Viral Pathogenesis, Attachment, and Entry (Vocabulary Flashcards)

Introduction

• Topic: Viral Pathogenesis, Attachment, and Entry (MIIM-512/MIIM-540)
• Author/Instructor: Dr. Michael Nonnemacher, Drexel University College of Medicine
• Purpose: Foundations of how viruses cause disease, how they attach to and enter host cells, and initial uncoating steps
• How to complete this topic (as per slides):
  • Read carefully through each section and take notes
  • Watch available videos and audio recordings
  • Complete the Check Your Understanding questions (periodically included; not graded)
  • Post questions/comments to the course forum
• Topic Organization (five sections):
  • 1) Introduction
  • 2) Viral Pathogenesis, Attachment and Penetration
  • 3) Uncoating
  • 4) [Let’s begin with viral pathogenesis] – broader context
  • 5) Summary/References (credits at end)

Learning Objectives (Section 2)

• Outline the seven steps to viral pathogenesis: 7 steps
• Define and illustrate key concepts: tropism, attachment vs entry, receptor/coreceptor interactions, and avidity
• Compare and contrast entry strategies for enveloped vs non-enveloped viruses
• Describe the three common uncoating strategies and their cellular locations
• Identify roots of viral entry into the body and factors affecting transmission
• Distinguish among abortive, lytic, and persistent infections (including the four types of persistent infections) and how susceptibility and permissiveness influence outcomes
• Connect determinants of cytopathic outcomes to viral pathogenesis
• Recognize common experimental readouts: eclipse period, plaque assays, and cytopathic effects
• Appreciate the immune response and viral immune evasion, and how inflammation can contribute to disease (immunopathology)

Basic Progression of Viral Disease (seven steps)

• Seven basic steps of viral disease progression: 7 steps
  1) Acquisition: entry of the virus into the host, crossing natural barriers
  2) Initiation of Infection: binding and entry into the primary host cell; driven by viral tropism (tissue specificity)
  3) Incubation Period: virus amplification; may be asymptomatic, prodrome, or symptomatic; possible secondary spread
  4) Replication: replication of genome and viral proteins; expansion of infection and symptom development
  5) Immune Response: innate/adaptive responses begin; inflammation can contribute to disease depending on virus strain/type
  6) Contagion: spread of virus within tissue and to other tissues/hosts
  7) Resolution: clearance or progression to chronic/latent infection
• Acquisition details: virus enters via natural barriers; skin breaks and inhalation are common routes; mucosal membranes provide protective barriers (tears, mucus, ciliated epithelia)

Acquisition & Initiation of Infection

• Inhalation and skin breaches are common routes for infection
• Skin as barrier; breaks (visible or not) enable entry
• Mucosal routes have protective features (tears, mucus, ciliated epithelia)

General Concepts in Virus Replication (overview of steps)

• Virus replication steps (general concepts):
  • Attachment (also adsorption)
  • Penetration (membrane crossing)
  • Uncoating (capsid disassembly to expose genome)
  • Macromolecular Synthesis: transcription/translation/programmed gene expression
  • Viral mRNA synthesis (if needed)
  • Viral protein synthesis
  • Viral genome replication
  • Assembly
  • Release and Maturation
• Virus entry comprises three sub-steps:
  • Attachment to target cell
  • Penetration across plasma membrane
  • Uncoating and genome access for replication
• Attachement specifics (enveloped vs non-enveloped):
  • Enveloped viruses use viral glycoproteins/spikes in the envelope to attach
  • Non-enveloped viruses use capsid proteins and sometimes spikes/fibers (e.g., adenoviruses)
  • Attachment is receptor- or co-receptor-mediated; defines tropism
  • Important distinction: attachment does not guarantee entry (e.g., HIV may attach to CD4 but requires a coreceptor for penetration/uncoating)

