pmb 114 mt3

Rabies (genome + disease)

(-)ssRNA enveloped virus, rhabdoviridae

Severe acute progressive encephalitis

transmitted via saliva through bite

neurotropic (attacks neurons), NOT in blood

virus travels to the brain via retrograde axonal transport

nearly 100% fatal in humans

Rabies (prevention)

Vaccinate the dogs!

bats also have it

Prevention = vaccination

- pre exposure prophylaxis (vaccine) given to high risk people

- Post exposure prophylaxis (PEP) (vaccine) given post bite before symptoms

No treatment once symptoms develop...

Cryptic rabies cases

associated with silver haired & easter pipistrelle bats

they have super small bites --> unnoticed

enhanced virulence + enhanced infectivity bc superficial route

they replicate in non neural tissue and at lower temps

(Sin nombre) Hantavirus

Hantaviridae

(-)ssRNA enveloped virus

causes hemorrhagic fever with renal syndrome (HRFS - old world)

hantavirus pulmonary syndrome (HPS - new world)

rodent host, people usually get it in camping locations

shed through urine/feces, gets aerosolized & inhaled

Infects epithelial cells, lungs can fill up with fluid --> Endothelial cells become "leaky"

in shrews and bats

Retroviruses (genome + virion structure)

(+)ssRNA

(+)ssRNA --> DNA intermediate (RT) --> mRNA

polyprotein method

env, gag, pol

env = envelope - surface + transmemnbrane

gag = core - protease, matrix, nucleocapsid, capsid

pol = enzymes - integrase, RT

Translation is NOT first, so protein has to be deposited into cell with genome

Not a segmented virus, "diploid" = 2 identical copies of viral genome

Retroviruses (simple vs complex)

simple = rna tumor simple genomes: Encode only unspliced and singly spliced mRNAs,

complex retroviruses encode multiply spliced mRNAs

Retrovirus Replication

Binding receptors on cell surface, fuse w plasma membrane to enter

RT of viral RNA in the core by RT --> dsDNA

core/capsid is starting to dissolve otw to nucleus

dsDNA and integrase enter genome (pre-initiation complex)

integrates enzyme into cellular genome → becomes provirus

Forever integrated into genome

Virus makes new progeny viruses through RNA poly 2 (host) or translation of spliced mRNA

proteins translated as polyproteins --> immature virus particles assembled and released via budding

maturation after virus released

Protease mediated Gag and GagPol processing causes major change in virion morphology

Gag made from gag-pol due to ribosome frameshifitng

Retrovirus Reverse Transcriptase (2 enzymatic activities

Polymerase Activity:

- Primer dependent (needs DNA or RNA)

- Template can be DNA or RNA

- Only incorporates dNTPs not rNTPs (only makes DNA but can use either as a template)

RNAseH acvitity:

- cleaves RNA when in duplex form (RNA:RNA, RNA:DNA)

error prone

Reverse transription process

RNa --> dsDNA

RNA has R for repeat, unique 5' end sequence (U5) , primer binding site (PBS)

3' end = polypurine tract, 3' sequence unique (U3), another copy of repeats

RT turns ssRNA into dsDNA --> ends are different from template, they have long terminal repeats which contain U3, R, and U5 (in that order)

dsDNA integrated, U3 has promoter --> transcription starts at R, rna transcript corresponds to genomic copy

retroviruses (rna to dsDNA details)

go watch the video LOL

cellular tRNA bound to PBS of rna = orimer to initiate reverse transcription (RDDP)

viral RT RNAseH degrades dna hybridized to DNA

newly synthesized DNA transferred and hybridized to repeat region at end of genome (where the tetris S shape comes from)

full length genome is copied by rev transc (RDDP)

viral RT RNAseH degrades RNA hybridized to DNA, except PT region (resistant)

DNA synthesis resumes using hybridized ppt RNA as primer (makes 2nd strand) (DDDP)

viral RNAseH degrades ppt primer and releases tRNA (now hybridized to DNA)

PBS primer hybridizes to complementary sequence at the start of newly synthesized DNA

both - and + DNA strands extended to create full lendth dsDNA genome for integration

Retrovirus Integration

integrase is in core, enters nucleus and is entered into target DNA sequence

duplication of host target sequence lets you see where it is

RNA poly transcription is only way out of the genome

HIV/AIDS (genome + transmission + immunity + origin)

(+)ssRNA

transmission as cell associated virus or free virus via bodily fluids: blood, sex fluids, breastmilk

13% infected don't know they have it

Most diagnoses made in early 20s, or 13-24

CCR5∆32 mutation are resistant to HIV infection --> can donate blood to help infected individuals

SIV, primate to human origin

HIV/AIDS pathogenesis (cellular)

Targets CD4⁺ T cells due to specific receptor interactions

Env gene encodes a glycoprotein → cleaved by viral protease into:

gp120 (SU): binds CD4 receptor

gp41 (TM): facilitates membrane fusion

gp120 binds CD4 → triggers conformational change → exposes coreceptor binding site

Coreceptors = CCR5 or CXCR4 (co-receptor is required for entry)

Virus may prefer one (CCR5-tropic or CXCR4-tropic)

After coreceptor binding → exposes gp41 transmembrane domain

gp41 is a class I fusion protein: promotes fusion of viral envelope with host cell membrane

Result: Viral RNA enters host T cell → infection begins

HIV/AIDS (t-cell infection)

Cytotoxic T cells (CD8⁺)

Interact with MHC I on infected cells

Kill infected cells directly

T-helper cells (CD4⁺)

Interact with MHC II on antigen-presenting cells (APCs) like dendritic cells, macrophages, B cells

Activate other immune cells, including:

B cells → become plasma cells → make antibodies

Cytotoxic T cells → enhance killing

HIV selectively infects CD4⁺ T-helper cells

Impairs both adaptive and innate immune response

MHC II signaling is disrupted → B cells can't activate → poor antibody production

Outcome:

Immune system can't respond to new threats

Leads to AIDS: death is usually due to secondary infections or cancers

HIV/AIDS infection

AIDS = your CD4 T-cells drops below a certain amount

Initially: HIV replicates immediately (integrating into the genome occurs, permanent)

Viral load stabilizes, virus still replicates actively but host immune system keeping it under control (good)

bad --> HIV wins, t cells decline until no immune response can be sustained, HIV replicates out of control, host subject to tons of infections.

