VIRO 3 | Models for Virus Replication

Explain general viral replication strategies

• All viruses need to hijack host protein synthesis machinery to produce viral proteins and new virions

• But replication strategies differ depending on type of genomes they carry: DNA, RNA, RT DNA/RNA

  • dsDNA = Class I

  • ssDNA = Class II

  • dsRNA = Class III

  • +ssRNA = Class IV

  • -ssRNA = Class V

  • dsDNA RT = Class VI

  • ssRNA RT = Class VII

Explain general DNA virus replication strategies, Herpesvirus as dsDNA virus (Class I)

• General DNA virus replication strategies

  1. Virus needs to transcribe mRNA, which can the be translated into proteins via host cell translation machinery

     1. dsDNA > mRNA > viral protein

  2. Virus needs to replicate its own genome

     1. dsDNA > dsDNA

  3. Some DNA virus can replicate in cytoplasm using viral DNA polymerase

     1. For those who can’t, host enzymes for mRNA synthesis & DNA replication are in nucleus; to avail of these enzymes, they must enter nucleus

• Herpesvirus = dsDNA virus

• Viral structure

  • Enveloped virus with linear dsDNA genome

  • Multiple protein layers ntv

    • At its core = nucleocapsid containing linear dsDNA genome

    • Protein tegument layer = viral proteins with regulatory functions

    • Viral envelope glycoproteins = can act as TFs for immediate early gene expression

• Genome features

  • Long

  • Terminal repeat sequences (L,S)

  • Unique sequences are separated by inverted repeat sequences (L,S)

• Entry & transport to nucleus

  • Once HSV binds to its receptor, viral fusion protein facilitates fusion of viral envelop + plasma membrane, allowing nucleocapsid + tegument proteins to enter

  • Nucleocapsid then uses host cytoskeletal elements (microtubules & motor proteins dynein & kinesin) to reach nucleus

  • Once at nucleopore, it inserts genome into nucleus, where it is circularized to prevent end replication problem & ensure complete genome replication

• Transcription initiation & immediate early gene expression

  • Some tegument proteins enter nucleus via NLS

  • These act as transcription factors for immediate early genes, recruiting host RNA pol II to transcribe viral mRNA

• Early gene expression & DNA replication

  • Immediate early genes trigger expression of early genes, including gene for viral DdDp

  • mRNA for DdDp is exported to cytoplasm for protein translation

  • then imported back to nucleus (via NLS), where it will then replicate viral genome

  • Virus replicates via rolling circle replication strategy, producing concatemers = long DNA strands with multiple genome copies linked together

  • This is later on cut and packaged into capsids

• Capsid assembly & genome packaging

  • Capsid & scaffolding proteins help in forming maturing nucleocapsids

  • Viral genome is packaged into nucleocapsids

  • Packaging signal in viral genome specify which length/segment is included

  • Assembly > nucleus, structural proteins must be imported into nucleus

• Envelopment & maturation

  • Assembled nucleocapsid first buds into nuclear membrane, acquiring temporary envelop

  • It enters ER, removing & re-acquiring envelop

  • Nucleocapsid buds into a vesicle, carrying envelop + viral glycoproteins

  • Vesicles moves into Golgi for further processing

    • Golgi = where it acquires viral glycoproteins

  • Virion is transported into host cell membrane; vesicle fuses with HCM, releasing mature enveloped virion out of host cell via exocytosis

• CPE = Syncytium formation

  • HSV causes syncytium formation (glycoproteins on surface of infected cells bind to neighboring cells > fusion) as CPE

Explain Parvovirus as ssDNA virus (Class II)

• General properties

  • “Parvo” = small; smallest known DNA viruses

  • Nucleocapsid + ssDNA genome

  • Genome = 9 viral genes encode 9 viral proteins (both structural & nonstructural)

  • Bc of its small genome, it relies heavily on host cell machinery

• Tropism

  • It targets rapidly dividing cells bc it needs active host DNA polymerase abundant during S phase of cell cycle

  • In dogs, they infect gut epithelial cells

  • In humans = precursors of blood cells (in bone marrow)

