Resting Membrane Potential (RMP)

Definition

• Neurons: approximately -65 to -70 mV (inside negative)
• Skeletal muscle: approximately -90 mV
• Cardiac muscle: approximately -85 to -95 mV
• Erythrocytes: as low as -10 mV

Ion Concentrations Across the Membrane

• Na+ is ~10x more concentrated outside
• K+ is ~35x more concentrated inside

How the RMP Is Generated - Three Contributions

1. Potassium Diffusion Potential (dominant factor)

• The Nernst potential for K+ = -94 mV (if K+ were the only ion)

2. Small Sodium Permeability (partially offsets K+ effect)

3. The Na+/K+ ATPase Pump (small direct electrogenic contribution)

The Nernst Equation

E_ion = (61 mV / z) × log([ion]_outside / [ion]_inside)

The Goldman Equation

V_m = (61 mV) × log [ (P_K[K+]_out + P_Na[Na+]_out + P_Cl[Cl-]_in) / (P_K[K+]_in + P_Na[Na+]_in + P_Cl[Cl-]_out) ]

E_m = (G_K / G_T) × E_K + (G_Na / G_T) × E_Na + (G_Cl / G_T) × E_Cl + ...

Equilibrium Potentials (Summary)

Role of the Na+/K+ ATPase - Two Roles

1. Direct electrogenic effect: pumps 3 Na+ out for every 2 K+ in, contributing ~-4 mV of negativity directly
2. Indirect (dominant) role: maintains the K+ concentration gradient that produces the large K+ diffusion potential, and maintains the Na+ gradient that will later power the action potential upstroke

Equilibrium vs. Resting Potential

• Small inward Na+ leak constantly depolarizes the cell
• The pump must continuously work to oppose this Na+ leak and restore gradients

Clinical Relevance

• Hyperkalemia (high extracellular K+): reduces the K+ concentration gradient, shifts RMP toward 0 (depolarization). This can cause muscle weakness and fatal cardiac arrhythmias.
• Hypokalemia (low extracellular K+): increases the gradient, hyperpolarizes the cell, making it harder to fire action potentials - also causing muscle weakness and arrhythmias.
• Local anesthetics (e.g., lidocaine): block Na+ channels, preventing depolarization from the RMP to threshold.

• Costanzo Physiology, 7th Ed., pp. 25-27
• Guyton & Hall Textbook of Medical Physiology, pp. 78-79
• Neuroscience: Exploring the Brain, 5th Ed., pp. 282-287

Viral Replication

Overview

1. Attachment (Adsorption)
2. Penetration (Entry)
3. Uncoating
4. Synthetic phase (Transcription, Translation, Genome Replication)
5. Assembly (Maturation)
6. Release

Step 1 - Attachment (Adsorption)

• Virion attachment proteins (VAPs) or spikes - proteins on the viral surface
• Receptors - specific molecules (usually glycoproteins) on the host cell surface; typically 10,000-100,000 per cell

Step 2 - Penetration (Entry)

1. Endocytosis - the cell engulfs the virion in a membrane-bound vesicle (endosome). Most common route.
2. Membrane fusion - enveloped viruses fuse their lipid envelope directly with the plasma membrane, releasing the nucleocapsid into the cytoplasm (e.g., HIV, herpesviruses).
3. Direct injection - bacteriophages inject their nucleic acid through the bacterial cell wall while the capsid remains outside.

Step 3 - Uncoating

• In the endosome (e.g., endosomal acidification triggers uncoating)
• In the cytoplasm
• At the nuclear pore (larger DNA viruses uncoat before entering the nucleus)
• Inside the nucleus (small DNA viruses like parvoviruses and papovaviruses enter the nucleus intact through nuclear pores, then uncoat)

Step 4 - Synthetic Phase

Where does replication occur?

4a. Transcription - Genome to mRNA

DNA Viruses

• Use the host's RNA polymerase II in the nucleus to transcribe viral DNA into mRNA
• Poxviruses are the exception - they carry their own RNA polymerase and replicate entirely in the cytoplasm
• Larger DNA viruses (herpesviruses, adenoviruses) also encode their own DNA polymerase - this is the basis of antiviral drugs like acyclovir (targets herpesvirus thymidine kinase + DNA polymerase)

(+) Positive-Sense RNA Viruses (e.g., Picornaviruses, Coronaviruses, Flaviviruses, Togaviruses)

• Genome directly acts as mRNA - translated immediately on entry
• One of the first proteins translated is RNA-dependent RNA polymerase (RdRp), which then:
  • Makes a (-) strand intermediate
  • Uses it as template to synthesize more (+) strand mRNAs and new genomes