CD8 T-cells get "exhausted" and die

10^10 virions per day made and destroyed

Increased likelihood of mutation in a virus that lets it escape immune system

rapid vs non rapid regressors (HIV/AIDS)

Rapid regressors are people who develop AIDS very fast

Non-progressors: maintain normal CD4 count at low levels for 10yrs without antiretroviral therapy (ART) (rare) not associated with attenuated virus

HIV/AIDS treatment

ART = antiretroviral therapy, works to keep virus levels low, not knock it out

PrEP and PEP:

PrEP = pre exposure, given to high risk individuals

Consistent use of it can reduce risk by 99%

Key for preventing virus from integrating

PEP = post exposure

Should be started within 72 hours

^ not effective if symptoms already occur

Access to these is super unequal

HIV/AIDS inhibitors

Attachment inhibitors:

Binds to GP120 surface of spike protein to prevent interaction with CD4

Mutations overcome it tho

Post attachment inhibitors:

Antibodies that bind once receptor binding domain binds with CD$, stops CD4 conformational changes

CCR5 antagonists

Molecules that bind to it and block interaction with GP120 after CD4 interacts

This wouldn't work for mutated individuals

Fusion inhibitors:

Fusions allows gp41 to insert into membrane and fold back on itself for fusion

Folding back requires interaction between sub domains HR1 and HR2

Took small fragment of hr2 and made a peptide of that which mimics HR2 so HR1 binds to it, then HR1 cant fold back on itself

separate? Lenacapavir drug:

for prep, Lenacapavir binds to hexamers so progression through breaking capsid can't happen

HepB (RT activity)

dsDNA, uses RT

• Viruses enter cell with ds DNA → becomes + RNA → proteins or - DNA to become dsDNA

• RNA template to dsDNA is where RT works

• You get a partially double stranded genome

Enzymatic activities of RT:

• RNase activity (Cleaves)

• Polymerase activity (RNA/DNA temp → makes DNA only)

Hep B P Protein

P protein in HepB has three key domains:

1. Terminal Protein – primes DNA synthesis by acting as a protein primer.

2. Reverse Transcriptase (RT) – copies viral RNA into DNA.

3. RNase H – degrades the RNA strand of RNA-DNA hybrids during reverse transcription.

referred to a lot as just hepB’s RT

HBV particles

Dane particles: They contain the viral envelope, capsid, and the HBV genome (partially double-stranded DNA).

• HBV makes huge numbers of non-infectious particles (envelope only)

• Decoy particles distract immune system

• Helps real virus evade detection and persist in the host

hep B genome

atypical: partially double stranded

• Polymerase (P protein) is covalently attached to the 5′ end of the (–) strand

• (+) strand has a 5′ cap, like eukaryotic mRNA

• 🔁 Both strands contain Direct Repeats (DR1, DR2)

  • These are short, identical sequences at specific sites

  • The 5′ ends of both DNA strands are near or within these direct repeats

  • DRs are important for genome replication and circularization

• RT does RNA to dsDNA, happens in confines of capsid → RT activity runs out of dNTP’s can't finish job → partially dsDNA molecule with some leftover RNA

RT in hepB vs retrovirus (dNTPs)

• Retrovirus: RT activity happens in capsid as its moving to the nucleus, capsid disassembly and brings in dNTPs

• Hep: reverse, RT as capsid assembled, runs out of dNTPs

HBV overlapping genes + consequences

4 of them (overlapping ORFs)

PreS1/s2 ORFs = surface antigen, makes S,M,L antigen proteins

ORFcore PreC = makes capsid

ORFP = encodes viral polymerase (P)

orf X = transcriptional activator (irrelevant?)

Overlapping genes = if theres a mutation in 1, it can mess up multiple (mutation in S can ruin P for ex)

or can mess up the polymerase

HBV replication cycle

Virions use receptor exclusively found in liver cells (liver tropic)

• Receptor mediated endocytosis

• HBV: Formation of cccDNA

  • Viral capsid delivers partially dsDNA to nucleus

  • Host enzymes repair DNA → cccDNA

  • cccDNA = stable, circular, viral DNA reservoir

  • Does NOT integrate into host genome (unlike retroviruses)

  • Acts as template for viral RNA + genome production

  • Host rna poly comes and transcribes mRNA and pregenomic RNA 

  • (pgRNA = template of rna converted to dna that gets packaged into virion)

HBV recycling pathway

Feature	Purpose
pgRNA + capsid in cytoplasm	Reverse transcribed to dsDNA
Capsids return to nucleus	Make more cccDNA early in infection
Envelope proteins missing	So virions aren’t secreted yet
10–30 cccDNA per cell	Boosts viral transcription + antigen expression
Liver cell division	Recycling maintains infection (no integration)

HBV gene expression / leaky scanning

HBV Gene Expression + Leaky Scanning

• RNA Pol II transcribes pgRNA + mRNAs from cccDNA

• Transcripts have repeats → help reverse transcription

• Leaky scanning lets ribosomes skip weak start codons
  → Produces multiple proteins from one mRNA

• Some are truncated or alternative-frame proteins

• Efficient way to control protein ratios (stoichiometry)

How HBV makes its relaxed circular DNA genome

DR - direct repeats

1. P binds ε on pgRNA → primes short DNA

2. Template switch: P moves to 3′ end

3. TP primes (–) DNA synthesis (RDDP)

4. RNAse H degrades RNA, except capped DR2

5. DR2 RNA primes (+) strand DNA (DDDP)

6. (+) strand goes DR2 → DR1

7. DR1 hybridizes to 5′ DR1 to circularize

8. (+) strand elongates → relaxed-circular DNA
   ⚠ Incomplete synthesis likely due to dNTP shortage

HBV spread + vaccine

mother to child

intravenous blood transfusions (before screening)

sexual transmission

IV drug use

needle sticks in hospitals

• Can be acute or chronic infection

• Acute: jaundice, high viral protein levels, immune kicks in and recovers 

• Chronic: proteins continually produced, immune system fails to clear viral DNA

• Likelihood of chronic vs acute is due to age

  • High risk for children, vaccine happens at birth

  • Individuals with chronic also have cirrhosis or liver cancer

  • hepB doesnt cause cell death, it messes up ur liver superbad

if infected, not curable

vaccine given at birth

Arenaviridae (genome + general)

enveloped, bi-segmented RNA genome:

• L segment: larger

  • has Z protein: lays a central role in the virus's assembly, budding, and replication regulation.

