Immunology 200 Midterm 2

30-90nm

• Viruses lack the essentials of life
• -They do not have the ability to take up energy and make use of it
• -They do not transport molecules out of cell or into cell
• -They do not reproduce themselves

-However viruses can evolve by natural selection and have protecting contents from external environment

Viruses are obligate parasites and they must have a host cell in order to reproduce

• -They need to be able to enter a host cell
• -They have the host cell replicate the viral genetic material
• -Have the host cell transcribe and translate the viral genese

A Virion is an infectious viral particle

Yellow fever was identified in 1901

• They are modified from Koch's postulates and they include:
• -Isolation of virus from diseased hosts
• -Cultivation of virus in host cell
• -Proof of filterability
• -Production of a comparable disease when used to infect experimental animals
• -Re-isolation of the same virus from the infected host
• -Detection of a specific immune response to the virus

-They package their genomes into a particle that mediates transmission from host to host

-A virus genome contains the information for an infectious cycle within a susceptible permissive cell

-All viruses are able to establish themselves in a host population so their survival is ensured

Hershey and Chase

-Genetic material is protected by the capsid  which makes them resistant to proteases, heat or temperature extremes

-They use as few proteins as possible

-The RNA assumes a helical configuration surrounded by protein capsid

-The information for assembly is contained within the protein structure itself (Self-assembly)

Information for assembly of coat is contained within the protein structure itself forming the capsid

• -Easier synthesis
• -Repeating structure

• -Attachment using a variety of receptors
• -Penetration of the membrane
• -Utilization of host cell proteins/enzymes needed for replication, transcription and translation
• -Biosynthesis of proteins
• -Assembly of progeny particles

An enveloped RNA virus

• Measles is an example of an enveloped RNA virus
• -The viral envelope membrane fuses with the host cell membrane to allow entry
• -RNA is transcribed and replicated using viral enzymes
• -Virus can leave cell by budding off with host membrane

The host cell that is taken with virus budding

-The envelope, which is host membrane, is of host origin

-It contains host proteins

Viruses synthesize a large number of new progeny and release the progeny at once

• -A dilution is mixed with bacteria and melted agar
• -Agar is poured onto a plate and phage plaques make holes
• -concentration is measured as plaque forming units

Plaque forming units per mL

• -The nature of the genetic material
• -Symmetry of the capsid
• -Presence of an envelope and the dimensions

Viruses can contain DNA or RNA, single or double stranded

• Polio
• HIV
• Influenza

• Herpes
• Pox

• -Sufficient virus at site of entry
• -Susceptible host cells that have
• a.)receptors required for viral entry and
• b.)they are permissive, containing factors needed for replication and dissemination
• -Local antiviral defense must be breached

• Conjunctive
• Respiratory tract
• Elementary tract
• Urogenital tract
• Anus
• Skin
• Capillary

Influenza

Through the air via aerosolized droplets

Fever, Loss of appetite, nausea, vomiting, abdominal pain, yellow colouring of eyes, dark urine

It is transmitted in infected blood

They are unable to clear the virus

The virus can persist and overwhelm the immune system at a later date in time

Herpes simplex 1

Hepatitis B

Herpes simplex I

Rash, Hypersensitivity reaction, fever, cough, conjunctivitis

Measles can develop into SSPE: Measles subacute scelrosing panencephalitis

• -Cytopathic effects on cells directly
•  o Cells may be lysed

-Viruses can be oncogenic

• -Immumopathology
•  o Damage as a result of local inflammation

- Cells lose contact inhibition and their normal interactions (Anchorage - independence) become immortal

- Can be transformed by virally encoded oncoproteins

-Viral disruption of host genes

Poliomyelitis caused by poliovirus

Cell rounding and lysis by a virus

Poliovirus infections may cause this

Cancer cells may lose their attachments and become mobile within the host

• Fusiform morphology
• Anchorage independence

Genes that regulate cell cycle, replication or activation of cellular process

Technically called proto-oncogenes until cancer is caused

A signalling molecule that phosphorylates proteins involved in signal transduction

When it binds to other proteins in the cell, causing conformational changes in the protein opening up the active site which becomes phosphorylated

Uncoating

Proteins, often glycoproteins

• Phagocytosis
• Endocytosis
• -Pinocytosis
• -Receptor mediated endocytosis

The particle is surrounded by plasma membrane by phagocytic cells.

They fuse with lysosomes and the particles are destroyed

• It is a type of endocytosis
• molecules enter directly into the cell

It is a type of endocytosis

A ligand of molecule binds to the receptor of the host cell and is internalized.