• Dependency: AAV & co-infection

  • Adeno-associated virus (AAV) = kind of parvovirus that requires co-infection w/ adenovirus to replicate

  • AAV = not free-living

  • Not pathogenic and thus widely used in gene therapy, used to deliver foreign genes safely into human cells

• Entry & transport into nucleus

  • Upon binding to receptors, Parvo is internalized via receptor-mediated endocytosis

  • Has phospholipase and thus degrades endosomal membrane, escaping into cytoplasm

  • Uses dynein & kinesin to reach nucleus

  • Bc of its small size, entire nucleocapsid enters nucleus

• Genome structure & rolling hairpin replication

  • Genome is flanked by ITR that forms hairpin structure (forms 3’OH) crucial for replication, as it is recognized by DNA polymerase to initiate synthesis of complementary DNA strand

  • Genome is not circularized and thus not directly used by host polymerases

  • Virus does “rolling hairpin” replication strategy, where ITRs provide 3’OH recognized by DNA polymerase to initiate synthesis of complementary DNA strand

  • 1 strand acts as template to generate more genomes

  • Both viral replication & transcription occurs in nucleus

• Gene expression & protein handling

  • Viral mRNA is exported into cytoplasm for protein translation

  • Nonstructural proteins > nickase cuts 1 strand of DNA to expose 3’ OH region for DNA synthesis

  • Structural proteins (capsid proteins) are then imported back to nucleus for several reasons:

    • Since viral genome replication occurs in nucleus, virion assembly = nucleus

    • Structural proteins risk degradation when left in cytoplasm

    • Cells treat DNA as threat in cytoplasm; degraded by nuclease

    • Importing proteins back to nucleus helps protect DNA & support virion assembly

• Virion assembly & exit

  • Once virions are assembled, these are released via lysis, bursting host cell & releasing viral particles

Explain overview of RNA virus replication strategies

• Classes

  • IV (dsRNA) = Rotaviruses, Reoviruses

    • dsRNA cannot function as mRNA

    • require RdRp in virion

    • bc dsRNA is PAMP (pathogen-associated molecular pattern; molecular structure broadly shared among pathogens but not normally found in host cells and thus would trigger immune response) and thus mRNA would need to be made within protected environment

  • V (+ssRNA) = COVID-19, dengue

    • +ssRNA can function as mRNA and thus would not need RdRp in virion

    • Rather, +ssRNA can act directly as mRNA and encode RdRp later for use

  • VI (-ssRNA) = Influenza, rabies

    • -ssRNA cannot function as mRNA

    • require RdRp in virion

    • RdRp is needed to make mRNA from negative strand

• RNA viruses do not have a DNA phase, unlike retroviruses

• RNA viruses bypass normal cell processes bc cells don’t usually use RNA as template to make more RNAs

  • Instead, they rely on RNA-dependent RNA polymerase, which is a viral protein that doesn’t have a cellular equivalent; only virus can encode it

Explain Rotavirus as dsRNA virus (Class III)

• Structure

  • Has dsRNA genome = PAMP and thus would trigger immune responses > TLR

  • Has multilayered capsid = inner, intermediate, outer protein layer to keep dsRNA hidden from host cytoplasm

• Entry & uncoating

  • Upon binding to receptors, Rotavirus is internalized via receptor-mediated endocytosis

  • Within endosome, outer protein layer is degraded, thus releasing a double-layered particle (DLP) into host cytoplasm

• Transcription & replication

  • Transcription begins in DLP to hide dsRNA from cytoplasm

  • Each RNA segment is associated with RdRp; mRNA is transcribed then extruded into cytoplasm for protein translation

  • +RNA transcript = template for protein synthesis; dsRNA replication happens within protected particle

• Assembly & maturation

  • Newly synthesized viral proteins & RNA segments are assembled into DLPs in viroplasm

  • dsRNA synthesis occurs in assembling DLPs

  • DLPs acquire another protein coat as it passes thru rough ER, which will be proteolytically cleaved as they exit host cell upon maturation

  • Design ensures dsRNA = never exposed to host cytoplasm

• Host defenses = RNA interference (RNAi)

  • Host combat viral infection via RNAi

  • RNAi = post-transcriptional gene silencing mechanism that’s highly sequence-specific

