viral Penetration : ex. Monkeypox virus C. Cellular Receptor LEGEND ⭐ : Important information 💬 : Good-to-know info from lecturer ➕ : Supplementary/Background Info ❗ : Exception RNA Viruses Genetic material is RNA Positive (+) single strand RNA virus can be ABBREVIATIONS immediately translated into protein upon entering DNA Deoxyribonucleic acid the cell ds Double-stranded Negative (-) single strand RNA virus must undergo Fc receptor Fragment crystallization receptor RNA replication to synthesize (+)ssRNA mRNA Messenger RNA ○ Use complementary (+)ssRNA strand for the RNA Ribonucleic acid synthesis of viral proteins RT Reverse transcriptase ss Single-stranded Table 2. Examples of RNA viruses Vc RNA Full-length complementary RNA RNA strand Examples dsRNA Rotavirus I. VIRUSES Yellow Fever virus Organized association of macromolecules Poliovirus (+) ssRNA Two major macromolecules: Dengue virus ○ Nucleic Acids Coronavirus Carries the blueprint for replication of progeny Measles virus (-) ssRNA virions Rabies virus ↪ When these start to multiply, it becomes Arenavirus infectious virus particles (±) ssRNA Some genera in the Bunyaviridae ○ Protein family Structural component of virus Viruses that use Reverse Transcriptase (RT) A. 3 Broad Classes of Viruses Special class of RNA viruses that can undergo reverse Based on genetic material transcription Table 1. 3 Broad Classes of Viruses Viruses that use Reverse DNA Viruses RNA Viruses Transcriptase (RT) dsDNA (+)ssRNA ssRNA-RT RNA virus that contains RNA as genetic material but ssDNA (-)ssRNA dsDNA-RT uses the enzyme reverse transcriptase for (±) ssRNA transcribing it back to DNA dsRNA DNA acts as intermediate and integrate into host genome DNA Viruses In proper conditions, DNA intermediate transcribed Subdivided into single-stranded DNA (ssDNA) or into RNA and translated into viral proteins double-stranded DNA (dsDNA) viruses Genetic material is DNA and follows the central dogma of molecular biology Page 1 of 15 | TH: DIONISIO | BSR/PSYCH TG 4 | CRISOSTOMO, MARTINEZ, SALANGUIT Checked and verified: TP LIM, A. GAMAD, MJ  BSR 1.04 Viral Invasion of Host Cells A. Initiation of Infection: Collision of Virions and Cells To initiate infection, viruses have to collide with host cell Collision is driven by: ○ Brownian motion DNA virus can be transcribed to RNA intermediate ○ Laws of diffusion and RNA is reverse transcribed back to DNA ○ Electrostatic interaction Table 3. Special Group of Viruses B. Finding the “Right” Cell Groups Example Viruses have specificity of infection ssRNA-RT Retroviruses (HIV) ○ Not all cells can be infected by a virus dsDNA-RT Hepadnaviruses (Hepatitis B) Major steps in finding the right cell: 1. Adhere to cell surface via electrostatic interaction TG Note: Examples of viruses under their respective groups have 2. When viruses are in proximity with the cell been summarized in the Appendix, in case you want to read on them, since Doc removed that part in the new PPT. surface membrane, attach to specific receptor molecules on cell surface ○ More than one receptor may be involved II. VIRAL INFECTION ○ Hallmarks of successful virus infection Virions are inanimate 3. Once virus has attached to its receptor, it can ○ No structure for locomotion penetrate the cell and transfer genome inside the Viruses are obligate intracellular parasites[Batch 2027] cell ○ They are not capable of surviving outside of the host cell for long. C. Cellular Receptors for Viruses Virus particles are too large to diffuse across plasma Essential for all viruses except: membrane[Batch 2027] ○ Yeast : no extracellular phases SUPPLEMENTARY INFO ○ Plants : enter cells via mechanical damage Consists of receptors and co-receptors General steps in virus infection of host cells: In 1985, one receptor identified: 1. Attachment and cell entry 2. Transcription of viral replicase ○ Sialic acid for Influenza virus 3. Genomic transcription and replication Progress stimulates monoclonal antibodies (mAbs) 4. Translation of structural proteins and molecular cloning 5. Virion assembly and release ○ Identification of several viral receptors and their structure to understand how they work ○ ➕ Allows progress in production of antivirals ➕ Function membrane proteins can act as viral receptors Different viruses can bind to same receptor Table 4. Viruses with same and different receptors VIRUS RECEPTOR Different viruses binding to same receptor Adenovirus SAME Coxsackievirus B3 Swine herpesvirus Pseudorabies virus SAME Human poliovirus Viruses from same family binding to different receptors Rhinoviruses (3) DIFFERENT Retroviruses (16) DIFFERENT SUPPLEMENTARY INFO Figure 1. Life Cycle of SARS-CoV-2 Criteria for Identifying Cell Receptors TG Note: This figure was included in the old PPT but was not Receptor binds virus particle discussed by Doc. Antibody to receptor blocks infection Receptor gene confers susceptibility ○ More than one receptor may be involved Page 2 of 15 | TH: DIONISIO | BSR/PSYCH TG 4 | CRISOSTOMO, MARTINEZ, SALANGUIT  BSR 1.04 Viral Invasion of Host Cells humans or why human influenza will not