Attachment

• Attachment is also called adsorption
• Determinants of attachment/tropism:
  • Viral surface proteins interact with host cell-surface receptors and co-receptors
  • Tropism is the specificity for a host tissue based on these interactions
• Enveloped viruses: attachment via viral glycoproteins/spikes in envelope
• Non-enveloped viruses: attachment via capsid proteins and/or spikes/fibers
• Key examples:
  • HIV-1: attachment to CD4 receptor via gp120; requires coreceptor (e.g., CCR5 or CXCR4) for penetration
  • Influenza A: HA (hemagglutinin) binds sialic acid on epithelial cells; receptor is a carbohydrate (not a protein)
  • Poliovirus: binds CD155 via a canyon on the capsid
  • Adenovirus: fiber knob binds CAR (coxsackievirus and adenovirus receptor); may require coreceptors such as α5β1 or α5β3 integrins for endocytosis
• Receptors vs co-receptors: coreceptors assist entry; ubiquity of receptor affects tropism and pathogenesis
• Receptor examples and concepts:
  • HIV: CD4 receptor + coreceptors (CCR5 or CXCR4)
  • Influenza: sialic acid; linkage type influences host range (α2,3 vs α2,6)
  • Adenovirus: CAR receptor; integrins assist internalization
  • Poliovirus: CD155 receptor with canyon binding
• Receptor characteristics:
  • Influenza HA recognizes sialic acid-linked receptors; receptor type can be carbohydrate rather than protein
  • Avidity: overall strength of virus–cell binding; affected by number of receptors and number of binding sites per glycoprotein; contributes to stable attachment
• HIV attachment video (conceptual): gp120 binds CD4; conformational changes permit coreceptor binding; gp41 fusion peptide insertion leads to membrane fusion

Penetration

• Penetration is the translocation of the virion across the plasma membrane
• Common mechanisms:
  • Receptor-mediated endocytosis
  • Fusion with the plasma membrane
• Endocytosis and fusion can be clathrin-dependent, caveolin-dependent, or clathrin/caveolin-independent depending on cell type and virus
• The role of receptor engagement in determining entry route (e.g., HIV in T cells vs. macrophages; influenza endocytosis via clathrin-coated pits)

HIV Penetration

• After gp41/hairpin conformational changes and fusion pore formation, the viral nucleocapsid enters the cytoplasm
• Uncoating occurs after fusion; capsid phosphorylation (potentially by PKA) and cyclophilin A facilitate uncoating and reverse transcription
• Three competing models for timing of uncoating in HIV:
  • Model 1: Uncoating proximal to the plasma membrane immediately after fusion
  • Model 2: Gradual uncoating during transport to the nucleus, linked to reverse transcription
  • Model 3: Uncoating at the nuclear pore after reverse transcription is complete

Endosomal/Plasma Membrane Uncoating Concepts

• Endocytosis steps (clathrin-mediated and endosomal maturation):
  • Clathrin-coated pit internalizes virus into an early endosome (pH ≈ 6.5–6.0)
  • Maturation to late endosome (pH ≈ 6.0–5.0) and fusion with lysosomes
• Endosomal pH and uncoating cues help determine viral genome accessibility
• Uncoating at plasma membrane vs endosome vs nuclear membrane depends on virus

Uncoating Within Endosomes in the Cytoplasm (Influenza example)

• Influenza entry: HA binds sialic acid; endocytosis (clathrin-coated vesicles common)
• Step 1: Endosome acidification (pH drops to ~5) triggers HA conformational change and fusion peptide exposure, driving fusion of viral and endosomal membranes
• Step 2: M2 proton channel pumps protons into virion; acidification primes vRNP dissociation from M1
• Step 3: Viral ribonucleoproteins (vRNPs) are released and imported into the nucleus via nuclear pore complex (NPC) using nuclear localization signals
• Key note: Influenza has a relatively low pH threshold for uncoating; uncoating occurs in late endosomes

Uncoating at the Nuclear Membrane (Adenovirus example)