(-) Negative-Sense RNA Viruses (e.g., Rhabdoviruses, Paramyxoviruses, Orthomyxoviruses, Filoviruses)

• Genome is antisense to mRNA and cannot be directly translated
• Must carry a virion-associated RNA-dependent RNA polymerase (transcriptase) packaged inside the virion
• This transcriptase immediately transcribes the (-) genome into (+) mRNAs upon entry

Retroviruses (e.g., HIV) - Special Case

• (+) sense RNA genome, but is not translated directly
• Virion carries reverse transcriptase (RNA-dependent DNA polymerase)
• Steps:
  1. Reverse transcriptase copies RNA genome → (-) DNA strand
  2. RNase H activity of reverse transcriptase degrades the RNA template
  3. (+) DNA strand synthesized → double-stranded DNA (dsDNA)
  4. dsDNA integrates into the host chromosome (provirus) via integrase
  5. Host RNA polymerase transcribes the integrated provirus → viral mRNAs + new genomic RNA

Double-Stranded RNA Viruses (e.g., Reoviruses)

• Must also carry a virion-associated transcriptase (host cells have no dsRNA polymerase)

4b. Translation

• Viral mRNAs are translated by host ribosomes
• Human cells require monocistronic mRNA (one protein per mRNA), unlike bacteria
• Viruses solve this by: polyprotein precursors cleaved by viral proteases (e.g., picornaviruses, HIV), subgenomic RNAs, or ribosomal frameshifting

• Early proteins: enzymes needed for genome replication (polymerases, kinases)
• Late proteins: structural proteins (capsid, envelope) needed for assembly

4c. Genome Replication

• New genomic copies are made using the appropriate polymerase
• RNA viruses encode their own RNA-dependent RNA polymerase (replicase) for this
• The same template is used for both transcription (mRNAs) and replication (full-length genomes) - these processes must be temporally separated
• DNA virus genome replication mirrors host DNA replication, but with varying degrees of dependence on host machinery:
  • Parvoviruses (smallest DNA viruses): fully dependent on host - require dividing cells (S-phase)
  • Polyomaviruses/Papillomaviruses: encode origin-binding protein, rest done by host
  • Herpesviruses/Adenoviruses: encode their own DNA polymerase + accessory proteins
  • Poxviruses (largest): encode all replication machinery themselves

Step 5 - Assembly (Maturation)

Step 6 - Release

Naked (non-enveloped) viruses

• Released by cell lysis (cell death)
• Cell death results from viral takeover of cellular machinery, triggering apoptosis
• Some viruses (e.g., picornaviruses) shut off host protein synthesis early; DNA viruses interfere with cell-cycle controls
• Some viruses encode proteins that delay apoptosis to allow more time for replication

Enveloped viruses - Budding

• Released by budding from a cellular membrane - coupled with final envelope acquisition

1. Viral glycoprotein spikes are synthesized and inserted into the plasma membrane (via Golgi pathway)
2. The matrix (M) protein coats the cytoplasmic side of the membrane at the spike cluster
3. The assembled nucleocapsid is attracted to the matrix protein
4. The membrane wraps around the nucleocapsid and pinches off, releasing the complete virion

• Most enveloped viruses: plasma membrane
• Herpesviruses: nuclear membrane (then exit via exocytic vesicles)
• Coronaviruses, poxviruses: cytoplasmic membranes (Golgi apparatus)

Outcomes of Infection

Clinical Relevance - Antiviral Drug Targets

• Sherris & Ryan's Medical Microbiology, 8th Ed., Ch. 6 (pp. 198-231)
• Jawetz, Melnick & Adelberg's Medical Microbiology, 28th Ed.

Viral Uncoating

What Is Uncoating?

• Enveloped or naked (non-enveloped)
• Entering by direct fusion or by endocytosis
• DNA or RNA, and where replication occurs

Mechanism 1 - Direct Fusion at the Plasma Membrane (Enveloped Viruses)

• Paramyxoviruses (measles, RSV, mumps): the F1 (fusion) protein mediates fusion at neutral pH at the plasma membrane. If the F0 precursor is not cleaved to F1, no fusion occurs and infection fails.
• HIV and some retroviruses: gp41 (fusion protein) drives fusion with the plasma membrane after gp120 binds CD4 + co-receptor (CCR5/CXCR4).
• Herpesviruses: fuse at the plasma membrane, releasing a tegument-coated capsid into the cytoplasm.

