Flu vaccines• WHO* specifies the contents of the vaccine each year to

contain the most likely strains of viruses that will attack the next year.

• Annually updated trivalent flu vaccine for 2008-2009 season consisted of H3N2, H1N1 and B influenza viruses. (But the vaccine H1N1 strain doesn’t confer protection to H1N1 swine flu.)

• Viruses are grown in hens’ eggs.

• Inactivated viruses are injected or live attenuated viruses are given as a nasal spray (not currently approved in children younger than 5).

*World Health Organization

H5N1 (Bird flu)• Most flu viruses infect birds. “Bird flu” refers to influenza in birds and in humans

in cases in which avian viruses cross the species barrier to infect humans.

• 2003-2004: Outbreaks of H5N1 in poultry in 8 Asian countries and 100 cases of human infections with 50% fatalities.

• Few (maybe none) cases involved bird-to-human transmission. A pandemic would require human-to-human transmission. A change of only two amino acids would allow H5 to efficiently recognize human cells.

• H5N1 viruses are being tested for making a vaccine, but mass production of an H5N1 vaccine would require safety and efficacy trials that might take longer than viruses would spread in a pandemic.

• Antiviral drugs oseltamivir (Tamiflu) and zanamivir, but not amantadine, are active against H5N1. Supply not adequate for a global pandemic. Antiviral resistance occurs in N1 viruses during Tamiflu treatment.

Response to bird flu threat in Asia

Protect domestic ducks Burn chicken carcasses Vaccinate chickens from contact with wild ducks

http://pr.caltech.edu/periodicals/CaltechNews/articles/v41/uneasypieces.html

A = Acquired = a virus received from someone else

I = Immune = an individual’s natural protection against disease-causing microorganisms

D = Deficiency = a deterioration of the immune system

S = Syndrome = a group of signs and symptoms that together define AIDS as a human disease

CDC* definition of AIDS

* (Centers for Disease Control and Prevention; Atlanta, GA)

What is AIDS?• HIV infection is not AIDS (is “HIV disease”)

•AIDS is umbrella term for 26 known diseases and symptoms

• AIDS diagnosis if meet three conditions:– Have one or more of known diseases/symptoms

See next two slides

– CD4 T cell count <200/µL What is CD4? What are T cells?

– Test positive for HIVWhat do HIV tests detect?

Figure 9-22Opportunistic infections and malignancies in AIDS patients

Symptoms of AIDS(each symptom can be caused by another disease; can’t rely on symptoms to diagnose

AIDS)

• Rapid weight loss• Dry cough• Recurring fevers, night sweats• Unexplained fatigue• Swollen lymph glands• Diarrhea that lasts more than a month• White spots on tongue, in mouth, or throat (thrush)• Pneumonia• Red, brown, pink or purplish blotches on skin or inside mouth, nose, or eyelids (Kaposi Sarcoma)• Memory loss, depression, other neurological disorders

DNA viruses follow the “Central Dogma”

DNA --> RNA --> Protein transcription translation

Most RNA viruses also follow part of the Central Dogma

RNA --> Protein

Clicker questionHoward Temin’s hypothesis

1) Hypothesize that actinomycin D also inhibits translation of proteins from RNA -- test for new protein synthesis after adding actinomycin D

2) Hypothesize that actinomycin D also inhibits viral RNA polymerases -- test for its effects on poliovirus RNA-dependent RNA polymerase

3) Hypothesize that actinomycin D kills the host cell and therefore the RSV -- test uninfected cell viability in the presence of actinomycin D

4) Hypothesize that DNA is an intermediate in RSV replication -- search for an enzyme that converts viral RNA into DNA

5) Contact the media and prepare for a trip to Stockholm6) All of the above

In the early 1960s, Howard Temin discovered something strange about Rous sarcoma virus (RSV; a RNA virus that causes malignant bone tumors in chickens). Temin noticed that RSV would not grow in the presence of actinomycin D, an antibiotic that inhibits DNA synthesis. What would you do if you made this observation?