• Adenovirus binds CAR via fiber; penton base interacts with integrins to trigger endocytosis
• Endosomal escape triggers release of protein VI; hydrophobic N terminus disrupts endosome membrane
• Capsid transported along microtubules to the nuclear pore complex
• Genome delivered to the nucleus; uncoating occurs at the nuclear membrane

Macromolecular Synthesis (overview)

• Second major step in replication: synthesis of macromolecules
• Six major types of macromolecular synthesis defined by genome type (viruses vary by genome type entering the cell)
• Key processes:
  • Viral mRNA synthesis (where needed)
  • Translation of viral proteins
  • Replication of viral genome
  • Regulation and timing depend on virus type

Assembly, Release and Maturation

• Final stages: assembly of virions and release/maturation
• Assembly often occurs spontaneously in many viruses
• Release mechanisms vary by virion type:
  • Non-enveloped viruses: typically released by cell lysis
  • Enveloped viruses: bud from the plasma membrane or via exocytosis; may involve a cell-associated phase
• Post-release maturation: additional maturation steps may occur, often requiring viral proteases packaged with the virion
• Budding overview (visualized in lecture): glycoprotein spikes insert into membrane; matrix proteins guide nucleocapsid to budding site; virus buds and may remain cell-associated

One-Step Growth Curve and Plaque Assay (infection quantification)

• One-step growth curve (single cell infected): eclipse period follows attachment when the virus is replicating but not yet producing extracellular particles; a later burst yields 100s–1000s of particles per cell
• Not all particles are infectious; many are defective
• Plaque assay: serial dilutions of virus plated on a confluent cell monolayer; each infectious particle creates a plaque by killing surrounding cells
• Plaques are counted to estimate infectious titer; multiply by dilution factor to express infectious units per volume

Cytopathic Effects (CPE) and Examples

• Cytopathic effects observed in infected cells include a range of morphological changes and inclusions
• Cowdry Type A intranuclear inclusions: HSV infection
• Negri bodies: Rabies infection (cytoplasmic inclusion bodies)
• Multinucleated giant cells (syncytia): common with enveloped viruses (e.g., SARS infection examples shown in slides)
• Macroscopic lesions: pox lesions, varicella, warts (examples of visible CPE)
• Mechanisms of cytolysis include: alteration of macromolecular synthesis, lysosome disruption, membrane perturbation by viral glycoproteins, viral protein toxicity, and induction of apoptosis/necrosis

Mechanisms of Viral Pathogenesis (general mechanisms)

• Common mechanisms include:
  • Altered cellular macromolecular synthesis and damaged DNA
  • Inhibition of host protein synthesis
  • Disruption of lysosomes and enzyme release
  • Altered membrane permeability via viral glycoproteins
  • Toxic effects of viral products (e.g., HIV gp120 promotes neuron apoptosis)
  • Chromosomal aberrations and enhanced necrosis
  • Induction of apoptosis or necrosis
• Immune-related aspects: immune responses can contribute to pathology (immunopathology) through inflammation and immune-mediated damage

Persistent Infection (four flavors)

• Four flavors of persistent infection:
  1) Chronic
  2) Latent
  3) Recurrent
  4) Transforming
• General types of persistent infection:
  • Chronic persistent infection: continual production of infectious virus with no obvious cytopathic changes (examples: retroviruses, hepatitis B virus, certain papovaviruses)
  • Persistent infection with continuous metabolic activity but no infectious virus: rare (Measles virus in CNS context)
  • Latent infection: no detectable infectious virus in cell-free material and little/no viral protein expression (classic example: HSV); can be reactivated to produce virus (recurrent infection)
  • Transforming infection: immortalization and oncogenesis; RNA viruses often productive; DNA viruses may not be infectious during transforming infection; mechanisms include activation of growth promoters, removal of growth suppressors, or inhibition of apoptosis

