Lecture 3 (cont.) Study Notes

Broad Overview of Viral Entry & Replication

• Speaker delivers a “broad-strokes” synopsis of how diverse viruses reach, penetrate, and replicate inside host cells.

• Emphasis: Receptor binding alone is sometimes sufficient, but many viruses need an extra lysosomal/endosomal step before genome release.

• Viral entry typically involves binding to receptors on the plasma membrane, which can trigger the formation of a clathrin-coated pit for subsequent internalization.

• After internalization, the virion enters an endosome that undergoes acidification (driven by ATP hydrolysis, $\text{ATP} \rightarrow \text{ADP} + \text{P}_{\text{i}}$), potentially progressing to a lysosome, before the viral genome is released.

• Once inside the cell, viruses utilize microtubules for intracellular transport:

  • Dynein motors move cargo from the plus (+) end to the minus (-) end (towards the nucleus), consuming ATP.

  • Kinesin motors move cargo from the minus (-) end to the plus (+) end (away from the nucleus), consuming ATP, ensuring directed movement of viral components.

Plasma-Membrane Attachment & Receptors

• All viruses covered bind an initial cellular receptor located on the plasma membrane.

  • Example receptors given:

  • $\text{sialic-acid}$ residues (influenza HA1 binds here).

  • $\alpha5\beta2$ or $\beta5$ integrins (classical icosahedral virus example—likely adenovirus).

  • For viruses such as the Paramyxoviridae, as depicted in the diagram, their surface fusion proteins, specifically F1 and F2 subunits, engage with host receptors. The fusion peptide within these proteins plays a crucial role in mediating the process.

• Binding consequences:

  • For many viruses, receptor binding triggers clathrin-coated pit formation, leading to internalization.

  • In the case of viruses like Paramyxoviridae, engagement with the receptor on the plasma membrane can lead to immediate membrane fusion, allowing the viral genome to directly enter the cytoplasm, while the capsid may remain outside the cell. The diagram illustrates this direct entry of the viral genome into the host cell.

Endocytosis

• After internalization the particle sits in an early endosome that progressively acidifies. This acidification is driven by V-type ATPase pumping $H^+$ ions inward ($\text{ATP} \rightarrow \text{ADP} + \text{P}_{\text{i}}$).

• pH drop (\approx 5–6) creates conformational changes in viral surface proteins.

  • Influenza HA: HA1 moves, exposing HA2 fusion peptide. As depicted, after HA1 adhesion to sialic acid and clathrin-mediated internalization, the virus is in an endosome.

  • Corona-/paramyxo-like SU \rightarrow TM rearrangement: SU “lifts away,” TM fusion peptide deploys.

  • Class IV (+)-Strand RNA Viruses (e.g., Semliki Forrest Virus): As illustrated, after receptor binding and endocytosis, the virus enters an endosome. Upon endosomal acidification, the viral capsid undergoes pH-triggered disassembly, allowing the $+\text{RNA}$ viral genome to be released directly into the cytosol, where it is immediately available for translation by host ribosomes.

  • Poliovirus: As shown in the diagram, Poliovirus first binds to a receptor on the plasma membrane. It is then internalized into an endosome, which acidifies. Within the endosome, a pore-forming event occurs in the endosome membrane, involving viral proteins like VP4 (and potentially VP2/VP3). This pore allows the (+)RNA genome, which is covalently linked to VPg, to exit the capsid and be released directly into the cytoplasm.

• Some viruses require an additional lysosomal protease step before uncoating (L3 protease example).

Lysosomal Uncoating (Reovirus Example)

• As shown in the diagram, Reovirus, initially comprised of outer layers ($\mu1, \sigma3$), an intermediate layer ($\sigma2$), and an inner core encasing its genome, enters the host cell and progresses to the lysosome.

• Within the lysosome, the acidic environment and host proteases facilitate the removal of the outer capsid layers ($\mu1$ and $\sigma3$).

• This uncoating process results in the formation of an Intermediate Subviral Particle (ISVP), which consists primarily of the $\sigma2$ layer and the viral genome. This ISVP (or further altered particle) then releases the genome into the cytoplasm for replication.

Microtubule-Based Intracellular Transport

• Viruses are too small for “random diffusion”; they hijack cytoskeletal motors.