The most important mechanism of entry for viruses

Receptor-mediated endocytosis

• -Ligand binds a cell surface receptor and diffuses into clathrin pit
• -Clathrin pit pinches off forming a coated vesicle
• -Clathrin uncoats the vesicle
• -the vesicle fuses with the early endosome
• -acidic environment of endosome releases ligand from receptor, and the receptor is returned to cell surface

Clathrin is a protein that plays a major role in the formation of coated vesicles

Viruses have evolved to use proteins on the host cell as receptors

Rhinovirus and poliovirus

PVR, or poliovirus receptor

PVR is the poliovirus receptor.

It is involved in dendritic cell and Nkappa interactions in normal host interactions

Icam-1

Rhinovirus

Intercellular adhesion molecule-1 is the receptor for Rhinovirus

It is involved in macrophage T-cell interactions in normal host interactions.

The presence of the receptor of which the ligand binds to

A direct interaction between the receptor and capsid

Because Icam-1 plays an essential role in macrophage t-cell interactions

• -Virus attaches to Icam-1 and enters by endocytosis.
• -Acidic environment of endosome causes uncoating
• -RNA is released into the cytoplasm

It facilitates destabilization of capsid

• Poliovirus is resistant to acid
• Rhinovirus is not

They block the binding of the receptor to the canyon

The interaction with PVR which causes the uncoating

The acidic environment inside the endosome causes uncoating in rhinovirus

In poliovirus, the interaction with PVR causes structural changes and release of RNA

Interaction with PVR causes structural change

• N termini of VP1 protein extends into
• membrane

May form a pore in which RNA can enter

Structural changes allow for a pore to form and RNA to cross membrane.

fusogen any agent, or set of conditions, that gives rise to fusion of membranes, including cell membranes

enveloped viruses encode their own fusogens and allow the fusion of the two cell membranes to escape

Sialic acid

Influenza virus hemagglutinin (HA)

Terminal sialic acid

In birds, alpha (2,3)

In human, alpha (2,6)

Fusogenic peptide is exposed by an acid catalyzed structural change

Virus acid is inside two membranes

uses fusogen to escape

Acidic environment of endosome

30%

Preliminary infection sometimes causing flu-like symptoms

Asymptomatic phase around 10 years

Symptomatic phase

AIDS

The depletion of CD4 T cells

• Carries envelope glycoproteins
• Each capsid carries two RNAs and reverse transcriptase

An enzyme that copies RNA into DNA

CD4 and coreceptor on T cell

At the cell membrane of the T cell

• HIV particles bind to CD4 and coreceptor on T cell
• Viral envelope fuses with cell membrane allowing viral genome to enter the cell

HIV infect cells that have CD4 and Chemokine receptors

chemokine

-CCR5

A mutation in CCR5, a chemokine receptor.

gp120

HIV gp120 binds to CD4 and cause conformational changes that expose CCR5 receptor binding site.

Binding to co-receptor induce second conformational changes exposing the fusion peptide

It copies the ssRNA into double stranded cDNA

• It inserts DNA into the genome
• -Viral cDNA enters nucleus and is integrated into host DNA

• Reverse transcriptase
• -tRNA serves as primer for first strand

RNase H cleaves

• DNA polymerase
• -Polypurin tracts (PPTs) serve as primer for second strand

• Base substitutions
• -Misincorporation mediated
• -Dislocation mediated

• Frameshifts
• -Misincorporation initiated
• -Slippage initiated

Infected cells must be activated

Transcription factor NFkB is turned on by activation of infected T cells or macrophages

• -T-cell activation induces low level transcription of provirus
• -RNA transcripts are spliced allowing translation of early genes tat and rev
• -tat amplifies transcription of viral RNA, Rev increases transport of singly or unsplised viral RNA to cytoplasm
• -Proteins Gag, Pol and Env are translated and assembled into virus particles which bud from the cell

• Gag:group specific antigen
• Pol: polymerase
• Env: envelope

• 1x10^10
• Around 240 infectious cycles

Based on how many steps to create mRNA

They can synthesize their own polymerases

5 prime to 3 prime

Because it replicates negative strand viruses

Poliovirus is a +strand RNA virus

The capsid is made up of around 60 copies of four proteins

• 1.Binding of virus to receptor and endocytosis
• 2.RNA associate with ribosomes
• 3.Translation
• 4.Polyprotein is made
• 5.Polyprotein cleavage
• 6.RNA synthesis occurs on vessicle membranes
• 7.+strand moves to vesicle
• 8.-strand copies are made
• 9.+strand copies are made
• 10.+strands move to vesice
• 11.Cleavage of the capsid precursors
• 12.Assembly into new capsid
• 13.Exit by lysis

Vacuoles or vesicles

• A polyprotein precursor
• RNA is translated into a single polypeptide
• -it is cleaved by two virally encoded proteases
• o 2Apro and 3Cpro