• S segment: smaller

• has IGR - intergenic region (loop) that separates ± regions

nucleocapsid coats RNA

old and new world

cause hemorrhagic fevers

some are non pathogenic

transmitted through mastomys rats —> rat to human —> human to human

• urine/feces, or bodily fluid/blood transmission

can be in mammals and snakes

Arenavirus genome expression

• Replicates in the cytoplasm.

• Ambisense RNA genome: Each segment (S and L) has two genes in opposite directions.

• Needs to make:

  • mRNAs for translation (to make proteins),

  • Antigenomes as intermediates for replication.

• Transcription Phase (Early Infection):

  • L protein (viral RdRp) binds the 3′ negative-sense ends of genome segments.

  • It transcribes subgenomic mRNAs for:

    • NP from S segment

    • L protein from L segment

  • These transcripts stop at a central intergenic region (IGR) — a structured RNA stem-loop that signals transcription termination.

• Replication Phase (Later):

  Once there's enough NP and L protein made:

  1. Full-length antigenomes (complementary to the genome segments) are synthesized.

  2. Then, the 3′ ends of the antigenomes are transcribed into subgenomic mRNAs for:

     • GPC (glycoprotein precursor → GP1 & GP2) from S segment

     • Z protein from L segment

timing of protein production (early/late) - arenaviridae

early: polymerase, nucleocapsid proteins

late (after viral rep): Z protein, glycoprotein

Arenaviridae replication & 3’ utr region & no polyad

Arenaviruses use ambisense RNA segments to control timing of gene expression — early proteins (NP, L) come first, then late structural ones (GPC, Z) after replication begins.

The 3′ UTR formed from the IGR helps control when and how much protein is made by influencing translation of viral mRNA — a key regulatory strategy for timing viral protein expression.

Arenavirus mRNAs are capped but not polyadenylated.

To make up for this, the virus relies on regulatory sequences in the 5′ and 3′ UTRs of each mRNA.

These sequences influence mRNA stability and translation, explaining why more NP (from strong UTRs) is made than GP (from weaker UTRs).

Reporter gene experiments confirm this difference in expression is driven by the UTRs themselves.

Arenavirus cap snatching + Z in viral assembly

cytoplasm, cap snatching occurs here

host derived sequences at 5’ end (characteristic of cap snatching)

carried out by 1 protein

late in infection when nZ produced —> time for packaging

Z plays a role in taking virus from translation to assembly, and also

• in viral assembly: interacts with glycoprotein GP, nucleoprotein NP, Polymerase L

• Z also dampens innate immune response

• Z protein from pathogenic can evade immune, non pathogenic cant

• Upregulates IFNs to get to antiviral state

Nucleocapsid protein and immune evasion - arenavirus

NP supresses type 1 IFN productoin (z does this too)

Crystal structure of LASV NP shows unique RNAse domain specific for dsRNA —> if it cleaves it , it cant be detected

Reovirused (Genome + general)

REO = respiratory enteric orphan virus

non-enveloped, icosahedral capsids

liner dsRNA genome

double layered capsid

replication in cytoplasm

incomplete uncoating of virions, which possess all the

enzymes required for ds RNA transcription

fecal/oral transmission

Reovirus entry and uncoating

• Multiple steps bc of multiple layers

• Transmitted fecal-oral, replicate in gut (acidic)

• Uncoating happens bc low pH environment of gut

• Binds with sialic acid or JAM-A (don't need to know names) receptor mediated endocytosis

Viral particle is double layered → acid induced proteolytic processing of outer capsid → creates intermediate subvirion particle (ISVP), has enzymatic activity, active when outer capsid protein taken away

Structural changes to capsid during entry - Reovirus

• Virion → ISVP → core

Think of reovirus like a locked canister:

• First, the shell is dissolved in the cell (sigma 3 removed),

• Then the docking arm pops out (spike extends),

• After docking, the lid of the canister twists open like petals (lambda 2 opens),

• Finally, the core inside is revealed, ready to release the viral genome.

Reovirus core as an “mRNA factory”

• Icosahedral internal core, at the base of each 12pt cortex is one part of mRNA

  • Has to be at base bc vertex is where RNA is extruded and copied and moved into cytoplasm

• Lambda 2 (flower blooming thing) is a pentamer than forms channel so mRNA can be transcribed off of dsRNA vGenome into mRNA extruded through these turrets

• Also has a capping activity enzyme in the turret, forms rna capping enzyme to put 5’ cap on. (like sending ur kid off to school LOL)

lambda 1-3

λ2: pentamers form the channels through

which mRNA is extruded; also is capping

enzyme.

λ3, μ2: RDRP subunits

λ1: RNA helicase; preps mRNA for capping

(removes terminal phosphate)

reovirus unique 4 channel structure

• Start with dsRNA molecule → separated using helicase → ssRNA portion of - sense template (for mRNA copying) → enters channel #1 → as copied, exits to cytoplasm (exposed to inside of core)

• In → copied → released again and cycled back to re-anneal with + sense mate

• Copied mRNA as synthesized comes out channel #3, gets capped, released into cytoplasm

• Channel #4 lets nucleotide triphosphates (NTPs) enter and are added on during copying.

Reovirus - replicase particles & secondary transcription

• Primary transcription = primary genome copied (each 10-12 mRNA segments extruded into cytoplasm, translated via ribosome → new viral proteins)

• All structural components in core are both structural and enzymatic

• As proteins built up + mRNA templates = viral replication compartments

  • Nothing can be done right until a new core is formed around each of 12 segments → once thats done then polymerase makes -sense copy of mRNA → dsRNA genome

  • Now u have a core that can be used to make more mRNAs, viral proteins to amplify process

  • Cores are assembling outer capsid as well at the end

  • Most 2ndary transcription in replication compartments from cores with newly replicated dsRNA genomes

takeaway:

Reovirus replication is tightly controlled and compartmentalized: it begins with primary transcription to make viral proteins, but full genome replication (dsRNA synthesis) only happens after a new core is assembled around all 12 mRNA segments—ensuring accuracy and coordination.

reovirus pathogenesis

• major cause of childhood gastroenteritis (stomach flu)

• Rotavirus death usually due to dehydration bc all water gone through diarrhea

• How does rotavirus cause huge fluid imbalance:

  • Caused by NSP4 an enterotoxin, protein, exposure of this to the gut causes fluid loss and diarrhea

  • NSP4 engages receptors on cells engaged in Ca signaling (important for chloride balance for inside/outside balance) 

  • If that's disrupted → net fluid excretion instead of homeostasis

  • NSP4 once in cells can disrupt junctions between cells (what keeps intestinal villi in close contact) → integrity of intestine villi weakens → atrophy → hyperplasia

  • Villi important for nutrient absorption, of those r gone then you can't take in nutrients / fluids

Non pathogenic reoviruses can enhance development of food sensitivities—> celiac disease

Reovirus vaccine

Live attenuated vaccine

-3 re-assortants, each with a different human gene encoding a specific serotype of VP4 or VP7 (viral capsid proteins), under the backbone of a rhesus monkey strain.