infect Disruptions of receptor gene blocks infection birds TG Note: Supplementary info was included in the old PPT but Currently, we have problems with avian flu in was not discussed by Doc. poultry but not in humans. Mutations occur and mutations lead to a new character and new characters lead to new modes of infection so III. REPLICATION CYCLE OF VIRUSES there may come a time where avian influenza A. Viral Attachment will affect humans. There are two general kinds of receptors: Neuraminidase cleaves sialic acid from galactose Accessory Receptor ○ Low affinity binding to virus ○ Not required for viral entry but may accelerate rate of binding and uptake of virus ○ Presence will aid in the binding and penetration of virus High Affinity Receptor Binding ○ Required for viral entry ○ Cells failing to express the appropriate receptor cannot be infected by the virus Answers question on why some cells are infected while others are not Ex. Rabies virus infects peripheral nerve cells and migrates to the nerve tract to go to the brain, but other cells (skin, lung, intestinal, etc.) will not be infected. ↪ This is because only nerve cells have the specific receptor that the rabies virus can infect Figure 3. SAα2,6-Gal (A) and SAα2,3-Gal (B) receptors highlight the binding pattern of linkage-specific lectins. Same goes for the Influenza virus which will As reflected in figure 3, there may be little difference infect the pulmonary system but not other in the attachment of the sialic acid to galactose but in systems. 3D models, the difference is emphasized. ↪ Human influenza will infect the lower respiratory tract while Avian influenza will Influenza Virus Surface Proteins not infect human cells ○ Nature of the receptors utilized by virus determines in part its host range and tissue tropism A. Influenza Virus Sialic Acid: Receptor for Influenza Virus Figure 4. Influenza virus surface proteins. Nucleocapsid protein (NP) protects the influenza virus’ genetic material Neuraminidase ○ A glycoside hydrolase Figure 2. Sialic acid located at the terminal end of glycoprotein anchored on ○ Enzymatic reaction with sialic acid results in the plasma membrane (left); ɑ(2,3) attachment to galactose (right). release of virions Sialic acids as a receptor for the influenza virus Hemagglutinin (HA) ○ N-acetylneuraminic acid (Influenza A,B) ○ Protein attached to a membrane that surrounds ○ 9-O-acetyl-N-neuraminic acid (Influenza C) the viral genome Attachment of sialic acid to galactose via: HA may bind to a specific receptor on the ○ α(2,6) - preferentially used by human strains surface of a cell ○ α(2,3) - used by avian strains ○ There are many existing types of HA based on Carbon number 2 in sialic acid sequence Carbon number 3 in galactose The HA and neuraminidase forms the type of ○ Explains why the avian influenza will not infect receptor and infection the influenza has Page 3 of 15 | TH: DIONISIO | BSR/PSYCH TG 4 | CRISOSTOMO, MARTINEZ, SALANGUIT  BSR 1.04 Viral Invasion of Host Cells ↪ H1 & H2 - human, pig, birds ↪ N1 & N2 - human, pig, birds B. SARS-CoV-2 ↪ H6 - birds only Enveloped (+) ssRNA virus ↪ Ex. H1N1 92-104 nm in diameter ○ Trimer: made up of 3 monomers (Fig. 5) Table 5. Component of a monomer and its characteristics Globular head Fibrous Stem Amino acid α-helix β-sheet configuration Sialic acid Attached to the binding-site: membrane that Component lodged in the surrounds the virus groove of the head Figure 7. SARS-CoV-2 with its spike proteins. Structural proteins: ○ Nucleocapsid (N) ○ Membrane (M) ○ Envelope (E) ○ Spike (S) Resembles the hemagglutinin of influenza Has its own receptor in the susceptible cell Receptor: Angiotensin converting enzyme 2 (ACE2) Co-receptors (AKA accessory receptors): ○ DC-SIGN & L-SIGN Examples of intercellular adhesion molecules ○ Heparan sulfate Figure 5. 3D structure of the viral receptor hemagglutinin elucidated using ○ Neuropilin DNA sequencing where its globular head (blue), fibrous stem (red) can be seen. Sialic acid binding site (green) is located in the globular head. SARS-CoV-2 Spike Protein Figure 8. S1 and S2 subunits of the SARS-CoV-2 Spike Protein reflecting the Figure 6. Hemagglutinin structural changes during infection. Cleavage site globular head and fibrous stem of Influenza virus. Carbohydrate residues (yellow) can also be seen. (brown) attached to the proteins in the S1 and S2 subunits. 