HIV is a RetrovirusRetroviruses do NOT follow the Central Dogma

• Retroviruses are a subset of RNA viruses that reverse usual flow of genetic information within host cell.

– Reverse transcription of viral RNA into viral DNA RNA --> DNA --> RNA --> Protein

• Reverse transcriptase makes double-stranded DNA from RNA.

– RT is a RNA-dependent DNA polymerase

ExpressionMembrane Targeting

Budding

Maturation

Nuclear transportIntegration

UncoatingReverse Transcription

BindingMembrane Fusion

Overview of Retroviral

LifeCycle

(For exogenous retrovirus start here)

(For endogenous retrovirus start here)

Joined Max Delbrück’s group at Caltech in 1949 to work

on bacteriophage. Switched to animal oncoviruses.

Joined Dulbecco’s group at Caltech in late 1950s.

Caltech PhD, 1959 (Animal Virology).

President of Caltech; 1997 - 2006. Now

Professor of Biology at Caltech.

1975 Nobel Prize

HIV structure (Beware: some inaccuracies in movie)

• Diameter ~100 nm (1000 Å)

• Outer envelope (lipid bilayer membrane) contains gp120/gp41 spikes ) and random cellular (host) receptors acquired during budding

gp = glycoprotein; # refers to its molecular weight in kilodaltons

• Capsid (Bullet-shaped core) – Protein arranged with symmetry related to an icosahedron – RNA inside core: 2 copies of positive sense RNA (~9500 bases)

• Problems with movie: i) only ~14 spikes per virionii) HIV does NOT contain an icosahedral matrix (other enveloped viruses, such as Herpes, do) http://www.lifehouseproductions.com/hiv_anim.html

Figure 11-24Genes and proteins of HIV

(we will discuss gag, pol, env, p24, tat, and rev)

HIV has an RNA genome flanked by two Long Terminal Repeats (LTRs), which are involved in viral integration and regulation of the viral genome.

Includes p24

Remember tat

Remember rev

HIV gag, pol, env• gag gene encodes structural proteins of the viral core as a poly-protein (cleaved into individual proteins by HIV protease).

– Matrix protein (p17)– Capsid protein (p24) -- assays to determine whether HIV is present look for this abundant HIV capsid protein– Nucleocapsid (nucleic acid binding protein) protein (p15)

• pol gene encodes enzymes involved in viral replication and integration (again, poly-protein cleaved into individual proteins by HIV protease).

– Reverse transcriptase– Integrase– HIV protease

• env gene encodes envelope glycoprotein gp160, which is cleaved by a host cell protease into gp120 and gp41.

–gp120 binds to CD4 (receptor) and CCR5 (co-receptor)– gp41 is responsible for fusion of viral and target cell membranes

Proteins often named as “p#”, where the number refers to the protein’s MW in kilodaltons

First steps of the HIV lifecycle -- more details

The use of CD4 as a receptor targets HIV to cells of the immune system

• HIV preferentially infects CD4 T cells because the cellular receptor for HIV is the T cell protein CD4. (Human macrophages and dendritic cells also express CD4.)

• CD4 T cells are a type of T lymphocyte (a white blood cell), which control other immune cells.

• HIV infection eventually results in low levels of CD4 T cells, resulting in opportunistic infections due to ineffective immune responses.

*Peripheral Blood Lymphocytes

*

What happens in an untreated HIV infection

• CD4 T cells are lost during HIV infection.-- Direct viral killing of infected cells-- Increased susceptibility to programmed cell death-- CD8 T cells (killer T cells) kill infected cells

• Below ~ 200 CD4 T cells/µl in peripheral blood, cell-mediated immunity is lost --> infections with opportunistic microbes.

-- Early infections include Candida species (thrush) and TB-- Activation of latent varicella zoster (chicken pox virus) --> to shingles. --

Epstein Barr Virus (infectious mononucleosis) --> B cell lymphomas

-- Herpes virus HHV-8 --> Kaposi’s sarcoma

-- Fungus P. carinii --> pneumonia

-- Co-infection with hepatitis C

-- Late stages often include cytomegalovirus or M. avium infections

Infection of a cell by HIV requires binding to CD4 and a coreceptor on host cells

• Spike protein on HIV virion is a complex of two proteins: gp120 and gp41.