Several oral, live attenuated rotavirus vaccines available

gram positive vs gram negative bacteria

Gram negative bacteria cells have an outer membrane then a thin layer of peptidoglycan then an inner membrane

Gram-positive bacteria have a thick cell wall composed of many layers of peptidoglycan and stain purple with a Gram stain.

Phages delivering viral genome

pierce envelope and deliver genetic material into cell envelope

• tail fibers trigger receptors → conformational changes → tail contracts, dsDNA genome into cell (like a syringe)

• Transfer rate and replication rate are fast

• picture is of this

unique case:

• Podovirus with short tail, core proteins act like a tail, they need to be revealed. Like a spring that gets unwound when receptors are engaged. Tail and fibers like a lock and key mechanism.

bacteriophages with no tail:

• they have H protein, virion binds to sugar molecules, unveils H protein which self-assembles into a nanotube that drives its way into the cell envelope and becomes a conduit for the viral genome. (like a drill)

• can also use their own internal membrane as a tunnel to deliver v genome.

Lytic vs Lysogenic Phages

• Lytic = predator-prey relationship with bacterial host, infect and kill it

  • Phage attaches to bacterial cell, delivers genome, takes it over to make viral copies/proteins, virions made, lyse out of cell

• Lysogenic = Lysogenic phages can either enter a lytic cycle, or enter a lysogenic state

  • After entry of genome, genome integrated into bacterial chromosome, maintained acquiescence state, bacterium which has this prophage Integrated vgenome), goes on to divide, progeny get this DNA

  • Goes on forever or until the virus is queued to enter the lytic cycle

  • queue is usually a stressor

Note: things like filamentous phages do “non lytic release” (make their viral genome and replicate then leave with no lysis)

Phage Mediated Lysis

Need to break through cell envelope

same first steps for gram - and +

• T4 complete infection cycle is 18-20 minutes

• holin = inner membrane spanning protein

• During cycle, holin localizes to inner membrane, phage produces endolysin (pac man), cleaves peptidoglycan but needs access to where peptidoglycan is → it accumulates when waiting

• Spanin = membrane spanning protein, fuses membranes

• all these proteins accumulate during infection cycle

• Holin triggers large holes in inner membrane, releases endolysin into peptidoglycan  → chews it up → once mesh barrier broken down, gram + lysed,

  • for gram - → spanin initiated to go conformational change: outer and inner membranes fuse = lysis

• Pinholin = makes tiny holes LOL, holes trigger endolysin

Phage-phage communication for lysis vs lysogeny

• One phage kills a cell → produces small peptide → accumulates as more viruses grow 

• If there's a high abundance of this peptide, this environment favors lysogeny → phages know there aren't many hosts left, they can't kill off all of them → they decide to chill out LMAO

• Communication = arbitrium = name of the signaling peptide

^ the peptide = AimR, it either is or isnt bound by aimP

Upon phage infection, phage comes in aimR = transcription factor which represses lysogeny = lysis chosen (left photo)

• For lysogeny: TF that represses AimR gets bound to peptide (aimP) → lysogeny (right side photo)

Jumbo phage - “nucleus” and compartmentalization

• GP105 builds a phage nucleus (protein shell) in center of bacterial cell.

• Inside shell: DNA, replication, and transcription machinery.

• Outside shell: translation occurs in cytoplasm via host ribosomes.

• Shell acts like a eukaryotic nucleus: separates mRNA production from translation.

• Purpose: immune evasion (blocks host enzymes), efficient genome processing.

• Shell is semi-permeable: lets mRNA and small molecules through, blocks folded proteins.

• Selective transport brings phage proteins (from cytoplasm) back into shell.

• Filament tracks and a spindle system keep nucleus centered.

• Capsids dock, load DNA from nucleus, move away to assemble tails.

Phages in human health / disease + challenges

Bacteriophages dont infect human cells —> we use natural capacity of phages to kill bacteria in our own fight against antibiotic resistance

• Positive: viruses are very specific to given hosts → can target just them with phages

• Challenge: narrow host specificity (reason why it prob won't become mainstream) → scientists have to find very specific phages to help treat people

bacteriophages as antibacterial agents (enzymes / process)

• Endolysin is a phage enzyme that degrades peptidoglycan, causing bacterial cell lysis.

• Works with holin, which creates pores in the membrane so endolysin can reach the cell wall.

• Researchers can apply endolysin directly to Gram-positive bacteria (no outer membrane), killing them externally.

• Doesn’t work on Gram-negative bacteria because their outer membrane blocks access.

• Endolysin is produced by phages and can be species-specific, targeting only certain bacteria.

lysogenic conversion

Alteration of the phenotype of a host cell by lysogeny

Host cell becomes immune to further infection by same phage

Other phenotypic changes can also occur

ex: pf4 virion phages can be used to alter genetic makeup of a bacteria that gets in wounds and ruins everything

^ an antibiotic might make everything worse by triggering the prophage to make more virions (lytic cycle)

bacterial defense against phages

• 1) bacteria can change their surface molecules so phages can’t infect those cells

• 2) intracellular barriers to phage replication (restriction modification systems & crispr/cas) most abundant

  • Sequence specific systems that degrade invading virus

• 3) mechanistically diverse systems evolved to detect viral infection, kill cell before virus can finish replication cycle 2nd abundant

  • Abortive infection systems

  • kill urself for the tribe :(

Surface modifications (bacteria and phages) to compete w eachother

• Bacteria alter expression of receptors, phages adapt to this

• Bacteria can mask receptors using glycans, phages can drill through it using enzymes

• Some phages have “tropism switching genetic cassettes” → they modify tail protein fibers to bind to more hosts

  • Done using diversity generating retroelement

Diversity generating retro element (DGE) - bacteriophages

• Phages evolve to keep up with bacterial defenses by changing their surface proteins, especially the tail fibers.