3 HA proteins = 3 globular heads = 3 sialic acid 2 subunits of spike protein (similar to Influenza’s binding sites globular head and fibrous stem): Cleavage site: located at the terminal N ○ S1 subunit ○ Important for the functionality of the virus protein β-sheet configuration ○ Virus binds to the host via HA → Protease cleaves Contains the receptor binding domain where at the cleavage site → HA configuration changes ACE2 enzyme will bind → globular head dissociates and exposes the ○ S2 subunit fibrous stem and the type I fusion proteins α-helix configuration Contains the stalk that attaches the S1 subunit Influenza Virus. Scan the QR code or click to the surface of the virus this link: The presence of an accessory receptor aids in the https://www.youtube.com/watch?v=oXzwtG attachment of the receptor to the binding site. FyBik Page 4 of 15 | TH: DIONISIO | BSR/PSYCH TG 4 | CRISOSTOMO, MARTINEZ, SALANGUIT  BSR 1.04 Viral Invasion of Host Cells Figure 10. Domain organization and pre-fusion structure of the SARS-CoV Figure 9. The SARS‐CoV‐2 spike pre-fusion and postfusion structures. The spike (S) protein. SARS‐CoV‐2 spike is a trimeric glycoprotein on the surface of the virus. The same colors are used for each domain and subdomains across the panels. Has 2 configurations ○ RBD down: not exposed to the environment FIGURE 9A ○ RBD up: exposed to the environment; can readily Trimeric glycoprotein structure of the spike protein bind to ACE2 receptor ○ Two of the protomers are shown with their molecular surface (dark and light gray), while the other is shown in a ribbon representation. Receptor binding domain labeled as RBD FIGURE 9B Domains in S1 subunit: ○ N-Terminal Domain (NTD) ○ Receptor binding domain (RBD) FIGURE 9C Fibrous structure found in the stalk (similar to Influenza’s HA) ○ α-helix configuration Protease cleavage sites in S2 subunit: ○ Furin cleavage site Figure 11. Change in the configuration of the RBD. Side view (A) and top Cleaved by host enzyme, furin view (B) of the spike trimers (dark grey, light grey, and white) with their RBDs Furin cleaves the S1 site → S2 (alpha helix) is changing from down to up configuration. Epitope heatmap of neutralizing exposed → allows binding with the ACE2 antibodies is also shown (yellow to red). Red areas are the most vulnerable binding sites for neutralizing antibodies. receptor ○ Fusion peptide (FP) Has the ability to change configuration during Cleaved by TMPRSS2 and Cathepsin B/L infection protease enzymes → releases the fusion ○ RBD configuration changes from down to up → peptide receptor binding site gets exposed → ACE2 enzyme binds with RBD → viral infection initiated TG/TP Note: The lecturer mentioned that TMPRSS2 and endosomal ⭐ RBD is one of the immunogenic proteins and a major cathepsins expose the RBD, though other sources target of neutralizing antibodies state that it releases the fusion peptide. ○ Neutralizing epitope: a portion of the RBD that can be recognized by the body to elicit immune Receptor Binding Domain (RBD) response Located at the S1 subunit Epitopes recognition → immune response Each monomer consists of an RBD recruitment ○ Neutralizing antibody domain Portion of the RBD where neutralizing antibodies bind → blocks RBD-ACE2 interactions Summary for Viral Attachment Upon collision, electrostatic interactions will bring the virus in proximity with the probable host cell ○ Leads to binding of the receptor to the viral Page 5 of 15 | TH: DIONISIO | BSR/PSYCH TG 4 | CRISOSTOMO, MARTINEZ, SALANGUIT  BSR 1.04 Viral Invasion of Host Cells protein 4. Protease digests at the cleavage ○ For influenza, site. The terminal sialic acid residues will bind to 5. This causes the globular heads to the HA protein of the virus bend, exposing the class I fusion ○ For SARS-CoV-2, protein. The ACE2 protein binds to the receptor binding 6. The hydrophobic terminal end of domain of the Corona spike protein the fusion protein allows it to penetrate the plasma membrane of IV. FUSION PROTEINS the host cell. A. CLASS I FUSION PROTEIN 7. Hairpinning occurs: Fusion protein bends and pulls the membrane closer together. 8. Fusion of virus cell and target cell membrane (principle: fluid mosaic model). 9. Viral genes are released in the host cell’s cytoplasm and are transported into the nucleus for replication. 10. Progeny viruses are produced which escape the cell to infect other Figure 12. Similarities among 5 five viral fusion properties. (A) Top view of target cells. the structures. (B) Side view of the structures. Perpendicular to membrane; resembles a spike on Hairpinning the cell membrane Mostly α-helix configuration Form trimers Responsible for attachment Influenza, Corona, Simian Virus 5, Ebola Virus, HIV type I, Moloney Murine Leukemia Virus Viral Fusion of Influenza Virus Figure 14. Hairpinning in the viral fusion of influenza virus. 14a: ○ Globular heads have been removed already (cleaved at step 5 of table 6) 14b: ○ Fibrous stem binds to the host cell membrane 14c: ○ There may be several trimers binding to the cell membrane. 14d: Figure 13. Viral fusion of influenza virus. ○ Shift in configuration, pulling the cell membrane to Table 6. Viral Fusion of Influenza Virus the virus membrane (the layer below) Diagram Steps 14e: 1. Virus approaches the target cell ○ Being a lipid bilayer, it can readily fuse and HA binds to the receptor. 