2Apro and 3Cpro

• RNA dependant RNA polymerase
• unique to virus
• specific for RNA template
• copies viral RNA not host RNA
• no proofreading activity so errors
• error rate limits size of virus

3Dpol

The sequence of the base pair

RNA strand links to 3AB via a tyrosine

-a protein

3AB takes RNA to membrane

RNA strand links to 3AB via a tyrosine

Primer starts synthesis on the tyrosine of 3AB

elongation

Cleavage of VPg off 3AB

• First step of translation is removal of VPg protein
• When capsid proteins are abundant, RNA genomes are packaged
• RNA recombination involves RNA polymerase template exchange

10-20

Virus for humans may be attenuated by passage in non-human cell lines e.g. monkey

If a reversion mutant occurs, the virus given in the vaccine may again become infectious to the human

• Parvo
• Papova
• Herpes
• Pox

• DNA dependent DNA polymerase (parvovirus)
• DNA dependent RNA polymerase in all DNA viruses

They encode their own

• 1. Template directed
• 2. Each strand is copied beginning at the origins
• 3. Uses a DNA dependent DNA polymerase

Bidirectional replication from an origin.

Bidirectional replication from an orgin

Primase

Synthesis of leading strand starts from replication origin on RNA primer

Lagging strand proceeds in short pieces as template becomes open

Open templates of RNA caused by the lagging strand of replication

Removal of the primer from the 5 prime end creates a gap that cannot be filled

• 1. Nick one strand
• 2. Replicate by continuous copying
• 3.Displace a strand
• 4. Complete the lagging strand
• 5. Double strand DNA templates result
• 7. Keep making more copies by continuing around again

They help package one genome

• Proteins bind to specific sequences at ends of strands
• Empty capsid binds to these proteins
• DNA is stuffed in
• A headful of DNA is stuffed in until proteins on other end are reaches
• Cleavage of DNA

Templates for the next replication cycle

Viral DNA polymerases may not be proofread

First proteins synthesized are transcriptional activators of genes

These transcripts make proteins needed later in synthesis

Late proteins are required for Capsid assembly and membrane proteins

• Virus attaches to PVR
• Uncoating at cell membrane
• Proteins are made as polyproteins
• Initial cleavage is autocatalyzed bu then proteases are released
• Capsid proteins autoassemble in a concerted assembly with RNA

fecal-oral transmission

a flavi virus

It can tell based on structural differences

It is the protein cap used for RNA stabilization

OH 3`

By making a capsid, the stabilization due to full capsid

Polyproteins are favoured

HIV-1

In the virion

HIV integrates into the genome and then uses host transcription machinery to make more copies and therefore can allow alternate splicing of transcripts.

• -Rough ER buds off into Golgi, Golgi buds off into rough vesicles that fuse with the cell membrane
• -Assembly of enveloped viruses frequently takes place at plasma membrane
• -Secreted or TM proteins are made on the Rought ER and secreted into ER lumen or left in ER membrane

T cell mediated killing of cells take effect later on in the infection and last longer

• Rhinovirus
• Rotavirus
• Influenza virus

• Antigens are recognized by innate immune system
• TLRs recognize viral capsids, viral RNA or DNA

Viruses that lyse the cell, or cause major disruptions to the morphology of the cell cause damage by direct cytopathic effect

• Cytopathic effects
• Inducing cell mediated immunity
• inflammation

• Neutrophils are the first to arrive at sites of infection
• They are phagocytic
• They are granules that contain antimicrobial peptides including defensins

Alpha defensins are potent antivirals

• Some are directly lytic to virus infected cells
• others bind to viral capsid and inhibit assembly

• Receptors on macrophages recognize patterns on viral antigens
• resident antigen presenting cells recognize the pathogen

Viruses are recognized by the presence of sugars on glycoproteins and surface TLR receptors recognize viral capsid or membrane proteins

• Internalization of antigens
• Detection by endosomal TLR receptors
• Deliver signals into the cell to activate macrophages
• Tells the macrophage what type of response to make

• They release cytokines to initiate immune respons
• -TNF-alpha, interleukin-1, IL-6 and interferon

• Interferon is an effector made by cells
• It is a warning protein made by leukocytes (especially dendritic cells) if they detect virus
• It bind to receptors on other cells and turns on antiviral program

They have local effects on other immune cells

-antiviral state is induced in neighboring cells

• 2`-5` oligoadenylate synthetase
• RNase L
• dsRNA activated protein kinase (Pkr)

It is a type 1 interferon and is activated to make aoligoadenylate which in turn activates RNase L

It is a type of type 1 interferon and degrades cellular and viral mRNA

It is a type of type 1 interferon and stops new protein synthesis in infected cells

Macrophages produce TNF-alpha to activated natural killer cells which produce interferon gamma

Proinflammatory cytokines act on hypothalamus

Most viruses cause fever or increased body temperature

They induce the liver to produce acute phase reactants which are effector proteins made in response to infection.  They bind directly to virus

They are effector proteins made from the liver in response to proinflammatory cytokines. They bind directly to virus particles in the host.