• Diverse population: Phage replication produces a heterogeneous population of viruses, each with different tail fibers.

• Receptor binding: Phages bind to specific bacterial receptors; if bacteria change their receptors, some phages can still infect with the right tail fiber.

• Stochastic process: Random chance ensures some phages with the right tail fibers can infect and replicate, even if most fail.

• DGE generates this diversity in the viral population, ensuring phages can adapt to evolving bacterial defenses and continue infecting.

 Bpp-1 DGR and mutagenic retrohoming - bacteriophages

• RNA to cDNA conversion: The Bpp-1 DGR uses RNA to form cDNA through reverse transcription.

• Errors during reverse transcription: Errors occur randomly at adenines, replacing the variable region of the gene in the phage.

• New tail fiber tip: This results in a new C-terminal portion of the gene, leading to diverse tail fibers.

• Mutagenic retrohoming: The diversity created allows the phage to maintain a diverse pool of viruses, enabling them to adapt and keep up with bacterial defenses.

same idea of random changes means bacteriophage is ready to attack / adapt to more things.

Restriction Modification systems - bacterial defense response

• Bacterial defense system: Bacteria produce both a methyltransferase (MT) and a restriction endonuclease.

• Marking “self”: The MT adds methyl groups to specific DNA sequences on the bacterial chromosome, marking it as "self".

• Identifying invaders: Viral DNA lacks these methylation marks.

• Defense action: The cognate restriction enzyme (CRE) recognizes unmodified (foreign) DNA and cleaves it, preventing viral replication.

How do some phages evade bacterial restriction enzymes?

• Chemical DNA modification: Some phages modify cytosine bases in their DNA to 5-hydroxymethylcytosine (5-hmC).

• Purpose: This modification prevents cleavage by bacterial cognate restriction enzymes (CREs), which normally degrade unmarked foreign DNA.

• Bacterial counter-response: Bacteria evolve new enzymes that specifically recognize and cleave 5-hmC-modified DNA.

• Phage’s next move: Phage adds glucosylated hydroxymethylcytosine (glu-HMC) to further shield its DNA — once again escaping host restriction.

Phages and CRISPR

• CRISPR-Cas = Adaptive immune system in bacteria

  • Made of repeats + spacers (memory of past phage infections)

  • Cas genes nearby encode machinery (like Cas9)

• During phage infection:

  • Bacteria capture snippets of phage DNA → insert into CRISPR array

  • Later, CRISPR array transcribed → processed into guide RNAs

  • Guide RNAs + Cas9 = CRISPR complex, which cleaves matching phage DNA

• How phages fight back:

  • Mutate their genome to avoid recognition

  • Produce proteins that inhibit Cas9 (anti-CRISPRs)

  • Physically protect their genome by replicating inside protein shells

• Bonus: There are CRISPR systems that target RNA, too!

basically crispr remembers phages and fights them so phages change their genome.

cGAS like pathway in bacteria + phages avoiding it

bacteria encode homologs of cGAS

• virus in, queue sensed (not dsDNA bc bacteria have that), sense viral infection, produce cyclic di-nucleotides → produce effectors and kick its ass

• Phages can circumvent this 

  • using enzymes with some similarity to what infects us

  • Bacteria use CDNs to signal viral infection (like human cGAS-STING).

  • T4 phage is resistant: produces ACB, an enzyme that degrades CDNs.

  • No CDN = no immune signal = bacterial defense is bypassed.

Smallpox progression

• 10-12 day incubation period

• Days 1-3 = sudden fever, prostration, nausea, vomiting

• Day 5: deep lesions and pustules all over body including mouth and larynx

• Scabs and bursting of lesions occurs over the next three weeks

• Scab material and lesions spread virus

Smallpox - Variola Major vs Variola Minor

• Variola major: hyper virulent, 30% individuals died

• Variola minor: 1% lethality

• Survivors are resistant to subsequent infection

WHO eradication program & vaccination strat

• Humans are only known host

• Vaccine highly effective against all known strains of the virus

• Vaccine easy to store and maintain

• Disease very easily recognized, no asymptomatic people

• Can easily determine who has been vaccinated

vaccination:

• Identify all individuals with disease

• Trace all contacts with diseased people

• Vaccinate all contacts to stop disease spread

• Vaccinate everyone in vicinity of outbreaks (ring vaccination)

• Advertise program to break chain of transmission

• Maintain world-wide surveillance to prevent pox reintroduction

• Vaccination in october (low point for smallpox)

• Prevention during this period could permanently destroy a smallpox chain

General Properties of Poxviruses & challenges

• Large, complex “ovoid” structures

• Font have 1 set morphology → amorphous “brick like”

• No defining symmetry

• They're dsDNA viruses but they replicate in the cytoplasm

• Smallpox challenges:

  • Genome replicates in a spot where its flagged as a PAMP

  • They can’t make use of any nuclear cellular machinery (they have to encode/package everything themselves)

Smallpox entry

• Macropinocytosis: Engage receptors, but trick cell into engulfing them

• Bind actin containing protrusions (filopodia), they track to the surface of the cell, once they each they induce signaling event → tricks cell into engulfment

• Induces plasma membrane “blebbing” 

  • Virus put phosphatidylserine (PS) on membrane 

  • PS is usually facing cytosolic side of cell except when cells undergo apoptosis

  • ^ signals to neighboring cells that it isn't a pathogen, but it's here to be engulfed

  • PS signaling dampens local innate immune response by telling phagocytic cells that its just a piece of an apoptotic cell 

smallpox genome organization + mini nucleus

• Linear dsDNA genome, no free 5’ 3’ ends

  • Each end is covalently closed

  • NOT circular

  • 10kb inverted repeats

• Genes expressed from both strands

• Large genome, encodes lots of proteins

• No introns in genes, prob bc virus isn't in nucleus 

• Vaccinia core is “mini nucleus” 41:45

  • Incomplete uncoating

  • Virus first enters, it expresses a set of genes (early genes) before it starts replicating genome, extruded from cores into cytoplasm for replication

  • Very similar to Reoviruses

DNA replication - smallpox

• Start with linear dna with its closed ends

• Virus makes nick near terminal repeats

• Hairpin unwinds

• Virus uses shorter end as a primer for DNA synthesis, extends till end, since its an inverted terminal repeat, it's made then fold back into a hairpin

• Then it keeps going using the rest of the strand to keep going

• “Strand displacement mechanism”

• Nickases and ligases to resolve them into unit length structures

• No free ends, terminal inverted repeats used to make long cacatomers of genome that flip back on each other to keep copying so there's never openings at the end and they can be resolved.