Mechanisms of Viral Transformation and Immortalization

• Balance of cellular growth activators (accelerators) and growth suppressors (e.g., p53, RB) governs cell cycle control
• Viruses can drive hyperproliferation and immortalization by:
  • Activating growth activators
  • Inactivating growth suppressors
• Consequences: increased cell growth, potential oncogenesis; transformation can occur via multiple pathways

Immune Response to Viral Infection

• Immune response comprises: innate (antigen-nonspecific) and adaptive (antigen-specific)
• Innate components: interferon, NK cells, macrophages
• Adaptive components: T cells, antibodies (humoral response)
• Viral immune evasion strategies and immunopathology are key to pathogenesis
• Immunopathology examples: interferon-driven systemic symptoms; delayed-type hypersensitivity T-cell responses; antibody-mediated effects (complement, ADCC, immune complexes)

Contagion and Transmission

• Contagion (virus transmission) involves virus shedding and release to sustain population-level infection
• Dead-end host concept: some infections do not transmit (e.g., rabies in humans)
• Shedding and release sites vary by infection: localized infections often shed from initial site; generalized/persistent infections may shed via blood, feces, urine, saliva, semen, genital secretions

Viral Epidemiology

• Modes of spread include aerosols, food, water, fomites, direct contact, sexual contact, birth, blood/blood products, organ transplants, zoonoses
• Five main factors affect transmission (cards explain each factor):
  • Stability of virion in the environment (e.g., drying, detergents, temperature)
  • Replication and secretion into transmissible aerosols/secretions
  • Disease and viral factors that promote transmission
  • Host factors (susceptibility, behavior, immune status)
  • Population and environmental context (crowding, social behavior)

Risk Factors for Infection and Transmission

• Age
• Health status and immune status
• Occupation (exposure to agent or vectors)
• Travel history
• Critical community size (seronegative populations)

Summary: Determinants of Cytopathic Activity and Outcomes

• Cytopathic activity determinants include: efficiency of replication, optimum replication temperature, cell permissiveness, cytotoxic viral proteins, disruption of macromolecular synthesis, accumulation of viral products/inclusions, altered cell metabolism (including immortalization)
• Types of infection outcomes (summary table):
  • Cytocidal (lytic): infectious virus produced and cell death; examples: HSV, picornaviruses
  • Abortive (non-permissive): no infectious virus produced; no effect on cell despite entry; no virus production
  • Persistent, productive: cell not killed and continues producing virus; examples: retroviruses, rabies virus
  • Persistent, nonproductive: cell not killed and no infectious virus produced; examples: Measles virus in SSPE (and other contexts)
  • Latent: no infectious virus detectable until reactivation; examples: VZV, HSV
  • Transforming (oncogenic): cell morphology changes, increased growth; examples: oncogenic DNA viruses (and some RNA oncogenic viruses)

Transmission Modes (Overview)

• Modes and examples (high-level):
  • Respiratory transmission: paramyxoviruses, influenza viruses, rhinoviruses, enteroviruses, etc.
  • Fecal-oral transmission: picornaviruses, rotavirus, reovirus, caliciviruses, norovirus, adenovirus
  • Contact transmission: HSV, rhinoviruses, poxviruses, adenovirus
  • Zoonoses (animals, insects): Togaviruses, flaviviruses, bunyaviruses, orbiviruses, arenaviruses, rabies, orf (pox)
  • Blood-borne: HIV, HTLV-1, HBV, HCV, HDV, CMV
  • Sexual transmission: herpesviruses, HPV, others
  • Maternal-neonatal transmission: rubella, CMV, B19, enteroviruses, HSV, varicella-zoster
  • Genetic (prions, retroviruses)