• gp120 binds tightly to CD4 on target cell.

• Binding to CD4 causes a conformational change that enables gp120 to bind to a coreceptor on the target cell. All coreceptors are members of the chemokine* receptor family.

• After binding of gp120 to CD4 and a chemokine receptor, gp41 inserts into the host cell membrane, causing fusion of the viral and host membranes.

*A chemokine is a small protein that activates or attracts (as in chemotaxis) leukocytes.

CCR5 or CXCR4

Fusion of viral and cellular membrane bilayers

Genetic deficiency of CCR5 confers resistance to HIV infection

• ESN (exposed seronegative): A small group of individuals who remain seronegative despite exposure are homozygous for a non-functional variant of CCR5. • Frequency in Caucasians of mutant allele is 10%. ~1% are homozygous. Allele not found in Western or Central Africans or Japanese.• No health problems in heterozygous or homozygous individuals.• Resistance to infection in these individuals confirms that CCR5 is the major coreceptor used by HIV to establish primary infection in vivo.• Possibility of using CCR5-blocking reagents prophylactically to prevent infection.

Usually infected with R5 viruses (use

CCR5 as coreceptor)

Often a switch to X4 viral phenotype (use

CXCR4 as coreceptor) -- more T cells are

infected

Macrophages become a reservoir for HIV after T cell depletion.

Figure 11-23HIV RNA is transcribed by viral reverse transcriptase into DNA

that integrates into the host genome

• Viral genome enters cell after fusion of viral and host cell membranes.

• Viral reverse transcriptase protein packaged together with viral genome transcribes viral RNA into viral cDNA (complementary DNA).

• Viral cDNA integrated into host cell genome by viral integrase.

• Integrated cDNA is called the provirus. • Analogous to integrated prophage in a phage lysogen

RT enters cell along with viral genome.

Integrase enters cell along with viral genome.

Consequences of integration of HIV genome into a host cell chromosome

• HIV DNA that is integrated into a chromosome is duplicated when cell divides, therefore all progeny of the infected cell will contain HIV DNA.

• Individuals with HIV in its latent, proviral form are healthy and show no signs of AIDS.

• HIV can be passed among healthy individuals because HIV can residue as a provirus in T cells. Healthy individuals whose T cells contain integrated HIV DNA can transfer HIV in blood or semen, both of which contain T cells.

Figure 9-15 part 3 of 4

NFB is a host cell transcription factor that binds to the viral LTRs to initiate transcription by the host cell’s RNA polymerase.

Tat and Rev promote viral replication in activated T cells.

HIV lies dormant in resting T cells and replicates in activated T cells

NFB is normally supposed to be used by the cell to transcribe genes related to host defense -- it enters the nucleus to transcribe genes only under conditions of stress (e.g., when T cells are activated).

Note that HIV is using the host cell’s normal defense mechanisms to transcribe its genes. Another example of evolution at work!

Tat is required for HIV-1 replication

• Tat = transactivator protein

• Tat protein binds to start of a new HIV RNA strand

• Tat binds to TAR: Transactivator Active Region

• Tat binds to TAR and activates transcription of HIV genes

Figure 9-15 part 4 of 4HIV needs to export unspliced, singly spliced, and multiply spliced mRNAs from the nucleus to the cytoplasm in order to make all of its proteins.

Eukaryotic cells normally prevent export of incompletely spliced mRNAs.

Fully spliced Rev mRNA leaves the nucleus and gets translated in cytoplasm. Rev protein then enters the nucleus and binds to a specific site on the viral RNA and to a host transport protein to force export of unspliced viral mRNA.

Final steps in HIV assembly and budding from host cell

,

Note there are some inaccuracies in this figure(maturation to a bullet-

shaped capsid occurs AFTER budding).