Vaccinia virions propelled from surface of cell by actin tails - smallpox

• virus expresses 2 proteins made early on plasma membrane, these proteins induce actin tails

• Actin tails are induced to be polymerized by virus

• Before virus finishes replication it has tails on the surface so when 

  • 1. it finishes it can propel away

  • 2. if there's another virus coming along, these proteins propel this virus away this cell (already infected) to a different one that isn’t infected.

SV40 (polio, hit and run tumorigenesis)

• SV40 was a contaminant of early 1960’s polio vaccines

• There ended up being no risk, bc it doesn’t cause cancer in humans

• “Hit and run model of tumorigenesis” = tumor inducing agent plays a casual role in initiation but not maintenance of the tumor

sv40 strength and uncoating

• SV40 is super strong bc VP1 protein (dont need to know name?), forms a pentamer which becomes extensively cross linked to itself via disulfide bond, like molecular chain mail

• How do you reverse this to get the viral genome out?

  • SV40 VP1 protein forms a pentameric capsid crosslinked by disulfide bonds — very stable, like molecular chainmail.

  • To escape, SV40 must uncoat its genome by reversing these crosslinks.

  • Enters the cell via lipid raft microdomains (not typical receptor-mediated endocytosis) → this sends the virus to the ER.

  • In the ER, protein isomerases break disulfide bonds, loosening the capsid.

  • SV40 mimics a misfolded protein, triggering the ERAD (ER-associated degradation) pathway, which exports it to the cytoplasm.

  • In the cytoplasm, the virus uses the microtubule network to travel to the nucleus, where infection proceeds.

vp2 and vp3 have DNA binding domains and nuclear transport signals, potentially used in delivering genome to the nucleus.

polyomavirus genome structure and organization

• Super tiny genome, not all DNA viruses are large

• Circular genome, packed with nucleosomes (cellular histones) from cell

• Don't make your own DNA organizing protein, steal it!

• All DNA viruses have kinetic organization of gene expression (early genes = transcribed before virus replicated, late  = genes transcripted after virus replicates)

  • Early are needed for cell setup (machinery cell encodes itself)

  • Late genes (structural capsid components)

• Early and late promoters

• T = tumor antigen (viral antigen)

• VP2 & 3 have nuclear localization signal

bidirectional DNA replication - polyomaviruses

Bidirectional DNA Replication in Polyomaviruses

• Polyomaviruses (like SV40) have a circular double-stranded DNA genome.

• Replication starts at a single origin of replication (Ori).

• From the origin, DNA replication proceeds in both directions — this is called bidirectional replication.

• This process creates a structure that looks like the Greek letter theta (θ) — often called a theta replication intermediate.

5’ end problem

• DNA polymerase can’t fill in the 5′ end after RNA primers are removed on linear DNA, leading to loss of sequence.

• But circular DNA avoids the 5′ end problem, allowing complete replication without losing genetic material.

regulation of S phase & how viruses hijack it

DNA virus can stay small bc they steal DNA replication from host cell

human regulation:

• Viruses push cells into S phase to access DNA replication machinery, often by inactivating Rb to free E2F, which turns on replication genes.

• (e2f in cytoplasm)

• Normally, Rb holds E2F inactive; CDKs phosphorylate Rb during proper cell cycle entry.

• p53 monitors for inappropriate replication and blocks CDKs or triggers apoptosis to prevent cancer — viruses often try to disable p53 to avoid this.

• P53 can block cyclin activity outside of its job, if that doesn’t happen it says apoptosis time. Kys. = no cancer

Virus overcoming:

• SV40 gets all this done with T bc it has rb binding domain, also has p53 binding domain to inactivate it

• Most of these don’t cause cancer bc cell dies

• Large T comes in, it inactivates RB and p53 → leads to S phase (involves turning on CDKs, they also phosphorylate T → signal to T that S phase successfully was turned on, turn ur activity to viral DNA replication/binding, triggers binding to DNA origin of replication and start replication

Polyomavirus - Large T antigen

Inactivates Rb (Retinoblastoma protein)

• LT binds to Rb → releases E2F → pushes cell into S phase → host cell starts making DNA replication machinery, which the virus needs.

2. Inactivates p53

• LT binds and inhibits p53, preventing apoptosis → infected cell stays alive long enough to make more viruses.

3. Binds the Viral Origin of Replication (ORI)

• LT recognizes and binds specific sequences at the viral ORI (origin of replication).

• hexamer binds it?

• Binding here is essential to start viral DNA replication.

4. ATPase/Helicase Activity → "Melts" dsDNA

• LT has ATPase activity (it uses ATP for energy) and acts like a helicase.

• This allows LT to unwind/melt the double-stranded viral DNA at the origin → creates a single-stranded region where host DNA polymerase can start replicating the viral genome.

Papillomaviruses: general

huge cause of cervical cancer

• Small, dsDNA genome

• Naked capsid

• Circular dsDNA genome (like polyoma)

• DNA is packaged using cellular histones as DNA organizing material (don't encode their own dna binding proteins)

• Huge diversity, can infect vertebrates

• Stays within species

• Infect epithelia, some have a preference for skin, some have a present for mucosal epithelia

• There are some cell and tissue specific viruses, some really only want to stay in one part of the body (bottom of foot wart for ex)

• Can be Low risk/benign and also cancerous

Papillomavirus genome structure & organization

10 ORFs

• E = early (DNA replication, viral life cycle, dna rep prep)

• L = late (capsid proteins, late stuff.)

• Control region (promoters, transcriptional regulatory elements)

basal lamina - Papillomavirus

• Infects basal cells via microabrasions

• basal cells are stem-like cells that divide and give rise to differentiated skin cells.

• Early proteins E1/E2 replicate DNA & repress E6/E7

• As cells differentiate, E6/E7 turn on to keep cells in S phase

• E6 and E7 slow down differentiation and keep the cells in S phase, so host DNA replication machinery stays active (benefiting the virus).