Mechanism 2 - Viropexis (Receptor-Mediated Endocytosis) + Low pH Uncoating

1. Virus adsorbs to the cell surface via receptor binding
2. The plasma membrane invaginates and engulfs the virion - enclosing it in a cytoplasmic endosomal vesicle
3. Cell surface receptors are recycled back to the membrane
4. The endosome is acidified by cellular vacuolar H+-ATPases (pH drops to ~5-6)
5. Low pH triggers a conformational change in a viral spike protein
6. This drives fusion of the viral envelope with the endosomal membrane, releasing the nucleocapsid into the cytoplasm

• Influenza virus: the hemagglutinin (HA) undergoes acid-induced conformational change in the endosome, driving fusion. Simultaneously, the M2 ion channel allows H+ to flow into the virion interior, disrupting matrix protein-nucleocapsid interactions and completing uncoating. This is the target of amantadine and rimantadine (M2 blockers) - blocking H+ influx through M2 prevents the matrix proteins from dissociating, so uncoating fails.
• Togaviruses (rubella), rhabdoviruses (rabies), coronaviruses: endocytic entry with pH-triggered fusion.

Uncoating of Naked Capsid Viruses via Viropexis

• Poliovirus and picornaviruses: low pH in the endosome causes capsid proteins to expose hydrophobic domains. These domains bind to the endosomal membrane, creating a pore through which the RNA genome is injected directly into the cytoplasm. Drug pleconaril and related compounds (arildone, disoxaril) bind to a hydrophobic cleft in the receptor-binding "canyon" on the picornavirus capsid and block this disassembly step.
• Adenovirus: taken up by endocytosis; endosomal acidification (half-life ~5 minutes) triggers escape of ~90% of particles into the cytosol. Uncoating is sequential and organized - it systematically breaks the stabilizing interactions built during viral maturation. Microtubules transport the partially uncoated particle to the nucleus, where final uncoating and DNA release occur, possibly at the nuclear membrane.
• Reovirus (unusual): the endosome contents are transferred to a lysosome, where lysosomal proteases strip away outer capsid proteins and activate virion-associated enzymes needed for transcription. The inner core particle (not the naked genome) is then released.

Mechanism 3 - Bacteriophage Injection (No Endocytosis)

• Tailed phages (e.g., T4) attach via tail fibers to the bacterial cell wall
• The hollow tail acts as a syringe, injecting only the nucleic acid genome into the cell
• The protein capsid remains outside, shed at the surface
• A small number of virion proteins tightly associated with the DNA may enter alongside the genome

Mechanism 4 - Two-Stage Uncoating in Poxviruses

• The virion envelope fuses with the plasma membrane (or endosomal membrane), releasing the viral core into the cytoplasm
• The core remains intact with its own internal enzymes

• The viral RNA polymerase (carried inside the core) transcribes ~half the genome into early mRNAs, which exit the core into the cytoplasm
• Among the early proteins translated is an "uncoating protein"
• This protein acts on the cores to complete uncoating and liberate the viral DNA from the core
• This step requires both RNA and protein synthesis - making it unique among viruses

Herpesvirus Uncoating - Nuclear Targeting

1. Envelope fuses with plasma membrane → tegument-coated capsid released into cytoplasm
2. Tegument proteins are shed
3. The capsid travels along microtubules to the nucleus
4. At the nuclear pore, the capsid docks and the DNA is injected into the nucleus
5. The viral DNA immediately circularizes upon release from the capsid
6. The empty capsid (ghost) remains at the nuclear pore

Summary Table

Clinical Relevance - Drugs That Block Uncoating

• Sherris & Ryan's Medical Microbiology, 8th Ed., pp. 208-213
• Jawetz, Melnick & Adelberg's Medical Microbiology, 28th Ed.
• Murray's Medical Microbiology, 9th Ed., pp. 455-456

Steps 4, 5, and 6 of Viral Replication - From the Basics

Quick Recap of Steps 1-3

• Step 1 - Attachment: virus stuck to the host cell surface
• Step 2 - Penetration: virus entered the cell
• Step 3 - Uncoating: the protein coat was removed, releasing the bare viral genome

STEP 4 - The Synthetic Phase (Making Viral Parts)

1. mRNAs (instructions for making proteins)
2. Viral proteins (structural + non-structural)
3. New viral genomes (copies of the viral DNA/RNA)

4A - Transcription: Making mRNA from the Viral Genome

For DNA Viruses (e.g., Herpesviruses, Adenoviruses, Papillomaviruses)

• The viral DNA travels to the nucleus
• The host cell's own RNA polymerase II reads the viral DNA and transcribes it into viral mRNA
• The mRNAs travel out to the cytoplasm where ribosomes translate them into proteins
• Exception - Poxviruses: they are so large they carry their own RNA polymerase inside the virion and replicate entirely in the cytoplasm, never needing the nucleus