14f: 2. Endocytosis is initiated, resulting ○ Formation of portal between host cell and virus in endosome formation. cytoplasm 3. H+ ions are pumped in via proton Allows entry of the viral genetic material to the pumps, creating an acidic host cell cytoplasm environment in the endosome. Acidic environment allows HA to undergo conformational change (cleavage), Page 6 of 15 | TH: DIONISIO | BSR/PSYCH TG 4 | CRISOSTOMO, MARTINEZ, SALANGUIT  BSR 1.04 Viral Invasion of Host Cells Viral Fusion of SARS-CoV-2 host cell membrane close enough to let the membrane fusion happen. Intervention Mechanisms using Viral Fusion Proteins The use of enzyme inhibitors prevent viral fusion by altering the configuration of proteins ○ Ex. Preventing the acidification of the endosome (Influenza) → prevents globular head dissociation ○ SARS-CoV RBD configuration change is still not fully understood Figure 15. Viral Fusion of SARS-CoV-2 Antibodies prevent binding of fusion peptides with host cells TMPRSS2 (coreceptor) enzyme cuts SARS-CoV-2 spike protein → ACE2-RBD attachment → ➕ Decrease in pathogenicity of SARS-CoV-2 and Hydrophobic amino acids in the spike becomes herd immunity along with immunizations allowed us exposed and attach themselves rapidly to the host to go on normally with our daily lives compared to 2 years ago cell → Hairpinning pulls both cell membrane closer to each other → Virus and host-cell membrane fuse → ○ ➕ “A good virus will not kill its host in order to Transfer of genetic material from virus to host survive” B. CLASS II FUSION PROTEIN Mostly β-sheet Form dimers Parallel to the membrane E.g. Dengue, Japanese Encephalitis Figure 17. (A) Influenza virus HA trimer with its globular head, hinge, and stem forming a trimer. (B) Flavivirus E dimer. Figure 16. SARS-CoV S glycoprotein-mediated membrane fusion process. Initially, the RBD is down → stimulus triggers the change in conformation of RBD → “up” RBD binds to ACE receptor → endosome cleave occurs exposing the fusion proteins → fusion proteins extends and penetrates the cell membrane → hairpinning occurs Figure 17. Flavivirus structure → lipid bilayer is now in proximity → host and virus’ Source: Journal of Clinical Virology (2012) membrane fusion occurs → transfer of virus genetic material to host Surface of Dengue virus (similar to Flavivirus above) ○ Envelope protein (Yellow protein) Composed of dimers Viral Fusion of SARS-CoV-2 [MDPI: Source of Doc’s Figure 15] ↪ 1 dimer: 2 envelope proteins Only the “up” RBD is able to bind the angiotensin-converting enzyme 2 (ACE2) receptor, and Has 3 domains ACE2 binding promotes the dissociation of the S1 ↪ Yellow, blue, red subunits from the trimer spike. Parallel to the membrane Dissociation of the S1 subunits exposes the S2′ cleavage sites and may lead to the extension of S2 subunits and release of the fusion peptide. After the insertion of the fusion peptides into the host cell membrane, the α-helical heptad repeat (HR1) regions of the S2 protomers fold back to interact with the HR2 regions to drive the approach of viral and cell membranes. The folded HR1s then interact with HR2s to form a six-helix bundle that brings the viral membrane and Page 7 of 15 | TH: DIONISIO | BSR/PSYCH TG 4 | CRISOSTOMO, MARTINEZ, SALANGUIT  BSR 1.04 Viral Invasion of Host Cells VIDEO SUMMARY Life cycle of the Dengue fever virus Scan the QR code or click this link: https://www.youtube.com/watch?v=3LhWua TRCME 1. Dengue virus RNA virus Outer surface is an envelope protein with a lipid bilayer Figure 18. Dengue virus cryo-EM reconstruction fitted with structure of ○ Contains capsid enclosing the RNA genome tick-borne encephalitis E protein Targets immune cells 2. Cell surface receptors for infection 1. Cognate receptor: Normal infection 2. Fc receptor: Antibody-dependent enhancement 3. Dengue virus replication process 1. Envelope protein binds to the cognate receptor 2. Triggers a receptor-mediated endocytosis ○ Virus is internalized in endosome 3. Formation of Endosome ○ Lowers the pH of interior ○ Virus changes arrangement to spike-like structures ○ Hydrophobic tips which allows them to penetrate the endosomes membrane and bend until its fusion 4. Releases the capsid into the cytoplasm ○ Capsid breaks apart and releases the viral RNA 5. Viral RNA travels to the rough endoplasmic reticulum ○ Positive sense strand Figure 19. Other structures of Dengue Virus ○ Can be directly translated into proteins 6. Ends of RNA bind to initiate the translation initiation VIDEO SUMMARY proteins ○ Complex attaches to the ribosome to initiate Dengue virus invades a cell translation Scan the QR code or click this link: ○ Whole viral genome is translated as a single long https://www.youtube.com/watch?v=nN1xJxX polyprotein chain Aptc 7. Capsid protein is at the cytoplasmic side of the endoplasmic reticulum while the envelope and 1. Aedes aegypti carries the dengue virus membrane protein are in the lumen side 2. Over 50 million people are infected each year, approximately ○ Activated by the host’s peptidase enzyme one in every thousand cases is fatal 8. In the cytoplasm, 3. As the female mosquito pierces the skin searching for blood, it ○ Protease enzyme activates all the other proteins in injects saliva with the dengue virus the polyprotein chain 4. Virus has made its way to the bloodstream, infecting and ○ These proteins aggravate to form the RNA replication reproducing in more cells 7 to 10 days later complex 5. Each time a dengue virus reproduces, it must release its RNA 9. Viral RNA is synthesized in