  • Uses Dicer protein that cleaves viral dsRNA into shorter segments, then loads it into RISC

  • In the RISC,

    • One strand is degraded; the other is used as guide to bind to complementary mRNA

    • Perfect complementarity = mRNA being cleaved & degraded

    • Partial complementarity = translation is repressed; ribosomes cannot bind

  • RNAi = a mechanism that prevents viral protein production & antiviral mechanism across many organisms

• RNA silencing therapeutic use

  • RNAi can be harnessed to target viral mRNA, causing either:

  • Post-transcriptional gene silencing

    • For COVID-19, scientists are introducing small interfering RNA (siRNA) that guide RISC to complementary viral mRNA, allowing it to be cleaved & degraded

  • Transcriptional gene silencing

    • For HIV, scientists are introducing synthetic dsRNA that activate histone deacetylases, causing DNA to tighten and not be read; thus mRNA is not produced

Explain +ssRNA (Class IV) viruses

• General properties

  • +ssRNA can immediately act as mRNA

    • Upon entry into cell host, ssRNA can serve as mRNA & be immediately translated by host ribosome into

    • Polyproteins 1a & 1b > cleaved into nonstructural proteins > RdRp

  • +ssRNA thus don’t need RdRp packaged in virion

    • Instead, they encode it into their genome, such that after translation > RdRp is synthesized

  • Once RdRp is synthesized, viral genome replication occurs

    • RdRp uses +ssRNA as template to create -ssRNA

    • -ssRNA is used as template to create more -ssRNA genomes

  • -ssRNA can be converted into transient dsRNA

    • Complementary - strand of dsRNA is used as template to produce more +ssRNA for genome amplification to ensure there’s enough viral RNA for virion packaging

  • +ssRNA has multiple roles in virus life cycle

    • act as mRNA for immediate translation

    • template for synthesis of -ssRNA

    • template for synthesis of more +ssRNA for new virions

• e.g., Picornavirus

  • “Pico” = small, RNA virus

  • Small, non-enveloped +ssRNA viruses

  • e.g., Poliovirus, rhinovirus

  • +ssRNA > mRNA transcript = no need for prepackaged enzymes

• e.g., SARS-CoV-2

  • enveloped +ssRNA virus with large single genome

  • Genome encodes sps

    • Structural proteins: spike, envelope, membrane, nucleocapsid

    • Polyproteins (1a, 1ab): cleaved into NS proteins

    • Subgenomic RNA (copies of viral genome; accessory & structural protein-coding region) for expression of structural & accessory proteins

  • Significance of subgenomic RNA = allows for separation of early non-structural (NS) protein production then later structural (S) protein synthesis

    • mRNA translation produces polyproteins 1a & 1ab, cleaved into nonstructural protein > RdRp used for genome replication

    • 6 nested subgenomic mRNAs with 5’ leader sequence produce structural proteins after replication & transcription begins

• Fusion & Entry

  • Fusion between viral envelope & host cell membrane occur in 2 ways:

    • At cell surface, when proteases like TMPRSS2 activates spike protein

    • Within endosome, after endocytosis & spike activation in acidic vesicles

  • Once inside cytoplasm, translation begins immediately bc +ssRNA > mRNA transcript

• Replication in DMV

  • Genome replication occurs in double-membrane vesicles, which has 2 critical functions

    • Immune evasion = DMV prevents host immune system from recognizing dsRNA intermediates

    • Efficient genome production = DMV concentrates replication machinery & templates within confined space, speeding up & increasing amount of viral mRNA synthesis for assembly

Explain -ssRNA viruses (Class V); Influenza viruses

• Before the COVID-19 pandemic, Human Parainfluenza Virus (HPIV) was predicted to cause next outbreak

• Influenza virus genome structure

  • IAV consists of segmented genome

  • 8 RNA segments, from longest to shortest

    3P H NNMN

    • PB2

    • PB1

    • PA

    • Hemagglutinin

    • Nucleoprotein

    • Neuraminidase

    • Matrix protein

    • Nonstructural protein

  • Each RNA segment is

    • packaged in nucleocapsid

    • capped w/ polymerase complex that includes endonucleases that snatches 5’ cap of host mRNA to prime mRNA synthesis