• In upper layers, late genes (capsid) expressed → virus assembles

• Virus sheds as cells die → avoids immune detection

• Warts persist due to continuous infection of basal cells

E6 and E7 - Papillomavirus

• Differentiated cells no longer divide so virus must induce S phase to hijack DNA replication factors

• HPV E7 binds retinoblastoma (RB) to displace E2F so E2F goes to nucleus and turns on DNA synthesis genes

• E7 also promotes CDK2 which phosphorylates RB further dissociating it from E2F

• Machinery on, if E2F released before planned it can get detected by p53 which will lead the cell to apoptosis

• Every virus that needs DNA replication needs E2F and also has to deal with p53 activation

• E6 targets p53 in papilloma → it induces proteolysis so p53 is degraded

  • Triggers E6ap ubiquitin ligase, transfers ubiquitin to p53 → degraded

3 phases of DNA replication. Papilloma

• 1) establishment phase: viral genome amplified to 100 copies in undifferentiated basal cells

• 2) maintenance phase: viral genome replicates only once per cell cycle as cell divides, 1 daughter cell remains, 1 differentiates. Gives cell an initial pool

• 3) productive phase: high levels of viralDNA per cell (1000 per cell) in terminally differentiated cells 

E1 and E2 origin binding proteins, induce binding of DNA

Dna linearization and cancer - papilloma

Rarely, the viral DNA linearizes randomly & integrates into host cell genome.

When the break occurs in E2 region, removal of the E2-mediated repression of E6/E7 predisposes cells to transformation (dysplasia): tumors express E6/E7 but usually not E1/E2!

Adenovirus Genome

• Medium sized

• Linear dsDNA

• Has Inverted terminal repeat sequences, used for DNA replication

• Covalently linked protein (terminal protein) = protein primer, used to avoid losing genetic info at extreme ends

• Early and late genes as well

  • (see photo)

• E1A and E1B genes = tumor antigens, coordinate entry into S phase, regulate transcription of other viral genes

• E3 = stealth region of genome, packed with genes that counteract adaptive and innate immune system

adenovirus ca be used in tumor cell killing and cancer immunotherapy

adenovirus attachment and internalization

1. Fiber protein binds to 1° receptors (CAR, MHC I, etc; widespread).

2. Penton base binds integrins (more selective), promoting virus uptake by receptor-mediated endocytosis. Pentons lost.

3. Entry into cytosol is pH dependent. Viral protease cleaves/ activates membrane-permeablizing protein present in virion.

4. Microtubules carry partially uncoated nucleocapsid to nuclear pores, where they bind cellular proteins that promote capsid disassembly and import of viral DNA into the nucleus.

E1A & E3 - adenoviruses

first protein to be made after virus entry and is required for

activating viral transcription and promoting viral DNA replication

no intrinsic DNA binding or enzymatic actvities, it coordinates dozens of celluar proteins

• 32 primary interactions with cellular hub

E1A is intrinsically disordered, and samples various conformations to maximize the number of interactions

• E1A have short linear interaction motifs (slims) = short stretch of amino acids that do adopt a particular structure, these are how it forms a series of connections and un-form them when they're done

• Has conserved regions too, same across all E1A proteins → these must be important & specific

  • 2 of these regions are similar to HPV’s E7 (binds RB), and SV40 (binds RB and p53)

Nucleates formation of the RNAPII preinitiation complex by binding promoter targeting transcription factors and co-activators. (kick off gene expression)

By freeing E2F, E1A activates transcription from a variety of genes containing E2F-binding sites in their promoters, including the adenovirus E2 early promoter

• Binds tumor suppressor Rb to induce S phase, viral & cellular transcription.

e3 = The E3 region encodes proteins that

(1) inhibit immune cell killing pathways and

(2) prevent MHC molecules from reaching the cell surface, shielding infected cells from detection by the adaptive immune system.

adenovirus inactivating Rb and p53

• E1b and e4orf6 use cellular ubiquitin ligases to tag p53 and send to proteasome for degradation

• E1B 55k, binds p53 directly and inhibits activity

• Bax = protein on mitochondria which does cell death when activated

• E1B 19K Bax,

  • Mimics Bcl-2 protein.

  • Inhibits Bax at mitochondria → prevents cell death.

• Methylation machinery can also be required to stop activation

adenovirus genome replication

• Start with dsDNA genome → start using terminal protein which is connected to DCMP nucleotide → position protein primer at extreme end through base pairing between C residue and G residue at end of genome → polymerase comes and primes off of this , copies genome, as copying happening strand displacement happening to strand not being copied → at end an entire strand is displaced and replaced → this is type 1 replication

• After this ^ 

• The ssDNA that got released coated with ssDNA binding protein so it doesn’t fold  back → inv terminal repeats allow it to form panhandle structure which looks like dsDNA end → this region is protein primed to start replication in the same way as type 1 → priming starts, replication starts → boom two dsDNA molecules → type 2

• Important: Protein priming replication, 2 stages of DNA replication

Herpesvirus (general)

Large, enveloped dsDNA viruses,

have cycles of latency and active / lytic replication

• really good at spreading through human population

• Once you get herpes you are infected for life

• can be asymptomatic

• Viruses can be spread with or without visible lesions, through saliva, mucous membranes, mother to child

• Super bad for immunocompromised people

• For most of us we have a balance where we have “co-evolved” with them

• Viruses are species specific, they get specialized for the host they infect

• Once in nucleus → can circularize: form episome = DNA not integrated but tethered to host chromosome, genome preserved till it gets a signal to replicate (silent state) or active

3 families; Alpha, beta, and gamma

Amorphous region between capsid and envelope = tegument

Packed with thousands of viral/cellular protein/RNAs delivered to host cell the second the virus fuses with host cell

Herpes encode their own DNA polymerases

Herpes entry machinery

• Has a ton of viral proteins that all regulate fusion and receptor binding they have to collaborate with each other in ways not fully understood

• G = glycoprotein shown on picture slide 6 (important in red box)

• Not talking about accessory factors

• Main set: 

  • gD = receptor binding undergoes conf change allowing it to bind gH and gL

  • gH & gL complex = acts as an intermediary between gD and gB, transducing signals from the interaction of gD with receptors.

