Viruses and host defenses How the successful virus

Viruses and host defenses How the successful virus beats the body

Learning objectives • Describe the role of interferons, natural killer cells, innate and adaptive immune system in recovery from virus infection • Explain how various viruses evade host immune strategies • Interpret data from experiments designed to determine virus evasion mechanisms

Host outcome • Recovery • Persistence • Rapid death

What prevents most viruses from causing disease? • Skin • Nonspecific innate defenses – Natural killer cells – Interferon – Complement – Apoptosis • Acquired immunity – Antibodies – Cytotoxic T cells – Lymphokines

How do we know the importance of each host defense mechanism in recovery? • • Infect and measure responses over time Use mutant animals/genetic defects in humans Infect and block specific defenses Do in vitro studies

Innate defenses: we survive most virus infections skin: impenetrable barrier unless insect vector or wound • mucous membranes: live target cells • inflammatory response: neutrophils • complement • macrophages – HIV integration/destruction; – Dengue virus immune enhanced uptake • cytokines

NK cells

Overcoming NK cells • CMV makes MHC homolog • How does it prevent cytotoxic T cells from killing virus infected cell?

Interferon: host proteins induced by virus in vertebrates that interfere with viral replication (type 1) • bind to receptor on host cells (determines specificity) • through signal transduction induces synthesis of antiviral proteins (AVPs) controlled by interferon-stimulated response elements (ISRE) which protect cell from viral replication

• What would happen if you microinject IF into a cell? • What would happen in presence of Actinomycin D? • Evidence for IF role in recovery from infection – Time – Place – Exogenous transfer experiments – Loss

Antiviral effects of IF-induced AVPs • Oligoadenylate synthetase activated in presence of ds. RNA – ATP-----> oligo. A up to 15 residues – oligo. A activates RNAase that cleaves host and viral m. RNA • Protein kinase (PKR) activated in presence of ds. RNA phosphorylates e. IF 2 needed for translation

Host cell responses are not always protective • Results of microarrays using high and low virulence strains – Red are upregulated – Green are downregulated – Black - no change • Increased inflammatory, apoptotic, oxidative damage responses

antibody • neutralization: antigenic shifts and drifts(variation) • complement mediated lysis: vaccinia makes homolog of inhibitor • HSV makes receptor for a complement component

cell mediated immunity: requires viral protein displayed in MHC 1 • downregulates transport of MHC to surface (Adenovirus; herpesvirus; measles) • Increases endocytosis of MHC • Neurons express little MHC • Mutation to new epitopes • latency

cell apoptosis • Blocked by viral proteins which may bind to host proteins promoting apoptosis • Produce mimic proteins that inhibit apoptosis

Multiple stolen genes of KSHV - HHV 8 • V-FLIP - FLICE inhibitory protein • Decoy receptor • • Bcl-2 - anti-apoptotic factor complement binding protein IL 6 -like cytokine three chemokines interferon regulatory factor D-type cyclin G-protein coupled receptor

How to make a killer virus • What characteristics should a biological weapon have? • How can it be constructed?

• Ectromelia virus causes mousepox • Recovery due to CTL death of infected cells via perforin pathway mousepox virus produces inhibitors of caspases • Vaccinia virus does not inhibit caspases so they are killed by two mechanisms • Il 4 skews immune response to ab production and shuts down perforin pathway

Ebola strategies • Releases soluble glycoprotein (portion of transmembrane protein) • Hypothesis: It may bind to a host cell and prevent its activity • How do you show binding to specific white blood cell? • Gel depletion

s. GP vs GP binding to neutrophils vs endothelial cells GP pseudotyped virus neutrophils endothelials Infection by GP pseudotyped luciferase virus

Ebola and IF induced gene expression • Northern blots • Which genes are affected by EZ? • How would you determine which EZ gene is responsible?

The Many Roles of Nef • Hypothesis: Downregulation of MHC 1 • How do you show this? • What can cause MHC downregulation?

CD 4 vs MHC downregulation • Nef genes from WT, nonprogressors, slow progressors and rapid progressors • What is advantage to virus of CD 4 downregulation? • How can you determine whether the same or different parts of the Nef protein are responsible for each activity?

Increases viral replication/budding by interacting with signal transduction and TCR

Nef binds to p 53 - what is affect on apoptosis? Nef binds to p 53. (a) A GST-Nef fusion protein specifically coprecipitates p 53. Purified recombinant GST-Nef (lane 1) and GST (lane 6) alone were incubated with purified recombinant p 53, affinity purified with glutathione-Sepharose beads, electrophoresed, and transferred to nitrocellulose. Purified p 53 was electrophoresed and transferred to nitrocellulose as a control (lane 7). Nitrocellulose membranes were then reacted with anti-p 53 in Western blotting. For competition of the GST-Nef-p 53 interaction by purified Nef protein, p 53 was incubated with purified Nef protein at 0. 3 - (lane 2), 3 - (lane 3), 10 - (lane 4), and 30 -fold (lane 5) molar excess before reaction with GST-Nef and processing as described above • How would you determine which proteins Nef binds to? • Nef also binds to several cellular signal transduction elements -What is role? How do you show affect on apoptosis? •

MOLT-4 cells which had been electroporated with purified recombinant fulllength Nef or GST or which had been mock electroporated were either exposed to a lethal dose of UV light (+) for approximately 30 s or were not exposed (-) and then returned to culture for 12 h. Hirt DNA was extracted from each of the samples and analyzed by gel electrophoresis for the presence of fragmented DNA.

The APOBEC enzyme family deaminates specific cytidine (C) residues in either DNA or m. RNA, converting them to uridine (U) residues • Viruses replicating in cells producing APOBEC 3 G need Vif gene for cells to be permissive • Vif- can grow in cells without APOBEC

Anti IF strategy of HCV • NS 5 a binds to PKR and inactivates • E 2 gene has 12 aa homology to autophosphorylation site of PKR and e. IF 2 a • How do IFres and Ifsens differ? • How might that help the virus?

Do PKR and E 2 bind? • • His tag binds to beads Isolate and run on gel Wt PKR K 296 = mutant in ATP binding domain • E 2 -C - no Phos site • Hn - cell protein control

Does E 2 interfere with PKR activity? • ATP- P 32 • PKR +/- E 2 and in presence of ds. RNA activator and substrate H 2 a

HSV blocks IF in several ways vhs degrades m. RNA, ICP 27 prevents splicing Virus dephosphorylates e. IF 2 a Virus destroys Jak 1, disperses ND 10 and disrupts PML