Uncoating: Endocytic Pathway, Plasma Membrane, and Nuclear Membrane Routes

• Recap of uncoating locations:
  • Endocytic pathway (endosomes): pH changes trigger uncoating for several enveloped viruses
  • Plasma membrane: direct fusion and genome release for some enveloped viruses (e.g., HIV)
  • Nuclear membrane: some viruses uncoat at or near the nuclear pore
• Influenza uncoating details (endosomal route):
  • HA binds sialic acids; clathrin-coated endocytosis common
  • Endosome acidifies (Step 1): pH drops to around ~5; HA conformational change drives fusion (fusogenic hairpin)
  • M2 proton channel (Step 2) acidifies virion interior; vRNP dissociates from M1
  • vRNP import into nucleus via NPC (Step 3)
  • Note: Influenza has a low pH threshold for uncoating; uncoating occurs in late endosome
• Adenovirus uncoating details (nuclear membrane):
  • Attachment via fiber to CAR; penton base interacts with integrins to trigger endocytosis
  • Low endosomal pH triggers release of protein VI, which disrupts endosome membrane
  • Capsid transported along microtubules to nuclear pore; genome delivered to nucleus
• HIV uncoating details (plasma membrane):
  • Fusion pore formation after gp120/gp41 interactions with CD4 and coreceptor
  • Capsid enters cytoplasm; uncoating likely involves capsid phosphorylation and cyclophilin A interactions
  • Competing timing models (three proposed): immediate near plasma membrane, gradual during transport/reverse transcription, or at nuclear pore after reverse transcription
• General endocytosis steps and endosome maturation recap:
  • Clathrin-coated pit forms vesicle -> early endosome (pH ~6.5–6.0) -> endosomal carrier vesicles -> late endosome (pH ~6.0–5.0) -> lysosome
  • pH changes and endosomal trafficking influence when and where uncoating occurs

Practical Concepts and Experimental Readouts

• Eclipse period (one-step growth curve): initial phase after infection during which no infectious virions are detected outside the cell, while intracellular components are synthesized
• Burst size: hundreds to thousands of virions per initially infected cell, many of which may be noninfectious
• Plaque assay: method to quantify infectious virus by counting plaques formed on a confluent cell monolayer; each plaque corresponds to infection initiated by a single infectious particle

Connections to Foundational Principles and Real-World Relevance

• Tropism and receptor usage link viral tissue specificity to disease manifestations
• Entry mechanisms influence pathogenesis, tissue tropism, and transmission potential
• Immune responses and immunopathology illustrate how host defense can contribute to disease symptoms
• Persistent and transforming infections connect to chronic disease states and cancer biology
• Understanding uncoating timing and routes informs antiviral strategies targeting entry or uncoating steps

Formulas and Numerical References (LaTeX)

• Seven steps in pathogenesis: 7 steps
• Endosomal pH ranges mentioned: pH \,\approx\,6.5\text{ to }6.0 (early endosome), pH\approx5.0 (late endosome)
• Endosomal maturation sequence and relative pH values are described qualitatively as above
• For clarity, all numeric ranges and constants are embedded in the notes with the appropriate LaTeX formatting as shown

References and Credits (as provided in slides)

• Textbook reference: Principles of Virology, Volume 1, 5th Edition (Flint et al.), ASM Press, 2020
• Additional references cited in slides include Adenovirus, Influenza figures, and various review sources
• Acknowledgement of credit and external figures/videos as noted in the original presentation

Key Takeaways for Exam Preparation

• Distinguish attachment vs penetration vs uncoating; know how enveloped vs non-enveloped viruses differ in attachment and entry
• Be able to describe three uncoating strategies and their cellular locations, plus HIV-specific timing models
• Recognize common virus examples for HIV, influenza, adenovirus, and poliovirus to illustrate attachment and entry concepts
• Understand the progression from acquisition to resolution, including the seven steps, and how host responses shape disease outcomes
• Recall the concept of tropism, receptors vs coreceptors, and avidity as determinants of infection specificity
• Be familiar with plaque assays, eclipse period, and cytopathic effects such as Cowdry Type A inclusions, Negri bodies, and syncytia

End of Notes