VIRO 2 | Entry (Adsorption, Penetration, Unocoating)

General steps to virus replication cycle

apunmr

1. Adsorption

2. Penetration

3. Uncoating

4. Maturation (assembly)

5. Release

_ refers to the process when virus particles attach to molecules on host cell surface

Adsorption

Host cell receptors can be _, serving various cellular functions, e.g., receptors, adhesion molecules, transporters rat

pcl

• Proteins

• Carbohydrates

• Lipids

The first virus receptor identified was the receptor of _

bacteriophage lambda (responsible for maltose uptake)

T/F: Some viruses do not require adsorption to infect

TRUE

Some viruses enter through cell damage and thus do not need to adsorb to host cell surface molecules

T/F: Virions can simply diffuse across host lipid bilayer

FALSE

Virions are too big and cannot make use of amphiphilic nature of lipid bilayer, protein channels, nor enter directly (unless they enter via cell damage)

T/F: There is usually specific virion-host receptor interaction

TRUE

T/F: Even if the host doesn’t have the receptor for a specific virus, the virus can still enter the cell, except for fungal/yeast and plant viruses

FALSE

Viruses generally cannot enter host cell if host cell doesn’t have receptor specific to virus

Host cell receptors can be proteins, carbohydrates, lipids, serving various cellular functions, including _

rat

• Receptors

• Adhesion molecules

• Transporters

There is usually a specific virion-host receptor interaction, except for _

• Fungal and yeast viruses bc they don’t have an extracellular phase and are just vertically/horizontally passed on from one fungal cell to another

• Plant viruses bc they enter via cell wall damage by arthropod vectors

T/F: All animal viruses require host cell receptors

TRUE

_ is one of the earliest identified receptor for animal virus, e.g., influenza

Sialic acid

Enumerate and explain some examples of attachment factors and receptors

• Attachment factors

  • Heparan sulfate proteoglycan

  • Sialic acid = glycoprotein

• Receptors

  • HIV = CCR5, CXCR4

  • Adenovirus = Car, Integrin

T/F: It is possible to have two receptors, one of which is a co-receptor required for attachment and entry of virus

TRUE

e.g., HIV

T/F: Galactose, sialic acid, GlcNAc, GalNAc, and glucose are some common components of host cell receptors

TRUE

The attachment of all influenza A virus strains to cells requires _ as receptors

sialic acids *terminal sugar attached to oligosaccharide on glycoprotein

_ is the one that recognizes sialic acid on host cell surface

Hemagglutinin (HA)

Explain the different sialic acids recognized by influenza viruses

• Avian influenza virus = a(2,3) linked sialic acids in duck gut

• Human influenza virus = a(2,6) linked SA in human respiratory epithelial cells, but a(2,3) linked is also found in ciliated epithelial cells in a minor population in human resp tract

• Porcine = both a(2,3) & a(2,6) in trachea; hence, a melting pot for emergence of new viruses

T/F: Humans can be directly infected by avian influenza virus

TRUE

Bc we have a(2,3) linked SAs recognized by avian influenza viruses, but it’s in ciliated epithelial cells in our respiratory tract, thus the chances are low bc most likely, this will have been combatted already by our immune system before it gets to respiratory tract

T/F: The attachment of all influenza A virus strains to cells require heparan sulfate proteoglycan as receptors

FALSE

The attachment of all influenza A virus strains to cells requires sialic acids as receptors

What makes pigs a melting pot for emergence of new viral diseases?

Pigs express both a(2,3) and a(2,6) linked SAs, thus allowing them to have receptors that can be recognized by avian and human influenza and subsequently making them a “mixing vessel” for influenza viruses, as this can facilitate reassortment and the potential emergence of new pandemic strains

How is a farm a very important surveillance point for influenza viruses?

This is where the interaction between 3 species with different sialic acid receptors happens, potentially facilitating reassortment and resulting in the emergence of new viral diseases

T/F: Highly pathogenic avian influenza (HPAI) viruses were found to directly infect cows and humans

FALSE

Bird > cow, cow > humans
Humans were not directly infected

T/F: There is evidence for avian influenza virus spreading from humans to humans

FALSE

Sequence of SARS-CoV-2, including its receptor-binding motif (RBM) that directly contacts _, is similar to SARS-CoV, suggesting that SARS-CoV-2 uses _ as its receptor

ACE2

While phylogenetic analysis indicates a bat origin of SARS-CoV-2, SARS-CoV-2 potentially recognizes _

ACE2 from various animal species, except from mice and rats (whose ACE2 receptors do not effectively bind SARS-CoV-2)