multiple steps: 6. The virus enters the host cell via receptor-mediated ○ Its ends fold up and forms a circle endocytosis and becomes enclosed within an endosome. ○ RNA attaches to the replication complex to start the Endosome: Piece of cell membrane with the virus first round of synthesis 7. Inside the endosome, the virus is surrounded by a protective ○ Using the virus’ positive sense RNA as a template, a shell of viral proteins. The environment inside the endosome negative sense copy is made becomes acidic (↓ pH), triggering viral proteins to rearrange and ○ The pair RNA strands form a double helix expose the viral membrane. ○ RNA then becomes a circle again, using the negative 8. The viral proteins form trimers, each with a fusion loop at its strand as a template to make a positive sense strand tip. These fusion loops stab the endosomal membrane ○ Many copies of the positive sense RNA strand are 9. The proteins fold back on themselves, pulling the two made through repeated cycles of RNA synthesis membranes together. This causes the outer and inner leaflets of Some of these strands are translated to make the membrane to fuse, creating a fusion pore. viral proteins 10. Through the fusion pore, the viral RNA is released into the Enough proteins are made to assemble new cytoplasm of the host cell. viruses 11. The released RNA hijacks the host cell’s ribosomes to begin 10. Envelope proteins aggregate at the lumenal side of the endoplasmic reticulum while the capsid protein 💬 synthesizing viral proteins. Similar to Influenza and Coronavirus aggravate at the cytoplasmic side ○ Viral RNA binds to the capsid protein → Packaged Both use fusion proteins to mediate the fusion of the viral into a new virus → Buds off to the endoplasmic membrane with the host cell membrane Page 8 of 15 | TH: DIONISIO | BSR/PSYCH TG 4 | CRISOSTOMO, MARTINEZ, SALANGUIT  BSR 1.04 Viral Invasion of Host Cells VIDEO SUMMARY reticulum ○ This virus is still immature, with its premembrane proteins cover the tips of the envelope protein to HIV Replication prevent premature fusion back into the cell Scan the QR code or click this link: Buds off and travels through the golgi apparatus https://www.youtube.com/watch?v=RO8MP towards the cell surface, while its premembrane 3wMvqg protein is processed to become mature 11. Virus becomes mature; new dengue virus are released Each step is important and therefore a potential target of from the cell to infect others antiretroviral drugs. Steps C. CLASS III FUSION PROTEIN 1. Infection of a suitable host cell (CD4 positive T-lymphocyte) Perpendicular to the membrane Entry of HIV requires cell surface receptors Mix of α-helices and β-sheets CD4 and co-receptors such as CCR5 and CXCR4 Form trimers ○ Interact with protein complexes imbedded in the virus Rhabdoviridae, Herpesviridae, Epstein-Barr virus, ○ Complexes are composed with two glycoproteins VSV virus Extracellular gp 120 and transmembrane gp 41 Attachment occurs ○ When HIV approaches a target cell, gp 120 binds to the CD4 receptors, initiating entry process ○ Promotes further binding to a co-receptor → results in a conformational change in gp 120 → allows gp 41 to unfold and insert its hydrophobic terminus into the membrane ○ gp 41 then fold back on itself → draws the virus towards itself → facilitates the fusion of their membranes Figure 20. Class III Fusion Proteins 2. Viral nuclear capsid enters the host cell and breaks open the two viral RNA strands and three replication enzymes Integrase Protease Reverse Transcriptase ○ Begins with viral RNA and has two catalytic domains: Ribonuclease H active site Polymerase active site ↪ Single stranded RNA is transcribed into an RNA-DNA double helix → Ribonuclease H breaks down the RNA ↪ Polymerase then completes the remaining DNA strand into a DNA double helix 3. Integrase cleaves a dinucleotide from each 3’ end of the DNA → creating two sticky ends → transfers it to the cell nucleus → facilitates its integration to the host cell genome Now contains the genetic information of HIV 4. Activation of the cell induces transcription of proviral DNA Figure 21. Viral entry with Class III fusion proteins into messenger RNA ○ Viral messenger RNA migrates into the cytoplasm, When looking for antivirals: [Trans 2027] where building blocks for a new virus are synthesized ○ Agent should be able to inhibit the transcription or Some of them have to be processed by the viral protease translation process unique to the virus ○ Protease cleaves longer proteins into smaller core If it is a general process (ex. DNA replication), proteins the host’s DNA replication will also be Crucial step in creating an infectious virus inhibited → killing both the virus and the host Two viral RNA strands and replication enzymes come cell together and core proteins assemble around them ○ Vital to know the pathway of viral entry in order to forming a capsid identify the unique point of virus-host interaction ○ Immature viral particle leaves the cell requiring a new envelope of host and viral proteins ○ Virus matures and becomes ready to infect others HIV replicates