  • Microtubule polarity: $\text{minus (–)}$ anchored near nucleus, $\text{plus (+)}$ toward plasma membrane.

  • Motor usage:

  • Dynein: plus \rightarrow minus (retrograde); pulls virions to nucleus.

  • Kinesin: minus \rightarrow plus (anterograde); used for egress or peripheral trafficking.

• Analogy: Mitochondria distribution is similarly “non-random” and microtubule-guided—underscores ordered intracellular logistics.

Social Context Mentioned

• Lecturer notes rise in anti-vaccine sentiment \rightarrow lowered childhood vaccination rates \rightarrow re-emergence of preventable infections.

  • Serves as real-world relevance for viral entry/epidemiology.

Influenza Virus (Orthomyxovirus) Specifics

• Structure

  • Matrix protein $M1$ lines inner envelope.

  • Ribonucleoprotein (RNP) contains genome + nucleocapsid protein (NP).

  • Envelope glycoprotein Hemagglutinin (HA) split into two chains:

  • HA1 = receptor binding; masks fusion potential.

  • HA2 = holds fusion peptide (becomes exposed at low pH).

• Entry Sequence

  1. HA1 attaches to sialic acid on host cell.

  2. Clathrin-coated vesicle internalizes virion.

  3. Endosomal acidification (mediated by $\text{M2}$ proton channel) \rightarrow HA conformational change.

  4. HA2 inserts fusion peptide into endosomal membrane \rightarrow membrane fusion \rightarrow RNP release, along with M1 monomers, into the cytoplasm. The RNP then moves towards the nucleus.

Classical Icosahedral Virus (Adenovirus-like) Example

• Capsid: textbook icosahedral architecture.

• Entry Pathway:

  • Initial Attachment: The virus binds to its primary receptors, such as $\alpha5\beta2/\beta5$ integrins, and also interacts with MHC or CAR molecules on the host cell surface, as depicted in the diagram.

  • Internalization: Following attachment, the virus is internalized into an endosome.

  • Endosomal/Lysosomal Maturation: The endosome progressively acidifies (driven by V-type ATPase pumping $H^+$ ions inward via $ATP \rightarrow ADP + P_i$), and the virus interacts with the lysosome. This acidic environment is critical for uncoating.

  • L3 Protease: This protease is activated by the acidic environment of the lysosome (as suggested by the $ATP$ dependent acidification in the endosome). Its activation correlates with lysosomal membrane disassembly, which facilitates the capsid's escape into the cytoplasm. Direct biochemical proof of membrane targeting by L3 is suggested but unconfirmed.

  • Microtubule Transport: Once in the cytoplasm, the virus hijacks host cytoskeletal motors. As illustrated, it uses dynein (a minus-end directed motor, consuming $ATP$ to $ADP+P_i$) to transport retrogradely along microtubules towards the nucleus.

• Genome Import:

  • The viral dsDNA genome enters the nucleus through the nuclear pore. As shown in the diagram, the virus lacks its own NLS but piggy-backs on host proteins (e.g., host histone H2 which carries an NLS, labeled "H2-NLS") to facilitate its passage into the nucleus. The nuclear pore complex, including nucleoporins like NUP214 (indicated in the diagram), is utilized for this import.

  • Uses a protein primer (“terminal protein”) covalently bound to the 5′ ends of its dsDNA genome to initiate DNA replication.

Class IV (+)-Strand RNA Virus (Picorna/Rhino-like) Section

• Defined as “Class 4” (Baltimore) \rightarrow $\text{+RNA}$ genome is directly infectious & translated.

• Cytoplasmic life cycle — no nucleocytoplasmic transport.

• Capsid undergoes additional pH- or receptor-triggered disassembly inside late endosome/lysosome \rightarrow RNA enters cytosol.

Coronavirus / Paramyxovirus-Style Fusion Notes

• Spike (S) glycoprotein organized into two functional subunits:

  • SU (a.k.a. S1) \rightarrow receptor attachment.

  • TM (a.k.a. S2) \rightarrow houses latent fusion peptide.

• Triggering event: conformational change in SU exposes TM fusion peptide \rightarrow inserts into host membrane.