T/F: No human to human spread of avian influenza virus (H5N1) has ever been detected in dairyhouse in US in 2019

TRUE

Bc there are very low chances for bird > human transmission

Explain the different interactions existing between viruses/virus surface proteins and their host receptors

• Virus (surface proteins) can recognize multiple host receptors

  • V1 → R1, R2, or R3

  • e.g., Herpes simplex virus type 1 (HSV-1)

• Different viruses can use the same receptor

  • V1, V2 → R1

  • e.g., Coxsackie & Adenovirus → Car (Coxsackie-Adenovirus receptor)

• Some viruses bind to more than 1 receptor (receptor + co-receptor)

  • V1 → R1 & R2

  • e.g., HIV → CD4+ & CCR5 (macrophage) CXCR4 (T-cells)

    • receptor + co-receptor

  • e.g., Zika virus → Axl & Gas

    • soluble protein + receptor

Zika virus is unusual because for it to bind to a host cellular receptor (Axl), it must first _

bind to a soluble protein (Gas6)

Explain adsorption receptor + co-receptor binding principle/process in HIV

• gp120 protein in HIV viral envelope specifically interacts with CD4 host cell receptor, causing a conformational change in gp120 that exposes a hidden binding site for a co-receptor

• Conformational change in gp120 leads gp120 to interact initially with CCR5 (macrophage-tropic), later on switching to CXCR4 (T-cell-tropic)

• Binding of gp120 with co-receptors then triggers gp41 to insert its fusion peptide into host cell membrane, facilitating fusion of viral envelope + host cell membrane

*1% of Caucasian human population carry 32-bp deletion in gene coding for CCR5, thus individuals homozygous for this deletion (32-CCR5 allele) = resistant to HIV infection

Explain the change in cellular tropism in HIV as disease progresses

• gp120 first binds to CD4 host receptor, triggering conformational change that allows co-receptor binding

• Initially (early in the infection), gp120 binds to CCR5 receptor in macrophages, thus B-chemokines can inhibit this binding

• Later on, gp120 binds to CXCR4 receptors in T-cells, causing severe immunodeficiency; a-chemokines inhibit this binding

_ inhibits binding of gp120 in HIV to CCR5 (macrophages)

B-chemokines

_ inhibits binding of gp120 in HIV to CXCR4 (T-cells)

a-chemokines

Sequence of SARS-CoV-2, including its _ that directly contacts ACE2, is similar to SARS-CoV, suggesting that SARS-CoV-2 uses ACE2 as its receptor

receptor-binding motif (RBM)

• _ that block the interaction of HIV-1 envelope with CCR5 or CXCR4 potently inhibit HIV-1 in vitro

• Pilot studies of _ have provided proof of concept for this novel drug class; phase III safety and efficacy trials are underway

• Chemokine receptor antagonists

• orally bioavailable small molecule CCR5 inhibitors

Enumerate three CCR5 receptor antagonists currently being developed

mva

• Maraviroc = Pfizer (2007)

• Vicriviroc = Schering Plough

• Aplaviroc = GlaxoSmithKline

What stabilizes virus-receptor interaction?

• Each virion has multiple sites that can bind to receptors (high avidity)

• Initially, attachment is reversible since the bond is noncovalent and there will only be a few attachment/interactions, but over time, as this increases, the virus-receptor interaction becomes more stable and eventually irreversible

Explain adsorption (n & e) viruses + virus proteins for attachment & fusion + host receptors

Viruses	Virus proteins (for attachment; fusion)	Host cell receptors
N: ~90% human rhinoviruses	VP1+VP3	ICAM-1
N: ~10% human rhinoviruses	VP1	LDL receptors
N: Polioviruses	VP1	CD 155
E: Murine leukemia	surface gp; transmembrane gp	Mouse cationic AA transporter
E: HIV-1	gp120; gp41	CD4
E: Influenza A & B	Hemagglutinin (HA); HA	Sialic acids with gp
E: Measles	HA; fusion protein	CD 150

1% of Caucasian human population carry _, thus individuals homozygous for this deletion are resistant to HIV infection

32-bp deletion in gene coding for CCR5

Explain how to gather experimental evidence to determine if a cell surface molecule is a virus (host cell) receptor

nmse

• If the normal ligand for cell surface molecule blocks virus binding / infectivity, e.g., excess CD4 ligands prevent HIV from binding to CD4, then this implies that HIV competes with CD4 ligands and CD4 is a receptor

• If the monoclonal antibody against cell surface molecule blocks virus binding/infectivity, e.g., anti-CD4 antibodies prevent HIV from binding to T-cells, then this implies that CD4 is required for HIV entry