billions of times per day, destroying the host’s immune cells and eventually causing disease progression Drugs which interfere with the key steps of viral replication can stop this fatal process Page 9 of 15 | TH: DIONISIO | BSR/PSYCH TG 4 | CRISOSTOMO, MARTINEZ, SALANGUIT  BSR 1.04 Viral Invasion of Host Cells ○ Entry into the host cell can be blocked by fusion The progeny virus assembles in the cytoplasm or inhibitors nucleus before it is released as an infectious virus particle ○ Inhibition of reverse transcriptase by nucleoside inhibitors or by non-nucleoside reverse transcriptase inhibitors is part of standard antiretroviral regimens ○ Action of integrase can be blocked ○ Protease inhibitors are part of standard antiretroviral therapy ○ Each blocked step in viral replication is a step towards better control of HIV disease V. VIRAL PENETRATION DNA VIRAL PENETRATION [Trans 2027] Viral genetic material will find its way to nucleus to replicate Once replicated, it can now assemble a provirus that will be released as viral particles RNA VIRAL PENETRATION [Trans 2027] Figure 23. Replication of an enveloped and plus-strand RNA virus All processes occurs in the cytoplasm (+) ssRNA viruses: directly proceeds to translation 1. After the virus attaches to a cellular receptor, fusion (-) ssRNA viruses: RNA replication first takes place of the virus envelope with the cell membrane or ○ Synthesized complementary RNA will be the endocytic vesicle releases the nucleocapsid into the template for protein synthesis cytoplasm 2. Viral mRNA is immediately translated on cytoplasmic ribosomes into the proteins required for RNA synthesis, because it is a positive-strand RNA virus. 3. The synthetase complex can both replicate the RNA to produce new genomes and synthesize viral subgenomic mRNAs from a minus-strand copy of the genome 4. The viral structural proteins are then translated from these subgenomic mRNAs 5. In the final maturation step, the nucleocapsid buds out through areas of modified membrane to release the enveloped particle Figure 22. General replication scheme for DNA virus TG Note: How doc explained this figure: Positive-stranded RNA such as the dengue virus infects 1. After a DNA virus attaches to a cellular membrane cells via a receptor Viral genome is released in the cytoplasm and mRNA is receptor, the virus DNA enters the cell and is immediately translated into proteins. transported to the cell nucleus Viral proteins assemble in the membrane forming the virus ○ Transcribed into mRNA by host RNA polymerase progeny, which is released into the environment 2. Viral mRNA are translated by host ribosomes in the cytoplasm and newly synthesized viral (structural and nonstructural) proteins are transported back to the nucleus 3. After the DNA genome is replicated in the nucleus either by the host DNA polymerase or by a new viral-encoded polymerase, progeny virus particles are assembled and ultimately released from the cell TG Note: How doc explained this figure: Depending on the genetic material, the DNA virus can replicate inside the nucleus then it undergoes transcription and translation Page 10 of 15 | TH: DIONISIO | BSR/PSYCH TG 4 | CRISOSTOMO, MARTINEZ, SALANGUIT  BSR 1.04 Viral Invasion of Host Cells the cytoplasm 4. Capsid precursor, protein, “Gag,” and RT are transported to the cell plasma membrane 5. Immature virions containing Gag, RT, and the genome RNA assemble near the modified cell membrane 6. The final maturation step involves proteolytic cleavage of Gag by the viral protease and budding to produce enveloped particles How doc explained this figure: Retroviruses are another class of viruses which has RNA as their genetic material: ○ RNA is first transcribed back into DNA via reverse transcription before it is transcribed into RNA and translated into viral protein Figure 24. Replication of a typical minus-strand RNA virus Basic dogma of molecular biology: Specific enzymes 1. After virus attaches to host cell, the nucleocapsid is make each virus unique released into the cytoplasm ○ By identifying the structure of the different 2. The mRNAs will be synthesized and are translated molecules, function can be elucidated into the viral proteins required for the synthesis of ○ Understanding the mechanism of action, able to the full-length complementary RNAs (vcRNAs) devise strategies on how to control if not prevent 3. These vcRNAs are the templates for minus-strand virus infection genome RNA synthesis 4. Viral mRNAs are also translated into membrane REFERENCES glycoproteins that are transported to the cell Ronald Matias MD. Notes from Virus Invasion of Host membrane Cells. 