• Fusion often requires \ge2 receptor engagements (co-receptor or dimerization requirement hinted).

• “Cyclotherm A” (likely cycloheximide or a cyclophilin inhibitor) can bind capsid proteins and modulate fusion efficiency.

Mechanisms Requiring Host Components (e.g., HIV)

• As shown in the diagram, certain enveloped viruses (like HIV, with its Reverse Transcriptase (RT), Integrase (INT), and two copies of (+)-RNA genome) exhibit entry and uncoating mechanisms highly dependent on host factors.

• Entry and Fusion: The virus attaches to host cell receptors, specifically CD4 and co-receptors (e.g., CCR4/CXCR4) on the plasma membrane. This interaction triggers direct fusion of the viral envelope with the plasma membrane, releasing the viral capsid/core directly into the cytoplasm.

• Uncoating and Host Factor Involvement: Once in the cytoplasm, the capsid undergoes uncoating, a process critical for genome release. Host components, such as cyclophilin A, are shown associated with the capsid and are essential for this initial uncoating step.

• Reverse Transcription: Following uncoating, the (+)-RNA genome is released, and the viral Reverse Transcriptase (RT) begins synthesizing a dsDNA copy of the genome, which is a prerequisite for subsequent integration into the host genome.

Nuclear Localization Signal (NLS) & Ran GTPase Review

• NLS

  • Short stretch of basic aa (e.g., PKKKRKV); either monopartite (one cluster) or bipartite (two clusters separated by 10–12 aa).

• Nuclear import sequence

  1. Cargo+NLS binds importin-$\alpha/\beta$ \rightarrow nuclear pore complex (NPC).

  2. Ran-GTP inside nucleus binds importin-$\beta$ \rightarrow cargo released.

  3. Export of importin-$\beta$-Ran-GTP \rightarrow cytoplasm.

  4. Ran GAP + RanBP1 + RanBP2 stimulate $\text{Ran-GTP} \rightarrow \text{Ran-GDP} + P_i$ .

  5. Ran-GDP returns to nucleus aided by NTF2 \rightarrow recharged to Ran-GTP by RCC1 (nuclear GEF).

• Viral genomes/proteins exploit this cycle for entry without coding their own import machinery.

Nuclear Export Signal (NES) Mention

• Leucine-rich NES mediates export via CRM1/Exportin-1; inhibition (e.g., leptomycin B) impedes viral RNP egress (implied but not extensively discussed).

Additional Technical/Molecular Details

• Tubulin dimer ratio \approx $1\,\alpha:!1\,\beta$ (transcript mis-states “2 to 1”).

• Microtubule polymerization mathematically represented:

  $\text{n\,}(\alpha\beta) \rightarrow (\alpha\beta)_n$ (plus-end elongation dominant).

• Stoichiometry of dynein/kinesin step size: ~8 nm per ATP hydrolyzed (not directly stated but helpful context).

• Lysosome acidification requires V-type ATPase pumping $H^+$ ions inward (ATP-dependent step referenced).

• Protein primer mechanism analogous to adenovirus pTP which uses serine O-link to 5′ dCMP of genome.

Ethical / Practical Implications

• Anti-vaccine trends can restore transmission routes for the very viruses under mechanistic discussion.

• Understanding entry pathways aids antiviral drug design (e.g., HA-inhibitors, integrin antagonists, fusion peptide blockers, V-ATPase inhibitors).

Conceptual Connections to Previous & Future Lectures

• Builds on earlier influenza session (HA cleavage, M2 ion channel, nuclear cap snatching).

• Foreshadows upcoming “structure chapter” where detailed polymerase/primer interactions will be covered.

• Upcoming coronavirus module will revisit SU/TM rearrangements, endocytosis vs. direct fusion.

Key Take-Home Points

• Receptor engagement \rightarrow endosomal maturation \rightarrow pH/enzymatic trigger \rightarrow membrane fusion/uncoating is the recurring theme.

• Cytoskeleton hijacking is essential for both inbound and outbound traffic.

• Viruses creatively exploit host nuclear transport machinery without encoding complete pathways themselves.

• Protein versus RNA primers exemplify the diverse replication strategies even among DNA viruses.

• Societal factors (vaccination rates) directly influence the epidemiology of the molecular events under study.