• If a soluble derivative of cell surface molecules blocks virus binding / infectivity, e.g., soluble CD4 proteins bind HIV and prevent its infection; CD4 is a receptor

• If the expression of gene encoding cell surface molecule in a virus-resistant cell causes that cell to be susceptible to infection, then this suggests that CD4 is required for HIV entry

_ is an inherent barrier to infection, e.g., mice and rats cannot get infected with SARS-CoV-2 because their ACE2 receptors do not effectively bind the said virus

Lack of membrane receptors

T/F: Murine models can be used for studying mechanisms of SARS-CoV-2

FALSE

This cannot be used because mice are resistant to SARS-CoV-2 because their ACE2 receptor do not effectively bind the said virus

_ is sufficient to make the cells susceptible to infection

Providing the receptor protein

Explain acquisition of phage sensitivity by bacteria through exchange of receptors (adsorption > membrane receptors = susceptible to infection)

• Phage-resistant (R) bacteria become lysed when co-cultured with sensitive (S) bacteria

• R cell lysis is triggered by phage lysins released from nearby infected S cells

• Occasionally, phages invade R cells only when co-incubated with S cells and this is because invasion is mediated by R cells gaining attachment molecules from S cells (most likely via HGT)

  *Gain of attachment molecules allow entry of phages that cause lysis

  *B. subtilis, lytic phage SPP1

_ refers to the phenomenon of resistant cells infected by phages in mixed cultures

acquisition of sensitivity (ASEN)

_ is when purified phage lysis proteins lyse cells from the outside, even in an interspecies manner

“Lysis from without”

T/F: The presence of phage-sensitive population within bacterial community could dramatically impact population dynamics via LO and ASEN

TRUE

Explain some therapeutic strategies conceptualized for 2019-nCoV

Introduce:

• Soluble components of RBD of SARS-CoV-2 to block its binding to receptor

• Antibody fragments specific to receptor to block virus binding

• Antibody fragments which bind to virus, blocking binding of virus to receptor

• Ligands binding to receptor and thus preventing virus from binding to receptor

_ are specifically designed to target the receptor binding domain (RBD) of SARS-CoV-2, preventing the virus from binding to the receptor and thus neutralizing it

Monoclonal antibodies

T/F: There is constant host-virus arms race

TRUE

How come mutations that prevent viral interaction with host haven’t predominated in the population?

• It’s because there is a limitation to the amount of mutations that virus receptors can undergo, given that these still have normal functions within the host body and thus cannot constantly undergo mutation

• Hosts typically have slower mutation rate compared to viruses

• Red queen hypothesis = co-evolution

T/F: Naked viruses typically have different proteins for attachment to receptors and fusion with host cell membrane

FALSE

Enveloped viruses typically have different proteins for attachment to receptors and fusion with the host cell membrane

_ refers to the human-specific receptor variant that confers partial protection from severest rhinovirus (RV-C)-induced asthma episodes

CDH3-Cys529

Influenza is an enveloped virus undergoing penetration and uncoating via _

entire virus taken up via receptor-mediated endocytosis > fusion & uncoating

_ is a disease that causes wheezing illness and asthma exacerbations

Rhinovirus (RV-C)

HIV is an enveloped virus undergoing penetration and uncoating via _

fusion of viral envelope + host cell membrane > uncoating of viral genome

All animals are known to maintain a receptor gene (for RV-C) with high level of sequence conservation known as _

CDH3-Tyr529

T/F: CHD3-Tyr529 increases risk of wheezing illness compared to human WT CDH3-Ser529

FALSE

CHD3-Tyr529 increases risk of wheezing illness compared to human WT CDH3-Cys529

_ refers to the entry of viruses into cells and removal of capsid

Penetration and uncoating

Explain penetration and uncoating strategies of enveloped viruses vs. naked viruses

• Enveloped

  • HIV = fusion > uncoating

    • Fusion of viral envelope with host cell membrane, leading to subsequent uncoating of viral genome, so that it’s transported to host cell

  • Influenza = endocytosis > fusion & uncoating

    • Entire virus is taken up via receptor-mediated endocytosis, then pH levels in endosome trigger fusion of viral envelope + host cell membrane, leading to uncoating of viral genome

• Naked

  • Poliovirus = partial degrading of capsid > extrusion of NA

    • Capsid is partially broken down, then NA is extruded into host cell

  • Adenovirus = endocytosis > partial degrading of virion > uncoating & delivery to nucleus