  • Each RNA segment encodes specific protein

    • Hemagglutinin = 18 subtypes & binds to sialic acid receptors on host cells

    • SA receptors are host-specific, affecting tropism

    • Important epidemiologically due to their role in host infection & immune recognition

• Influenza pandemics & immunity

  • Multiple influenza pandemics have occurred, starting with 1918 flu

  • Caused by lack of natural immunity to new HN subtypes

  • Even slight changes in subtypes > immune evasion & outbreaks

• Segmented genome & infectivity

  • Infectivity requires all RNA segments to be present

  • Packaging signals ensure correct assembly, enabling RNA-RNA & RNA-protein interactions

    • RNA-structural proteins

      • RNA-capsid proteins

      • RNA-envelop proteins

  • These interactions are important bc during budding, these ensure that all segments are included

  • Nucleocapsid are assembled perpendicular to host plasma membrane to ensure

    • Proper genome packaging

    • Correct interactions with envelope proteins

    • Efficient budding

• Why segmented genomes matter > Reassortment

  • Bc segmented genomes can lead to reassortment that allow rapid evolution, particularly

    • If host is infected by 2 influenza viruses, the genome segments (H1N1, H5N1) can mix and result in strain that can evade immune recognition & cause outbreaks

  • Pigs are critical “mixing vessels”

    • Can be infected by both avian & influenza virus, thus reassortment may result in new strain that is transmissible to humans & can evade immune recognition

• Antigenic shift vs. drift

  • Shift

    • Occurs when 2 strains infect same host cell, leading to reassortment & creation of new virions that can evade immune system > outbreaks

  • Drift

    • Involves random mutations over time due to error-prone RdRp that lacks proofreading abilities

    • Occurs in both segmented, non-segmented RNA genomes

    • Reduce overall antibody effectiveness

• Multihost ecology of H5N1 (highly pathogenic influenza virus)

  • Reservoir host: aquatic birds; virus circulates stably among them

  • Spread to: poultry, seabirds, mammals, humans (occasionally)

  • Recently detected in: mink, cows, sea mammals, goat, cats

  • Global spread

    • Australia = only avian virus-free continent

• Recent H5N1 developments in US

  • Cattle outbreak (2013)

    • Spillover from wildlife to cattle

    • Cattle-cattle transmission > shared mill equipment

    • Farm-farm > animal transport

    • Domesticated animals

  • Not highly pathogenic in humans, except immunocompromised

  • Still no sustainable human-human transmission

  • Surveillance is critical, especially in cattle

  • Hypothetical concern: If US farmer gets infected with both H5N1 and seasonal H1N1, this could lead to reassortment & create strain capable of human-human transmission

• Avian influenza H7N9

  • Detected first in China

  • Infects both birds & humans

  • Limited person-person transmission occurred

• RNA viruses & mutation rates

  • RNA viruses tend to have high mutation rates due to lack of proofreading of RdRp, leading to:

    • genetic drift & loss of immune recognition

    • reassortment (segmented genomes)

    • recombination, causing large-scale genetic change (genetic shift)

Explain overview of RT viruses

• Viruses that use reverse transcription

  • Class VI (ssRNA-RT) = RNA is reverse transcribed into DNA

  • Class VII (dsRNA-RT) = Has gapped DNA genome that undergoes reverse transcription during replication

• Reverse transcriptase

  • Enzyme that synthesizes DNA from RNA template

  • Highly conserved across species

  • Has catalytic site where nucleotide triphosphates are added to growing DNA strand

  • Not unique to viruses; exist in many organisms

• Activities of RT

  • RdRp

    • Synthesizes DNA strand from RNA template

    • Primer-dependent

    • Produces RNA-DNA hybrid

  • RNase H

    • Degrades RNA strand in hybrid

    • Endonuclease function

  • DdDp

    • Synthesizes complementary DNA strand, making a dsDNA

• Broader importance of RT

  • RT is found in all domains of life

  • In humans, telomerase is a RT that solves the end-replication problem in linear chromosomes; carries an RNA template that extends 3’ ends of DNA to prevent shortening