2027 Trans (2023). Virus Invasion of Host Cells. How doc explained this figure: For a negative-strand RNA virus, the RNA must first be BOOKS replicated into a positive-strand RNA. After this, the Moore, K.L., Dalley, K.F., Agur, A.M. (2017) virus proceeds with replication and protein synthesis in the same manner as a positive-strand RNA virus. ONLINE SOURCES Heinz, F.X. & Stiansy, K. (2012). Flavivirus and their antigenic structure. Journal of Clinical Virology. https://www.sciencedirect.com/science/article/abs/pii/ S1386653212003435. Accessed on 08/2024. Wang, Y. & Xiang, Y. (2020). Spike glycoprotein-mediated entry of SARS Coronaviruses. MDPI. https://www.researchgate.net/publication/346 851784_Spike_Glycoprotein-Mediated_Entry_of_SAR S_Coronaviruses. REVIEW QUESTIONS VIRUSES Figure 25. Replication of a retrovirus 1. Viruses can be divided into three broad classes based 1. After entering the cell, the retrovirus RNA genome is on their genome. The following are the said classes reverse transcribed into double-stranded DNA by RT except: present in the virion A. Plasmid viruses 2. DNA copy migrates to the cell nucleus and integrates B. DNA viruses into the host genome as the “provirus” C. RNA viruses 3. Viral mRNAs are transcribed from proviral DNA by D. Viruses that use reverse transcriptase host cell enzymes in the nucleus. Both spliced and 2. Which enzyme is used to convert RNA back to DNA? unspliced mRNAs are translated into viral proteins in A. RNA transcriptase Page 11 of 15 | TH: DIONISIO | BSR/PSYCH TG 4 | CRISOSTOMO, MARTINEZ, SALANGUIT  BSR 1.04 Viral Invasion of Host Cells B. DNA transcriptase A. Ebola virus C. Reverse transcriptase B. Dengue virus D. RNA convertase C. Coronavirus 3. DNA viruses specifically Poxviruses replicate in which D. Influenza virus organelle: 12. Class II fusion proteins have both α-helices and A. Cytoplasm β-sheets. (T/F) B. Nucleus C. Ribosome VIRAL REPLICATION D. SER 13. In the replication process of the dengue virus, an INITIATION OF INFECTION endosome increases the pH and changes its arrangement to spike-like structures. Its tips are 4. The following mechanisms drive viral collision except: hydrophilic which allows them to penetrate the A. Brownian motion endosomes membrane and bend until its fusion B. Laws of diffusion A. First statement is correct, while the second C. Electrostatic interaction statement is wrong D. Flagellar locomotion B. First statement is wrong, while the second 5. Viral cells do not have specific receptors and will statement is correct infect all cells in the body. (T/F) C. Both statements are correct 6. This type of receptor is needed for viral entry. D. Both statements are wrong A. High Affinity Receptor 14. Positive strand RNA can be directly translated, unlike B. Low Affinity Receptor a negative strand RNA. (T/F) C. Accessory Receptor 15. All DNA viruses replicate in the nucleus except for:** D. Modified Receptor A. Paramyxoviridae B. Poxviruses REPLICATION CYCLE OF VIRUSES C. Flaviviruses D. Arboviruses 7. In influenza virus, how does virus bind to the host? A. When nucleocapsid is cleaved *** End of Review Questions *** B. When neuraminidase cleaves sialic acid from galactose TG Note: All questions with “*” are lifted from 2027 Trans C. Through hemagglutinin RATIONALE D. When the globular head dissociates 8. Which of the following statements regarding SARS-CoV-2 spike proteins are false? 1. [A]. Recall A. It has 2 subunits 2. [C]. Recall B. Its subunits are similar to influenza virus’ globular head and fibrous stem 3. [A]. Most DNA viruses replicate in the nucleus, but for C. Both of its subunits contain beta sheet structure Poxvirus, it specifically replicates in the cytoplasm D. S1 subunit contains the receptor binding domain where ACE2 enzyme will bind 4. [D]. Viruses are inanimate and does not have their own 9. What is the portion of the RBD that can be structure for locomotion recognized by the body to elicit immune response? A. ACE2 5. [F]. Viruses exhibit specificity wherein its configuration has to B. Neutralizing epitope match that of the surface receptor to initiate infection and enter the cell. Thus, not being able to infect all cells in the body C. Amino acid D. β-sheets 6. [A]. High affinity receptor is needed for viral entry since cells that do not have the appropriate receptor cannot be infected by VIRAL FUSION PROTEINS the virus whereas accessory receptor is not required but may accelerate the uptake of the virus. 10. What occurs when fusion protein bends and pulls the membrane close together? 7. [C]. Recall A. Hairpinning B. Neutralization 8. [C]. S1 subunit: β-sheet configuration; S2 subunit: α-helix configuration C. Viral attachment D. Cleavage 9. [B]. Recall 11. The following viruses have class I fusion proteins, except:** Page 12 of 15 | TH: DIONISIO | BSR/PSYCH TG 4 | CRISOSTOMO, MARTINEZ, SALANGUIT  BSR 1.04 Viral Invasion of Host Cells 10. [A]. Recall Receptor Receptor Binding 11. [B]. Dengue virus is a class II fusion protein Binding affinity Low High Virus Entry Not Required Required 12. [F]. Recall How does viral attachment occur? 