    • Naked virus is taken up via receptor-mediated endocytosis

    • Inside cell, partially degraded virion is directed to nuclear envelope where genome is uncoated and delivered to nucleus

_ refers to the phase when virion is uncoated and nucleocapsid is released into host cell, thus no intact virions are detected inside the cell, lasting for as long as there is still no new assembled viral particles in the host cells

Eclipse phase

Give 3 examples of viruses that penetrate host cell via direct fusion of viral envelope with host plasma membrane

phh

• Paramyxoviruses

• Herpesviruses

• HIV

Elaborate on virus penetration via direct fusion of viral envelope with host plasma membrane (in enveloped viruses)

afn

• Occurs after attachment to host cell receptor

• Fusion is promoted by fusion protein

• Nucleocapsid is directly released into cytoplasm

e.g., Paramyxo, herpes, HIV

_ facilitates the fusion of viral envelope with host plasma membrane

Fusion proteins

• e.g., transmembrane proteins in murine leukemia,

• gp41 in HIV

• HA in influenza A & B

• fusion protein in measles

T/F: Fusion sequence of fusion proteins is mostly made up of hydrophobic sequences

TRUE

So it can insert itself into target membrane

Why is fusion protein normally hidden away?

*Normally (in HIV), fusion protein is only accessed after binding of attachment viral protein (gp120) to co-receptor (CCR5, CXCR4)

• For the fusion process to be regulated because if you just have a series of hydrophobic AA sequences exposed, then virion can just fuse to any nearby lipid bilayer

T/F: Surface subunit (SU) is gp120 and transmembrane subunit (TM) is gp41 in HIV; both are viral proteins but only gp120 interact to cell receptors

TRUE

Explain how fusion occurs in an HIV-1

• Binding of HN protein to its cellular receptor induces conformational changes, which triggers also a conformational change in fusion protein, exposing fusion peptide and making protein fusion-competent

• Binding of gp120 (SU) to CD4 exposes a high-affinity chemokine receptor-binding site on SU

• SU-CCR interaction leads to conformational changes in gp41 (TM) that exposes fusion peptide and permits it to insert into cell membrane, catalyzing fusion

In HIV-1, the conformational change that exposes fusion sequence is triggered by _

virus binding to a receptor

_ leads to conformational change in TM (gp41) that expose fusion peptide, allowing it to be inserted into cell membrane and catalyzing fusion

SU(gp120)-CCR interaction

• In HIV-1, _ exposes a high-affinity CCR5-binding site on gp120

• _ interaction leads to conformational change in gp41 that exposes fusion peptide and permits it to be inserted into cell membrane, catalyzing fusion

• Binding of gp120 (viral attachment protein) to CD4 (host cell receptor)

• gp120-CCR5

T/F: During the fusion process in HIV-1, the C-terminal region (HR2) anchored in viral membrane undergoes B-helical transition and runs alongside outside of core

FALSE

During the fusion process in HIV-1, the C-terminal region anchored in viral membrane undergoes a-helical transition and runs alongside outside of core

A minimum of _ are necessary to achieve membrane fusion

Two gp41 trimers

In hemifusion intermediate in HIV-1, only _ mix

outer leaflets of cell and viral membranes

What happens during complete fusion in HIV-1?

• Both inner and outer leaflets mix

• Viral contents penetrate the cytosol

Give 1 strategy through which gp41-mediated fusion may be inhibited in HIV-1

• By introducing peptide inhibitor T-20, which occupies the site alongside the trimer core and prevents the a-helical transition of the C-terminal helix

• 5m038 also blocks a-helical transition

Explain what occurs in penetration via endocytosis of virion by host cell

• Host cell plasma surrounds whole virion and forms a vesicle

• Low pH causes virion envelope to fuse with endosome membrane of vesicle via fusion proteins

• Nucleocapsid is released into cytoplasm

*Occurs in influenza viruses, rhabdoviruses

Give examples of viruses that undergo penetration via endocytosis of virion by host cell

Enveloped viruses

• Influenza viruses

• Rhabdoviruses

T/F: Sperm-egg fusion proteins are homologous to viral class I fusion proteins

FALSE

Sperm-egg fusion proteins are homologous to viral class II fusion proteins

Penetration (enveloped): Clathrin-mediated vs. Caveolin-mediated endocytosis

• Clathrin-mediated

  • Virus binds to specific host receptor, triggering formation of clathrin-coated pits

  • Pit buds inward, forming clathrin-coated vesicles that transport vesicle into cell

  • Vesicle uncoats, fuses with early endosomes, undergoes acidification (via proton pump), which triggers release of viral genome into cytoplasm

