HIV Virology Lecture2!3!29!12!1

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HIV/AIDS: Virology

Danuta (Danka) Kasprzyk, PhD

HIV/AIDS: Issues and Challenges

March 29, 2012



HIV/AIDS: Virology

Danuta (Danka) Kasprzyk, PhD

How do you pronounce my name?

First name: Dun-ku

Surname: Kus-pshick

No vowel sounds between the r-z



HIV: Koch’s Postulates (1890)

Are a method for establishing that specific

microbes cause specific diseases

Designed to experimentally establish a

causal relationship between a microbe

and a disease

Established when Koch was trying to show

the causes of Tuberculosis and Anthrax

Use an animal model for establishing this

relationship





HIV: Koch’s PostulatesExceptions:

Some diseases have unequivocal signs and symptoms

(i.e. tetanus; HIV)

Some individuals can carry disease, not get ill

(asymptomatic carriers; typhoid, HIV, polio, cholera,

HSV)

Some diseases, may be caused by a variety of microbes

(i.e. pneumonia)

Some pathogens cause several different diseases or cause a syndrome (HIV triggers multiple diseases)

Some viruses and some bacteria cannot be grown on

artificial media

Certain pathogens, cause disease in humans only (HIV)



Koch’s Postulates revised

Scientists are establishing ‘Koch’s postulates’ to take into account molecular science

Molecular postulates = set of experimental

criteria that must be satisfied to show that agene found in a pathogenic microorganism

encodes a product that contributes to the

disease caused by the pathogen

Based on virulence of pathogens

Currently difficult to establish for all

microorganisms



Koch’s Postulates revised

Testing a candidate virulence generequires a relevant animal model of the

disease

The ability to genetically manipulate themicroorganism that causes the disease

Animal models are lacking for many human

diseases Currently scientists cannot manipulate

many pathogens genetically…..



HIV: Pathogenesis

By 1982-19D83 a virus was suspected for the cluster of symptoms reported

Similar epidemiology to Hepatitis B

Effects similar to animal retroviruses

scientists were studying

Similar effects to feline leukemia virus

(HTLV)

Possible retrovirus

Human retrovirus hypothesized



HIV is a lentivirus

Slow growing virus Genus of the retroviridae

family

Produce multi-organ

diseases Characterized by long

incubation periods and

persistent infection

Infect a wide range of primate hosts, as well as

some non-primate

mammals



Lentivirus Family: Retroviridae

Retroviruses deliver their own genetic informationinto host DNA

Lenti = Latin for slow

Long incubation period Five sero-groups

Each infecting

vertebrate hosts Sheep and goats

Horses, Cattle

Cats

Primates



HIV-1: Pathogenesis (origin anddevelopment)

HIV = Human Immuno-deficiency

Virus

HIV is a lentivirus Belongs to a family of retroviruses

that have RNA genomes contained

in a lipid envelope

Retroviruses use reversetranscriptase (RT) for replication

One of the most primitive and oldest

organisms on earth

Graphic by: Russell

Kightley



What is HIV?

Any of several retroviruses thatinfect and destroy helper T cellsof the immune system

Retrovirus

Contains an RNA genome

To get its genetic materialinto host performs reversetranscription

Changes RNA to viralDNA

Gets incorporated into

host cell DNA



Establishing HIV as a cause

1982-1983:Established infection routes

Transmitted via sexual intercourse

Transmitted via bodily fluids

Unsterile injections

1983/1984: HIV isolated by two

independent labs 1985: First antibody test licensed

1989: First test that could detect virus



HIV-1: How does it work?

Is a retrovirus with two strands of RNA, surrounded by capsid

3 structural and 6 regulatory

genes encode 15 viral proteins

Has envelope receptors that bind

on cells with CD4 + receptors

Macrophages and T-lymphocytes

Requires the enzyme reverse

transcriptase (RT)

RT causes frequent basechanges, and recombination

Single strand of RNA prevents

“double-checking”, and lets

mutations occur Harvard AIDS Institute

Library of Images



HIV-1

RNA genome (Two strands)

Protease (enzyme)

Capsid (p24)

Env (gp41)

Matrix (MA)

Envelope protein

(gp120)

Reverse transcriptase (RT enzyme)

Integrase (enzyme) Harvard AIDS InstituteLibrary of Images



Three lines of defense protect us from invaders of

varying types

Immune System is key

Overview of the body’s defenses



Basic Components of the ImmuneSystem

Immunology: cells and tissues involved in

recognizing and attacking foreign

substances in the body e.g. bacteria,viruses, fungi and parasites.