  • Retrotransposons = fossil evidence of RT activity in genomes

    • Present in archaea, supporting idea of UCA

• RT & RNA world hypothesis

  • RNA preceded DNA

    • RNA = both catalytic & information functions

  • RT may have bridged RNA-based life to DNA-based life

  • RNA can catalyze its own replication & protein synthesis, e.g., ribozyme activity of rRNA in ribosomes

  • DNA became permanent genetic storage due to stability of double-stranded structure

Explain ssRNA-RT (Class VI); HIV

• Structure

  • Enveloped virus

  • 2 +RNA copies ver virion

  • Infects CD4+ T cells & macrophages

• Entry

  • Binds to host receptors

  • Fusion protein mediates entry

  • Capsid uncoats in cytoplasm

• RT

  • 1st strand synthesis = uses host tRNA primer bound at pbs for RT DNA from RNA template

  • RT degrades RNA; synthesizes complementary DNA strand

  • End up with linear dsDNA with long terminal repeats imported to nucleus for integration

• Integration

  • Integrase inserts viral DNA into host genome

  • Permanent / cannot be excised

  • Can now be transcribed to produce

    • Viral proteins

    • RNA genome

• Transcription & assembly

  • Alternative splicing produces different regulatory/accessory proteins unique to HIV

  • Common

    • Gag = capsid protein

    • Pol = RT+integrase fusion

    • Env = env protein

  • Gag-Pol fusion protein may form

• Maturation

  • HIV are not infectious right after budding

  • Proteolysis of polyproteins are required postbudding to mature

  • Proteases inhibitors = target of HIV therapy

• Why is HIV diploid?

  • 2 RNA genomes linked via kissing loop complex

  • Only 1 RNA template is used for RT

  • Template switching allows

    • Recombination during DNA synthesis

    • Increased diversity

  • High mutation + high recombination = highly diverse virus population within 1 host

• Molecular steps in RT

  • Host tRNA primer binds near 5’ end; extension space is short

  • Extension occurs, switching to 3’ end due to sequence similarity

  • Purine-rich tract (from degraded RNA) serves as primer for +strand synthesis

  • Template switching again as synthesis proceeds

  • End up with linear dsDNA with LTRs > promote integration

• Consequences of integration

  • U now have permanent viral DNA in host genome

  • Retrovirus can be passed onto next generation if it infects germ cells > endogenization

• Endogenization = provirus becomes part of germline DNA

  • Over time, they lose their ability to replicate

  • Endogenous retroviruses = ancient, non-replicating viral DNA

  • Retrotransposons = ancient RT elements, lacking envelop and cant form virions

  • 42& of our genome = retroviral

• Disease & oncogenesis

  • If integration occurs near cell cycle genes, this may activation oncogenes > tumor formation (Rous Sarcoma, Murine Leukemia)

• HIV progression & therapy

  • Initially = high viral load + sharp CD4 drop

  • Latency period follows

  • AIDS onset occurs after gradual CD4 depletion

  • Antiretroviral therapy

    • Reduce viral production

    • Prevent CD4 depletion

    • Prevent immunosuppression

  • HIV drug targets ripf

    • Reverse transcriptase

    • Integrase

    • Protease

    • Fusion protein

Explain dsRNA-RT (Class VI); Hepatitis B virus

• Structure

  • Enveloped virus with partial dsDNA genome (gapped)

  • + strand = incomplete, linked with short RNA with 5’ cap

  • - strand = covalently linked to viral polymerase

• Why genome is gapped

  • RT happens inside virion after capsid is closed during assembly

  • RT begins after packaging and does not finish > gapped dsDNA

• Entry

  • Viral entry > import to nucleus

  • Host enzymes in nucleus repair/seal gap, forming relaxed circular DNA > template for transcription

• Vesicle production in HBV infection

  • Infected cells release many non-infectious vesicles that display surface proteins of virus but do not contain genome

  • This outnumbers infectious vesicles, distracting immune system and assisting in immune evasion

Explain viroid as subviral element

• Structure

  • Only naked RNA, circular, single-stranded, noncoding

  • Does not code any proteins

  • High secondary structure, which makes them stable & capable of ribozyme-like activities