For Positive-Sense (+) RNA Viruses (e.g., Poliovirus, Coronavirus, Hepatitis C)

• The genome IS already mRNA - it has the same polarity (+ sense = readable directly)
• As soon as it enters the cytoplasm, ribosomes attach directly to the genome and translate it immediately - no transcription step needed first
• The first proteins made include RNA-dependent RNA polymerase (RdRp) - this is the enzyme that will later copy the RNA genome

For Negative-Sense (-) RNA Viruses (e.g., Influenza, Rabies, Ebola, Measles)

• The genome is the opposite polarity to mRNA - it cannot be read by ribosomes directly
• The virus solves this by carrying its own RNA-dependent RNA polymerase (RdRp) already packaged inside the virion
• As soon as uncoating occurs, this pre-packaged polymerase immediately begins copying the (-) strand genome into (+) strand mRNAs
• These mRNAs are then translated by host ribosomes

For Retroviruses (e.g., HIV)

• The genome is (+) RNA, but retroviruses do NOT translate it directly
• Instead, the virion carries reverse transcriptase - it copies RNA → DNA
  • Step 1: RNA → single-stranded (-) DNA
  • Step 2: RNA template degraded by RNase H activity
  • Step 3: (+) DNA strand made → double-stranded DNA (dsDNA)
• The dsDNA enters the nucleus and is integrated into the host chromosome (provirus) by the enzyme integrase
• Now the host's RNA polymerase transcribes the integrated provirus - producing both mRNAs AND new genomic RNA

Temporal Regulation: Early vs. Late Genes

4B - Genome Replication: Making Copies of the Viral Genome

• DNA viruses: DNA replication in the nucleus; uses viral DNA polymerase (or borrows host polymerase for smaller viruses). Follows the same biochemical rules as host DNA replication but requires a primer.
  • Small DNA viruses (parvoviruses): fully depend on host - require cells that are actively dividing (in S phase)
  • Medium DNA viruses (papillomaviruses, adenoviruses): encode origin-binding protein; use a mix of host and viral machinery
  • Large DNA viruses (herpesviruses): encode their own complete DNA polymerase + accessory proteins
  • Largest (poxviruses): encode everything themselves
• RNA viruses: use the RNA-dependent RNA polymerase (RdRp) = replicase to copy RNA → RNA
  • (+) strand viruses: genome copied to a (-) strand intermediate, then back to many (+) strands
  • (-) strand viruses: genome copied to a full-length (+) strand (antigenome), then back to many (-) strands for packaging
• The host cell has no RdRp - it cannot copy RNA from an RNA template. This is why all RNA viruses must bring or make their own polymerase.

4C - Translation: Making Viral Proteins

• Polymerases / replicases
• Proteases (cleave polyproteins into individual proteins - e.g., HIV protease)
• Kinases, integrases
• Proteins that shut off host cell protein synthesis or immune defenses

• Capsid proteins (VP1, VP2, VP3... in picornaviruses)
• Envelope glycoproteins (inserted into ER membrane, processed through Golgi, travel to cell surface)
• Matrix (M) proteins (link envelope to nucleocapsid)
• Nucleoproteins (coat the viral RNA)

The Monocistronic Rule

• DNA viruses: splice a single large precursor RNA into many separate mRNAs (using nuclear splicing machinery)
• Segmented RNA viruses (influenza, rotavirus): each genome segment = one gene = one mRNA
• (-) strand RNA viruses: polymerase starts a new mRNA at the beginning of each gene
• (+) strand RNA viruses (picornaviruses, flaviviruses): translate one giant polyprotein, then a viral protease cuts it into individual functional proteins

STEP 5 - Assembly (Packaging / Maturation)

Four Basic Rules of Assembly

1. Self-assembly - the proteins have complementary shapes and naturally come together (like puzzle pieces)
2. Stepwise and ordered - cannot skip steps; each piece must attach before the next one can
3. Pre-formed capsomeres - individual protein subunits first cluster into intermediate capsomere units, which then join to form the full capsid
4. Packaging signal - the viral genome contains a specific sequence called a packaging site that the assembling capsid recognizes, ensuring only the correct genome gets packaged (not random cellular RNA or DNA)

Assembly of Helical Capsids (e.g., TMV, Influenza, Paramyxoviruses)