Immunity: the condition of being immune.

Immunity can be innate or the result of aprevious exposure.

Antigen: any substance capable of

triggering an immune response.



Basic Components of the Immune

System Lymphatic vessels and nodes: designed to trap

and destroy antigens and play a critical role in

fighting all infections including HIV Phagocytes: “scavengers” of the immune system

By digesting/processing antigens, their role is to initiate

the immune response by presenting antigens to the

lymphocytes. Serve a secretory function critical to mounting the

inflammatory response and regulating immune

responsesGrimes D. and Grimes R.: AIDS & HIV Infection

St. Louis, Mosby, 1994.



Barriers Skin

Epithelial cells (mucosal)

Defense Recognition of an invader

Destruction of invaders

Cells

Dendritic cells

Macrophages

Natural killer cells

The Immune System



Cells Lymphocytes (B-cells)

Generate antibodies

Antibodies

• Alert the immune system into a response by bindingdirectly to invaders and signaling a response

• Directly neutralize invaders

Lymphocytes (T-cells)

T killer cells (CD8): bind to cells with foreignantigens

T helper cells (CD4): help B-cells and T killer cells

attack

The Immune System



Recognizing the Invaders

LYMPHOCYTES

Are white blood cells found

most often in the lymphatic

system

Produce the immune response

Originate from stem cells in thebone marrow

Two major types

The Immune System



Two types of lymphocytes

B cells- Blood Defenders

Formed in the Bone marrow

Produce antibodies after

exposure to antigen

Humoral immunity- in theblood

Bacteria and viruses in

blood

T cells- Cell Defenders

Processed in the Thymus

Two types: CD4 and

CD8

Cell-mediated immunity-in cells

Destroy body cells

infected by bacteria,

viruses, or cancer cells



T-Cells: two types

Subtype 1 (CD4) (T helper cells): Regulator cells

Known as helper cells

Recognize “invaders”

Sound the alarm

Summon armies of cells by producing

cytokines

These mount a direct attack on invader

Subtype 2 (CD8) (T killer cells):

Fighter or effector cells

Known as cytotoxic cells

Bind directly to antigen (invaders) and kill it



CD4 cells: Two types

Two types of CD4 cells: Memory cells:

Programmed to recognize a specific antigen after it has been previously seen

Naïve cells

non-specific responders

CD4 cells replicate 100 million times a day

The average person has between 800 & 1500CD4 cells per cubic millimetre of blood

CD4 cells are the target cells of HIV

Bartlett, J.: The Johns Hopkins Hospital 2002 Guide to Medical Care of Patients with HIV Infection



HIV and the Immune System

When HIV enters the body it must enter a cell to live and reproduce.The HI virus attacks CD4 cells, eventually killing them

The newly produced HIV then moves into new CD4 cells and infectsthem. The body’s immune system tries to replace the lost CD4 cells,but over time it is unable to keep these levels up.

HIV infects: CD4 (memory), CD8 (rarely),

Dendritic, Macrophages, Natural killer cells

HIV

HIV

HIV

CD4

Enters CD4 Cells

HIV

HIV HIV

HIV Replicates Kills CD4

Cells

CD4HIV




Finds a break in the

epithelium (mucosal

surface)

Dendritic cells bind to

HIV’s gp120

Dendritic cells

migrate from mucosal

surfaces to regional

lymph nodes where:

HIV forms a

reservoir

And binds to CD4

protein molecules

on T-cells

Neutralization of Virus Bound by Dendritic Cells

Mucosalepithelium

LaminaPropria

Intra epitheliallymphocytes

2.

DC-SIGN+

Dendriticcells

virus bearing dendritic cellsmigrate to the paracorticalregion of the lymph node

1.

3.