13. [D]. lowers the pH, hydrophobic Upon collision, electrostatic interactions will bring the virus in proximity with the probable host cell 14. [T]. Negative strand must be converted first to positive ○ Leads to binding of the receptor to the viral strand before it can be translated protein 15. [B]. Poxviruses replicates in the cytoplasm ○ For influenza, the terminal sialic acid residues will bind to the HA protein of the virus SUMMARY ○ For SARS-CoV-2, What are viruses? the ACE2 protein binds to the receptor binding Organized association of macromolecules (nucleic domain of the corona spike protein acids and proteins) RBD configuration changes from down to up Needs a host cell for replication → receptor binding site gets exposed → What are the three broad classes of viruses? ACE2 enzyme binds with RBD → viral infection Needs a host cell for replication initiated ⭐ Groups Example What is the receptor for Influenza virus? ssRNA-RT Retroviruses (HIV) Sialic Acid dsDNA-RT Hepadnaviruses (Hepatitis B) ○ N-acetylneuraminic acid (for Influenza A,B) ○ 9-O-acetyl-N-neuraminic acid (for Influenza C) How does viral infection initiation occur? ○ ɑ(2,6) - human strains; ɑ(2,3) - avian strains Collision of a virion with a potential host cell must ○ Neuraminidase - cleave sialic acid from occur because virions are: galactose ○ Inanimate What are the steps in fusion of Influenza virus with the ○ Driven by Brownian motion, laws of diffusion, host cell membrane? electrostatic interaction Protein binds to receptor → endosome formation → ○ Obligate intracellular parasites H+ influx leads to conformational change → ○ Too large to diffuse across the plasma globular heads bend → penetration and hairpinning membrane occur → fusion of viral and host cell membranes Step 1: Adhere to cell surface (by electrostatic interaction) What are the different classes of viral fusion proteins Step 2: Attach to specific receptor on the cell Class I Class II Class III surface Perpendicular to Parallel to cell Perpendicular to Step 3: Transfer genome inside the cell cell membrane membrane cell membrane Mix of both ɑ helix Mostly ɑ helix Mostly β sheets What are cellular receptors for viruses? and β sheets Form Trimers Form Dimers Form Trimers Essential for all viruses, EXCEPT yeast and plants Flavivirus (Dengue Rhabdoviridae & Influenza Virus VIRUS RECEPTOR & Japanese Herpesviridae SARS-CoV-2 Different viruses binding to same receptor Encephalitis) Adenovirus SAME Coxsackievirus B3 Swine herpesvirus Pseudorabies virus SAME Human poliovirus Viruses from same family binding to different receptors Rhinoviruses (3) DIFFERENT Retroviruses (16) DIFFERENT What is the difference between accessory, receptor, and high affinity receptor binding? Accessory High Affinity Page 13 of 15 | TH: DIONISIO | BSR/PSYCH TG 4 | CRISOSTOMO, MARTINEZ, SALANGUIT  BSR 1.04 Viral Invasion of Host Cells FREEDOM WALL individuals Table 8. Group 2: ssDNA viruses Family Virus Disease Respiratory disease Bocavirus Diarrhea in children Parvoviridae Transient hemolytic Human anemia parvovirus Aplastic anemia Table 9. Group 3: dsRNA viruses Family Virus Disease Gastroenteritis Human Reoviridae Diarrhea rotavirus Table 10. Group 4: (+)ssRNA viruses Family Virus Symptoms Flu-like illness Hand-foot-mouth disease Coxsackievirus Myocarditis Meningoencephalitis APPENDIX Hepatitis GROUPS OF VIRUSES Picornaviridae Enterovirus* Flu-like illness Table 7. Group 1: dSDNA viruses Aseptic meningitis Family Virus Disease Echovirus Severe myocarditis Infections of the Hepatitis respiratory tract Poliovirus Polio Human (pharyngitic, Adenoviridae Adenovirus pneumonic) Rhinovirus Common cold Conjunctival Togaviridae Fever Gastroenteritis Chikungunya Alphavirus* Severe joint pains Mononucleosis and Togaviridae German measles Neoplasms Rubella Rubivirus* Hodgkin lymphoma Coronaviridae SARS virus Burkitt lymphoma Epstein-Barr Nasopharyngeal Acute and chronic carcinoma hepatitis Hepatitis C virus CNS lymphoma Hepatocellular Various autoimmune carcinoma Herpesviridae diseases Mild fever Herpes Cold sores Dengue Flaviviridae simplex type Dengue virus hemorrhagic fever 1 Dengue shock Herpes Genital herpes syndrome simplex type Japanese Encephalitis 2 encephalitis Varicella- Chickenpox virus Zoster virus Astroviridae Astrovirus Gastroenteritis Warts *is a genus and not a family Human Papillomavirid Genital warts papilloma ae Squamous cell Table 11. Group 5: (-) ssRNA viruses virus carcinoma Family Virus Disease Progressive multifocal Encephalitis JC polyoma Polyomaviridae leukoencephalopathy in Paramyxoviridae Nipah virus virus immune compromised Page 14 of 15 | TH: DIONISIO | BSR/PSYCH TG 4 | CRISOSTOMO, MARTINEZ, SALANGUIT  BSR 1.04 Viral Invasion of Host Cells Measles virus Measles Mumps virus Mumps Mild respiratory Parainfluenza infection Respiratory Pneumonia Pneumoviridae Syncytial Virus Rabies Rhabdoviridae Rabies virus Hydrophobia Filoviridae Ebolavirus Hemorrhagic fever Hemorrhagic fever Bunyaviridae Hantavirus with renal syndrome Orthomyxoviridae Influenza Table 12. Group 6: ssRNA-RT viruses with DNA intermediate Family Virus Disease Adult T-cell leukemia/lymphoma Human T-cell Human myelopathy Retroviridae lymphotropic virus condition (Retrovirus) (HTLV-1) Tropical spastic paraparesis Human AIDS Immunodeficiency virus (HIV) Table 13. Group 7: dsDNA - RT virus Family Virus Disease Acute hepatitis Chronic hepatitis Hepadnaviridae Hepatitis B Cirrhosis Hepatocellular carcinoma Page 15 of 15 | TH: DIONISIO | BSR/PSYCH TG 4 | CRISOSTOMO, MARTINEZ, SALANGUIT