• Protein subunits are pre-formed into doughnut-shaped disks
• These disks attach to the viral RNA at a specific packaging site
• They add stepwise in both directions along the RNA like beads on a string
• The RNA and protein together coil into a helix
• The process automatically stops when the end of the RNA is reached
• For segmented helical viruses (influenza), each genome segment is assembled separately into its own nucleocapsid, then all segments are gathered together during final virion assembly

Assembly of Icosahedral (Cubic) Capsids (e.g., Adenovirus, Picornavirus, Herpesviruses)

• Individual capsid proteins first cluster into pentamers (groups of 5) and hexamers (groups of 6)
• These capsomeres condense together to form an empty hollow capsid shell
• The viral genome is then threaded into the empty shell (DNA viruses) - OR
• A small nucleation complex of capsid protein + genome forms first, then the rest of the capsid assembles around it (some RNA viruses)

Where Assembly Occurs

Enveloped Viruses - Acquiring the Lipid Envelope

1. Viral envelope glycoproteins are synthesized by ribosomes on the rough ER
2. They travel through the Golgi apparatus where they are glycosylated (sugar groups added) and processed into their mature form
3. They are inserted into the desired membrane (plasma membrane, ER membrane, nuclear membrane, or Golgi membrane depending on virus)
4. The nucleocapsid recognizes and migrates to these membrane patches
5. The membrane wraps around the nucleocapsid → budding → complete enveloped virion

STEP 6 - Release (Exit from the Host Cell)

Release of Naked Capsid Viruses - Cell Lysis

• The virus hijacks and shuts down normal cellular functions (e.g., picornaviruses block host protein synthesis)
• Viral proteins may disrupt the cell membrane or interfere with cell-cycle control
• This triggers apoptosis (programmed cell death) - the cell essentially destroys itself
• Some viruses encode anti-apoptotic proteins to delay this (to maximize virion production before the cell dies)
• Cell lysis releases hundreds to thousands of new virions into the extracellular environment at once

Each infected cell can produce up to 100,000 particles - though only 1-10% may be fully infectious (defective particles result from assembly errors).

Release of Enveloped Viruses - Budding

1. Viral glycoprotein spikes cluster in a region of the plasma membrane (delivered there via the Golgi pathway)
2. The cytoplasmic face of that membrane patch becomes coated with matrix (M) protein
3. The completed nucleocapsid is attracted to and docks with the matrix protein
4. The membrane curves around the nucleocapsid and pinches off, releasing a complete enveloped virion outside the cell
5. The cell membrane is repaired - the cell survives and can continue budding out more virions

• Most enveloped viruses (influenza, HIV, paramyxoviruses): plasma membrane
• Herpesviruses: nuclear membrane (then transported out via exocytosis)
• Coronaviruses, poxviruses: cytoplasmic/Golgi membranes

Special Case - HIV (Cytopathic Retrovirus)

• Accumulation of unintegrated viral DNA in the cytoplasm (toxic)
• Toxic effects of certain viral proteins (gp120, Vpr)
• Alterations in cell membrane permeability
• Induction of apoptosis
• Cell-cell fusion (syncytia formation)

Summary Diagram

STEP 4 - SYNTHETIC PHASE
     |
     ├── Transcription: Genome → mRNA
     |      • DNA viruses: host RNA pol II (in nucleus)
     |      • (+) RNA: genome = mRNA, translate directly
     |      • (-) RNA: virion-packaged RdRp makes mRNA
     |      • Retroviruses: reverse transcriptase RNA→DNA→integrated→mRNA
     |
     ├── Translation: mRNA → Proteins (by host ribosomes)
     |      • Immediate early proteins (regulatory)
     |      • Early proteins (polymerases, enzymes)
     |      • Late proteins (capsid, glycoproteins)
     |
     └── Genome Replication: New copies of viral genome
            • DNA viruses: DNA polymerase in nucleus
            • RNA viruses: RdRp in cytoplasm
                
STEP 5 - ASSEMBLY
     |
     ├── Capsid proteins self-assemble stepwise (capsomeres → capsid)
     ├── Genome packaged into capsid via packaging signal
     ├── Enveloped viruses: glycoproteins processed through Golgi →
     |   inserted into membrane → nucleocapsid recruits matrix protein →
     |   membrane wraps around nucleocapsid
     └── Maturation complete: virion ready

STEP 6 - RELEASE
     |
     ├── Naked viruses: cell LYSIS → all virions released at once
     └── Enveloped viruses: BUDDING → continuous release without
                                      immediate cell death

• Sherris & Ryan's Medical Microbiology, 8th Ed., pp. 213-231
• Murray's Medical Microbiology, 9th Ed.
• Harrison's Principles of Internal Medicine, 22nd Ed., Ch. 19