• 2 families

  • Pospovirioidae

    • Rod-like shape

    • Replicates in nucleus

    • Has central conserved region

  • Avsunviroidae

    • Hammerhead ribozyme (self-cleaving structure)

    • Replicates in chloroplast

• Transmission & replication

  • Entry: mechanical damage (plant wounds)

  • Spread: cell to cell via plasmodesmata; long-distance via phloem

  • Transmitted vertically thru seeds/pollens; horizontally

• Replication mechanism

  • RNA → RNA via RdRp from host

  • DdRp

• Example

  • Potato spindle tuber viroid – causes stunted, spindle-like potato plants

  • Cadang-cadang

Explain satellites & satellite viruses as subviral element

• Subviral agents that require “helper virus” for replication

• Cannot replicate or form capsids independently

• 2 forms

  • Satellite nucleic acids (NA)

    • Do not encode capsid proteins

    • Instead, they’re packaged inside helper virus’ capsid

    • Some encode nonstructural proteins

  • Satellite viruses

    • Have their own capsids (complete virions)

    • Still require helper virus for replication enzymes

• Difference from defective viruses

  • Defective viruses have homology with parent virus; they’re mutated forms of original viruses

  • Satellites are unrelated to helper virus genomically

  • e.g.,

    • Satellite = phage P4

      • Helper = phage P2

    • Satellite = AAV

      • Helper = Adenovirus

Explain AAV as subviral element

• Naturally dependent on adenovirus for replication

• Without adenovirus, it will be integrated into host genome

• Upon future adenovirus infection, integrated AAV will be rescued & resume replication

• Gene therapy

  • AAV is engineered

    • Replication genes replaced with transgenes

    • Helper functions are provided in packaging,

    • making AAV an efficient vector for gene delivery

Explain virophages as subviral element

• Infect protist hosts and require co-infection with giant viruses (NCLDV – nucleocytoplasmic large DNA viruses)

• Examples:

  • Helper Virus: Mamavirus, CroV

  • Virophage: Sputnik, Mavirus

Key Features

• Replicate in host cytoplasm

• Do not replicate inside helper virus, but depend on its transcription machinery

• Reduce replication efficiency of the helper virus → beneficial to host survival

Evolutionary Implications:

• Related to DNA transposons like Polintons/Mavericks

• Possibly originated from virophages integrated into host genomes

• These elements can be vertically transmitted, leading to diversity and expansion in host genomes

Explain Hepatitis Delta Virus as subviral element

• Small circular RNA virus requiring Hepatitis B Virus (HBV) as helper

• Co-infection with HBV leads to more severe disease

• Like viroids, it is RNA-only and doesn’t encode a polymerase

• Uses the envelope and coat proteins of Hepatitis B in order to complete its replication cycle

Current Mystery:

• Other delta-like agents have been found via transcriptomics in different species

• Their helper viruses are unknown or possibly not required

Explain prions as subviral element

• Discovered by Stanley Prusiner

• Protein-only infectious agents—no nucleic acid

• Cause neurodegenerative diseases called Transmissible Spongiform Encephalopathies (TSEs)

Mechanism of Action

1. Normal prion protein (PrPᶜ) changes conformation into disease form (PrPˢᶜ)

2. PrPˢᶜ is stable, resists degradation, and induces others to misfold

3. Leads to accumulation of amyloid plaques in the brain → neurodegeneration

4. No immune response is mounted

Diseases

• Scrapie – first TSE in sheep

• Bovine Spongiform Encephalopathy (BSE) – Mad cow disease, spread via contaminated feed

• Kuru – in humans due to cannibalism

• Creutzfeldt-Jakob Disease (CJD) – human TSE; variant CJD (vCJD) linked to BSE

Types of TSEs

• Infectious

• Familial (inherited mutation in prnp)

• Sporadic (random conversion to PrPˢᶜ)

Transmission & Entry

• Prions can cross epithelial barriers

• Taken up in gut (Peyer’s patches) and transported to the brain

Current Concern (USA):

• Chronic Wasting Disease in cervids (deer family) linked to prion-contaminated soil