  • gB = gets put into fusogenic state by gL and gH, primary membrane fusion protein, physically fuses the viral and cellular membranes, so virus can enter host cell

tegument proteins (virus entry)

• VHS, VP16, PML bodies, PP71, IE1

• Tegument factors are setting up favorable environment in host cell

• Example of tegument proteins: 

  • VHS = virion host shutoff factor, a nuclease, cuts up RNA

    • Frees up ribosomes so there's no competition, creates an overall immune evasion phenotype, nuclease degrades cellular RNA = kills antiviral encoding transcripts = dampens innate immune responses

  • VP16: transcription activator, kick starts production of immediate early genes which turn on early genes

Some tegument proteins prevent permanent silencing of viral genomes by targeting PML bodies

• PML bodies (host) = substructures in nucleus, contain epigenetic modifiers, silence DNA by putting restrictive methylation marks to condense it, makes it inactive = intrinsic restriction

Evading PML bodies: pp71 tegument protein goes into nucleus and causes dissociation of 2 of epigenetic modifiers present in PML bodies

ie1 = causes dispersal of PML to prevent repressive activity.

Herpes Viral Replication

Blocks the cell in G1 = not known why

DNA replication strat is rolling circle mechanism:

• nick made, DNA synthesis from 3’OH end (discontinuous), keeps going, resulting in 2 double stranded DNA circles?

Virally encoded proteins for genome replication:

✓DNA polymerase (2 subunit)

does NOT encode rna poly

late gene trascription coordinated by a protein mimicking tata binding protein

• The virus makes its own version of TBP (a TBP mimic).

  This mimic can directly bind to viral late gene promoters and bring in RNAPII right away.

  So, it bypasses the need for the rest of the host’s transcription machinery.

• more “Streamlined”

Herpes —> packaging viral genome

viral genome concatemer + procapsid + terminase

terminase binds inverted terminal repeats on viral DNA, takes it to portal on icosahedron (empty procapsid), ATP dependent process —> translocates the DNA, feeds it in until next terminal repeat, nuclease activity cleaves it, portal sealed, DNA packaged icosahedron matured

Herpes exiting the cell

Has to disrupt nuclear lamina to escape into the cytoplasm to capture the tegument

Buds into the nuclear membrane on the nuclear side, Buds out on cytosolic side

Catches tegument factors, buds into transgolgi network, gains its lipid envelope and is they exocysed out of cell

The nuclear membrane is hard to break through, but herpesvirus hijacks CDK activity, just like in mitosis, to weaken it so the virus can escape the nucleus.

herpes replication cycle (overview)

attachment

fusion

microtubular inwards transport

genome injection into nucleus

circularization

latency replication switch

latency

OR

theta replication

Rolling circle replication

Capsid assembly

DNA packaging

nuclear egress

microtubular transport outwards

budding at golgi

release by exocytosis

Herpesvirus latency (general)

Gamma is the only family that is oncogenic (causes cancer)

Alphas establish latency in neurons:

• it traffics along sensory neurons to deposit genome in a quiescent state, no viral proteins expressed.

• They hang out in non dividing cells

  When the host is stressed → triggers virus to reactivate, travels back down axon, releases viral particles, causes local lytic reactivation.

• Cold sores: those are where herpes is latent in

• Beta HV: quasi latency state, hang out in monocytes and macrophages

• Gamma HV: humans pathogens (epstein barr, sarcoma) establish latency in your B-cells, oncogenic. Most of us infected with EBV

B cell differentiation

Virus infects naive B cell

shows version of antibody it will make on cell surface, if it recognizes smt with antibody → becomes active

Virus drives naive b cells to go to germinal center and proliferate as if they did find an antigen → creates a stable long lasting reservoir for the viruses (bc b cell was made into a memory b cell)

From infection to proliferation:

EBV infects a B cell.

LMP1 (a viral protein) gets expressed in the B cell membrane.

LMP1 mimics CD40, a normal receptor that usually requires T cell interaction (via CD40L).

But LMP1 doesn’t need CD40L — it's always “on.”

LMP1 recruits TRAF proteins (TNF receptor-associated factors).

Result: B cell thinks it’s being told by a T cell to grow, divide, and stay alive.

This allows the virus to maintain latency and expand infected B cells without immune activation.

Move out of germinal center and into memory cell

Memory cell divides, viral genome divides with it so it doesn't get diluted out

Doesn’t express viral proteins → undetected

Back into lytic cycle: when b cell gets differentiated into plasma cell which happens bc b cell comes in contact with its matching antigen

how does cancer happen - herpes

Virus needs to drive cell progression in absence of approporate signals

latency program encodes viral proteins with growth stimulating potential

• Oncogenes

Activation of the ‘growth program’ by EBV during B cell activation

poses a cancer risk, but is usually counteracted by the virus and the

immune system

main note: lots of latency genes have oncogenic activity

Epstein–Barr Virus (EBV) and Multiple Sclerosis (MS)

MS = Autoimmune disease, Eats away myelin sheath on nerves → causes tons of symptoms

EBV is main / only driver of MS

This is because of an autoreactive antibody produced during initial burst

Some antibodies against EBNA-1 end up attacking GlialCAM which is what coats the neurons/myelin sheath bc it looks the same

These antibodies end up cross reacting with myelin sheath protein and attack it.

Normally NK cells and T cells kill reactivated EBV infected plasma cells that produce autoreactive antibodies

Sometimes, individuals infected with EBV have immune cells that present a weird form of MHC-1 (HLA-E)

This form (HLA-E) can inhibit NK cell activity when loaded with another peptide from EVB protein LMB-1 (CD40 mimic)

Baculovirus Biology

Used to kill insects, environmentally friendly pesticide, mammalian cell transduction vectors.

liquifies insects…

2 forms:

• Occlusion is released when insect is liquified, this is the one that hits other insects

  • have this extra outer layer for extracellular environment, gets degraded to become budded.

• Budded virus is cell to cell transmission

Budded form buds off from plasma membrane, occlusion bodies carry virions in a crystallized structure which contain a lot of nucleocapsids.

Note:

budded viruses made during early replication, occlusion viruses made during late replication.

Baculovirus Pathology

Infects insects

Insects eat crystals, gets into the gut of the insect, infect epithelial cells in gut of insects, either replicate in epithelial cells (nucleocapsid makes more nucleocapsids) or they pass through epithelial cells into deeper tissues of insect

Budded form and occlusion forms made

(complex life cycle slide ^)

Actin in baculoviruses

Actin facilitates viral movement: Actin filaments help the baculovirus virions move within the host cell by using actin polymerization for intracellular transport.

Virus egress: Actin is involved in the cell-to-cell spread of the virus, helping newly formed virions move toward the cell membrane for budding or cell lysis.

Cell-to-cell transmission: During infection, actin-mediated movement helps viral particles move through the cytoskeleton and spread between neighboring cells, ensuring continued infection.