• Caveolin-mediated

  • Virus binds to caveolae, then transported via caveolin-coated vesicles into compartments

  • Does not require acidification for viral genome release

For fusion to take place in influenza virus (enveloped), _

• Low pH of endosome can directly alter conformation of fusion protein OR

• it may activate a cell protease that cleaves the fusion protein

Enumerate 3 circumstances that cause conformation change in the fusion protein, allowing it to be inserted into the host cell membrane

• Binding of virus to receptor gp120-CCR5 interaction (in HIV-1)

• Low pH of endosome directly altering conformation (in influenza)

• Activation of protease that cleaves fusion proteins (in influenza)

T/F: Hemagglutinin is a type-1 fusion protein because it is parallel to viral envelope

FALSE

Hemagglutinin is a type-1 fusion protein because it is perpendicular to viral envelope

Explain penetration of ebola virus

• Ebola virus doesn’t have a very specific host receptor and most likely taken up by pinocytosis

• Once inside endosome, there’s cleavage of lipoproteins, exposing it to endosomal-membrane receptors, allowing fusion that releases the nucleocapsid into the cytoplasm

• Different bc acidic conditions of endosome don’t change viral receptor but digests mucin cap, hiding receptor from binding to host cell receptor

*Dengue virus has similar mechanism

Envelope proteins of dengue is type _ because it is parallel to viral envelope

2

*Type 1 = perpendicular, Type 2 = parallel

_ is homologous to viral class II fusion proteins (parallel)

Sperm-egg fusion proteins

_ refer to fusion proteins essential for sexual reproduction and exoplasmic merger for plasma membranes

Fusexins

Fusexins are fusion proteins essential for _

sexual reproduction and exoplasmic mergers for plasma membranes

_ is a common origin and evolution of sexual reproduction, enveloped virus entry into cells, and somatic cell fusion

Superfamily FUSEXINS

_ refer to distinct variations within a species of virus or bacteria, classified based on differences in their surface antigens

Serotypes

Explain how immunity to 1 dengue virus (DENV) serotype can increase disease severity in humans upon subsequent infection with another DENV serotype

Immunity to 1 DENV serotype can increase disease severity in humans upon subsequent infection with another DENV serotype bc serotype cross-reactive antibodies facilitate DENV infection of myeloid cells by promoting virus entry into Fcy receptors—a process known as antibody-dependent enhancement

Why can’t hydroxychloroquine be used as treatment for COVID-19?

• Plasma membrane protein facilitates fusion of host plasma membrane + viral envelope

• Because cell lines they used didn’t have this plasma membrane protein, initially, it was thought that hydroxychloroquine can be used to treat COVID

• However, because humans have this plasma membrane protein that provides a separate pathway for fusion of viral envelope + host cell, which cannot be blocked by hydroxychloroquine, hydroxychloroquine is then rendered ineffective against COVID

Explain 2 ways through which penetration and uncoating occurs in naked viruses

• Poliovirus = partial degrading of capsid > extrusion of NA into cytoplasm

• Adenovirus = endocytosis > low pH of endosomes lead to lysis/release of viral genome into cytoplasm > uncoating and delivery to nucleus

The unique shape of bacteriophages not only facilitate their attachment to host cell receptor but also _

cause ejection of their viral genome into host cell through their tail tube / channel

Explain what happens during viral genome ejection in naked viruses through host cell envelope via tail tube

• Tailed bacterial viruses (bacteriophages) enter host cells by piercing host envelope with their tail

• Delivery into cytoplasm requires ejection of viral genome from icosahedral capsid through tail tube and/or channel

Explain 2 viruses that can do pore-mediated penetration of viral genome into host cell

• Caudovirales

• Poliovirus

_ refers to penetration method through which viral genome is extruded into host cell without endocytosis or bypassing vesicular transport and instead forms a pore through which genome is released

Pore-mediated penetration, e.g., Caudovirales, Poliovirus

3 methods through which penetration may occur in naked viruses

• Ejection of viral genome via tail tube in bacteriophages

• Pore-mediated penetration

• Permeabilization (break down) of host membrane

Explain unique penetration strategy of Reovirus (that increases its infectivity)

• Reovirus has 2 icosahedral protein layers

• Taken in via endocytosis, but lowering pH degrade only the outer coat

• Virus with partially degraded single coat can then permeabilize endosomal membrane, being released into cytoplasm

• It’s crucial that the virus still has 1 coat because it contains an unusual dsRNA gene that does mRNA synthesis inside to prevent host cell from recognizing presence of foreign body inside it, thus evading immune recognition