Have CD4 proteins



HIV acquisition

Entry points

Lymph nodes

Gut/intestines-90% of T-memory

Cells reside in gut

Bone marrow

Brain

Entry points




T-lymphocyte cell with CD4molecules being infected withHIV

CD4 protein molecules arethe primary receptors for HIV

Lymphocytes andmacrophages have co-receptors that HIV needs;CXCR4 and CCR5

HIV only has 9 proteins of its

own, borrows from us ~13% Northern Europeans

(1% of Caucasians) havegenetic defect and no CCR5receptors on T-cells



HIV-1: Life cycle

Viral attachment

Entry into host cell

Viral replication needs Reverse Transcriptase (RT)

Produces a DNA copy of RNA called a provirus

Integrates provirus into host cell DNA

Infected cell is activated

Viral transcription occurs

Translation of virus

Virion assembly

Budding



HIV Life Cycle

Retrovirus penetrates hostcell.

Virion penetratescell and its DNA isuncoated

The new viral DNA is transportedinto the host cell’s nucleus andintegrated as a provirus. Theprovirus may divide indefinitelywith the host cell DNA.

1

2

3

DNA

Transcription of theprovirus may also occur,producing RNA for new

retrovirus genomes andRNA that codes for theretrovirus capsid andenvelope proteins.

4

Matureretrovirus leaveshost cell,acquiring anenvelope as itbuds out.

5

Capsid

Reversetranscriptase Virus Two identical strands of RNA

DNA of host cell’schromosomes

Provirus

Host cell

Reversetranscriptase

Viral RNA

RNA

Viral proteins

Identicalstrands of RNA

Integrase

Protease

http://www.hopkins-aids.edu/hiv_lifecycle/hivcycle_flsh.html



Electron microscope picture of an HIV-infected cell

Courtesy of Matthew A. Gonda, Ph.D., Chief Executive Officer, InternationalMedical Innovations, Inc.




HIV surface gp120 protein

binds to CD4 molecules on

T-cells (docking)

To enter cell, HIV uses one

of two chemokine receptors

CCR5, CXCR4

Allow fusion of HIV to the

cell membrane

Once HIV has fused with

membrane, it releases the

capsid into cytoplasm (the

cell interior)

Or CCR5

coreceptor




Uncoating of the capsidtakes place

releasing viral RNA

releasing viral proteins generating viral reverse

transcriptase (RT)

Generates an RT

complex Docks with micro-

filaments—and

Enters the cell nucleus




RT transcribes:

Viral RNA into a

complementary DNA and

generates a protein

integration complex (PIC)

The PIC gets integrated

into the DNA of the cell

nucleus using integrase

The PIC gets integratedinto many different

chromosomal locations

within the cell




Once the cell’s DNA has

been altered in this way,

it is known as proviral

DNA (part virus/part cell)

It begins the process of

producing virus

Reproduces, generates

pro-virus

Pro-virus goes on to

formulate additional virus




The CD4 cell is nowprogrammed to be an ‘HIVfactory.’

Long viral protein chains

are produced which arethen cut into the necessarypieces to produce moreHIV.

This process is activated

by the viral protease enzyme.

Protease splits the longprotein chains into particles






Electron-microscopephoto of HIV budding

HIV generates 109 (B)viral particles every

day Within a month 100M

viral copies per 1 ml of blood

Over the course of 10years generates 100sof thousands of generations



HIV infection movie

http://www.youtube.com/watch?v=RO8MP3wMvqg

http://www.youtube.com/watch?v=9leO28ydyfU&feature=rela

ted

http://www.freesciencelectures.com/video/hiv-replication-

and-life-cycle/

Factors affecting transmission or


http://www.youtube.com/watch?v=9leO28ydyfU&feature=related


http://www.freesciencelectures.com/video/hiv-replication-and-life-cycle/
















Factors affecting transmission or acquisition of HIV

Mode of transmission (direct vs indirect) Type and duration of sexual activity

Use of condoms or other barrier methods

Circumcision

In addition: Viral load of infected individual

In Phase I and Phase III

Gender

Co-Factors Sexually transmitted infections

Genetic factors

Delta 32 mutation, HLA-B57 mutation

Immune system function of receptive partner?