It is an acute phase reactant.

It binds to mannose of viral glycoproteins and acts as an opsonin.

• It is on many viruses that are made in animal hosts
• Primates to have this enzyme
• The sugar is on many bacteria so we make antibodies to it

Terminal galactose is found in viruses and we create antibodies to it.

It caused too much inflammation which caused pathology

Around one week

They kill the infected cell by the delivery of a signal inducing death or apoptosis

They recognize viral peptides presented on MHC class 1

Newly synthesized proteins

Resistance to a particular virus depends on the MHC of the individual

The allelic composition of MHC

• Antigen presentation and costimulation
• IL2

Antigen is internalized and presented on MHC class 2 of antigen presenting cell. Costimulation is also required

Helper T cells proliferate and produce IL-2 which activates Cytotoxic T cells.

Activation of Naive CTL requires costimulation by APC

Infected dendritic cells can present the naive T cell costimulation to generate CTL effector cells

Influenza, measles and HIV

Memory cells are made only if CTL is primed by IL-2 from a helper T cell

They are series killers

They are not damaged in the killing process

The engagement of MHC class 1 by natural killer inhibitory receptor inhibits killing

Nk cell inhibitory receptor binds with MHC 1 on normal cell to prevent killing

VIruses often down-regulate surface expression of MHC which makes them a target of NK cells

• Monoclonal antibodies recognize 1 epitope
• Polyclonal antisera has many specificities

Monocolonal antibody

Polyclonal antisera

• Polyclonal antisera
• they have many antibody specificity

An in vitro test in which antibodies bind to virus and prevent the infectious cycle

Free virus particles are susceptible to neutralizing antibodes

Neutralizing antibodies neutralize viruses

• Possible mechanisms:
• Steric interference with receptor
• Fix capsid so pH dependent uncoating is blocked
• Capsid stabilization so that uncoating is blocked

many antibodies are useless, and HIV can mutate away from good neutralizing antibodies so they stop working.

• Measles
• HIV

Western blot to detect anti-HIV antibodies in patient serum

The structure of HIV-1 envelope glycoprotein contributes to evasion from the immune system

Variable regions cover the conserved regions

Important epitopes are hidden within the molecule such as the fusion peptide

Envelope glycoprotein

It is an evasion strategy

Important epitopes of gp120 are hidden until fusion, where they are only exposed for a moment during fusion process

A loop structure on HIVs gp120 has the same shape as part of the chemokine RANTES which binds to CCR5 chemokine receptor

This is an evasion strategy

Changes in gp120 glycosylation

mutations in the location of glyosylation sites means that HIV is making an evolving glycan shield

Some neutralizing ab block fusion process

Low affinity Ab binding to complement receptors can help HIV get in

Regions of gp120 look like self, Ab are usually not made to self antigens

Interfering Ab bind to HIv but do not block entry but block the action of neutralizing antibodies

opportunistic infections and disease start

Macrophages and responding T helper cells are the first infected. 

Dendritic cells bring antigen in lymph node.  Dendritic cells can be infected by HIV

Infected dendritic cells present antigens on MHC class 1.  The CD8 cytotoxic t cell kills them with signal and costimulation

TH cells

Intracellular proteins

three

It is a linear 9 amino acid sequence from within the protein

Viral peptides can be presented if they fit

Each peptide varies except and anchor residues

CTL immune response tends to focus on a few epitopes originally seen

The transfer of a pathogen from non-human animals to humans and subsequent spread of the pathogen between humans

Chimpanzee

+ strand RNA viruses

Neuroinvasive disease, west nile encephalitis, meningitis or west nile poliomyelitis

Humans are dead end hosts because viral titre is not high enough to transmit from us to other animals

incubation period is 2-12 days, the symptoms include fever, muscle soreness, cough, hypoxemia and diarrhea

Super spreaders, infected many people and hosted many strains of this RNA virus

We use Koch's postulates and infect a healthy animal to see if illness occurs

They can be infected with almost anything, RNA viruses, but they are often not harmed

-They can host many things

RNA dependent RNA polymerase does both transcription and replication, if removed replication can not occur

They are named for their HA (Hemagglutinin) and NA (Neuraminidase) types

The spanish flu

Most people who died from the virus were young adults and otherwise healthy

It is a bird flu

It encodes an NS1 (Non structural gene 1) variant that shuts down the interferon response

NS1 is Non structural gene 1.

It is an inhibitor of interferon, and if it is strong it shuts down the interferon response meaning the virus wins