HIV: Transmission risk per

exposure Without ART prophylaxis

Blood transfusion: ~ 9 in 10

Male to male, receptive partner: ~ 1 in30-100

Sharing needles: ~ 1 in 150

Occupational exposure, needle stick: ~1in 200-300

Male to female: ~ 1 in 700-2000

Female to male: ~ 1 in 1000-3000

HIV T i i i k



HIV: Transmission risk per exposure

Maternal-to-child transmission Overall about 1 in 3 infants are infected in the

absence of anti-retrovirals

In utero: ~ 10%

Intrapartum: ~10-20%Breast feeding: ~10-20%

Approximately 1 in 3 child infections are due tobreast feeding

Acquisition of infection depends on mothers’ stage of AIDS

Children exposed to ‘mixed feeding’ (breast andsolids) are at highest risk for HIV acquisition

HIV 1 N t l Hi t



HIV-1: Natural History

6-12weeks Duration: 1-15+ years Duration: 2-3+ years

Infection

CD4+ T-cell count

Plasma viremia

1º infection

syndromeClinical latency

Constitutional

symptoms

AIDS and death

Opportunistic

infections



Three Stages of HIV Infection

A. Stage I:

Asymptomatic or persistent lymphadenopathy

~ 50% of individuals will develop a febrile, flu-like illness

Onset of illness is generally 1-6 weeks

following exposure and can last 1-3 weeks

“ Acute Retroviral Syndrome” is often mistaken

for the flu

HIV Pathophysiology



B. Stage II:

Persistent thrush (Candida albicans) infections

Other rashes, skin eruptions, fevers, night sweats

Immune system actively and successfully fighting

HIV replication Can last for 1 to 15 years

C. Stage III:

Clinical AIDS, lasts about a year Opportunistic infections with a variety of

organisms: fungi, bacteria, viruses, parasites

Pneumocystis, cryptococcus, toxoplasmosis,

Kaposi's sarcoma, etc.

HIV Pathophysiology



1

10

100

1,000

10,000

100,000

1,000,000

10,000,000

P

l a s m a H I V R N A

MAC, CMV, PML, PCNSL, Cryptococcus, Microsporidia, Toxo

PCP

CD4 Cells

4-8 Weeks Up to 12 Years 2-3 Years

CD4 Cell Count

1,000

500

CD4 Count and OpportunisticInfections

200

100

HIV

CD4

Bacterial Pneumonia, TB, HSV, Cryptosporidiosis

Thrush, lymphoma, KS



HIV-1: Treatment

1987: First compounds acting against RT tested 1987: AZT found to block Reverse Transcriptase

1990s: anti-retrovirals targeting other aspects of

HIV replication besides RT search begun

Protease inhibitors

1994: ACTG 076, shows transmission from

mother to fetus reduced to 8% with 12 week

course of AZT 1996: David Ho reports at Vancouver BC

International AIDS Conference triple combination

therapy suppressed HIV to undetectable levels



- Prevent binding

- Prevent fusion

- Preventassembly

and

maturation

- Prevent

transcription of

RNA to DNA

- Prevent

insertion of viral

DNA to host

DNA



HIV/AIDS treatment

Treat OIs with variety of anti-bacterial anti-fungal/parasiticdrugs, and anti-cancer drugs

Highly Active Antir etroviral

Therapy (HAART) Nucleoside/nucleotide reverse

transcriptase inhibitors (NRTI)

Non-nucleoside reversetranscriptase inhibitors (NNRTI)

Protease inhibitors (PI)

Fusion inhibitors

Usually 1-3 HAART drugs per day—between 2-4 times a day



0

2

4

6

8

10

12

1992 1993 1994 1995 1996 1997 1998

I n c i d e n c e

/ 1 0 0

p e r

s o n - y e a r s

Disseminated MAC

Esophageal candidiasis

Kaposi’s sarcoma

CMV retinitis

CMV disease

Cryptococcosis

Toxoplasmosis

Incidence of Opportunistic

Infections and Malignancies

J.E. Kaplan et al. CID 2000;30:S5-S14

PCP Protease inhibitors



Hypothesized patterns of HIV Dependency Factors (HDFs): >

250 proteins HIV depends on to replicate (Brass, et al. 2008)



Documents

HIV Virology Lecture2!3!29!12!1