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1

Retroviruses

HIV

HTLV-1

Gene Therapy

XMRV (not really studied)

HIV causes aids, one of th emost important infectious diseases in the world

HTLV-1 (human T cell leukemia virus type 1), causes an adult T cell leukemia, rare in the US

Gene Therapy
- ability to integrate, retroviruses are sometimes used as gene therapy vectors to stably introduce genes into human cells

XMRV- link between this and chronic fatigue syndrome and prostate cancer but this virus was a lab contaminant so ignore this

2

Part 1: Retrovirus Basics

Enveloped virus

Spread by body fluids

Single enveloped spike protein (Env) RNA genome (single virally encoded glycoprotein called envelop protruding from the viral membrane)

Positive sense RNA

Two copies per virion

Lentivirus - type of virus that can infect non dividing cells, has 6 additional genes that control viral gene expression or subvert the host immune response (in addition to gag, pol and env)
- have to get into nucleus and most simple retroviruses cant get into nucleus until it breaks down during replication/ division but a lentivirus can

3

Schematic of a retroviral genome and proteins involved

LRT (U3 R U5) — gag — pol — env — LTR (U3 R U)

Gag: Group specific Antigen - polyprotein that is processed by viral protease to make the viral matrix and capsid proteins that surround the genome
- large protein cleaved by protease to produce proteins that form the nucleocapsid

Pol: polyprotein that contains the enzymes reverse transcriptase (RT) integrase and protease
- polymerase, produces a polyprotein that contains the three enzymes made by retrovirus: protease integrase and RT

Env: makes the envelope protein that protrude from the viral membrane

LTR: long terminal repeats - repeated sequences at the end of the 10 kB genome that help the virus replicate, play a role in replication and in gene expression

4

What makes Retroviruses Unique

Feature 1

Why is this important?

Retroviruses can make DNA out of RNA

Single Stranded RNA viral genome—> Viral reverse transcriptase enzyme (RT) —> double stranded DNA copy

Important bc
1. RT is a drug target
2. RT makes mistakes—> high error rate gives rise to drug resistance (because it has no proof reading ability) —> HIV can quickly evolve and adapt to selective pressures from antiviral drugs or immune system

5

What makes retroviruses unique?

Feature 2

Why does it matter

Retroviruses integrate their DNA into the host chromosome

Viral DNA + host cell DNA—> integrated viral DNA (via viral integrase enzyme)
- covalent insertion of the virus’s double stranded genome into the host genome via integrase

Important bc
1. Integrase is a drug target
2. Once integrated the viral dna becomes a permanent part of the cell, it can only be eradicated by killing the cell which makes HIV a life long infection and good for gene therapy
- cannot clear the virus infection from a cell without killing the cell, retrovirus infections persist for the life of the organism

First integrase inhibitor was Raltegavir

6

How are retroviruses used clinically

As gene therapy vectors

Integration results in stable expression of the transgene but integration can also lead to tumor formation

Eg 11 male infants with SCID
- bone marrow cells collected infected with mouse retrovirus expresses gene for IL2 receptor—> give cells back to patient —> SCID was cured but they developed leukemia
- murine retrovirus liked to integrate into transcriptional start sites and in 4 pt it activated expression of a gene linked to leukemia

7

Where does HIV preferentially integrate?

At active genes, but not at transcriptional start sites

- integration sites cluster around gene rich regions
- chromosome 13 and 18 which are gene poor have few integrateion events while gene-rich chromosomes have many integrations bc HIV likes to integrate within active genes not start sites (also makes it safer to use for gene therapy than simple retroviruses)

8

How has gene therapy changed to reduce risks associated with integration

Retroviruses—> lentiviruses (can integrate into non-dividing cells )

Eg CAR-T therapy
- genetically modified HIV expressing Chimeric Antigen Receptor in T cells
- HIV integrated and CAR expressed, enabling T cells to bind to cells expressing protein CD19 (B cells) and kill them (have body kill Emily’s ALL)

Because lentiviruses have a lower prob to interfere with expression of genes involved in cellular transformation, they are being used in gene therapy trials

9

Transfection

Introduction of any DNA or RNA into cells by a method other than using a virus

10

Transduction

Delivery of a cellular gene to a new cell by using a virus

11

What is the enzyme other than RT and integrase that Retroviruses use

Why is it important?

Protease!!!

Important bc it is an important drug target (protease inhibitors)

GAG/POL polyprotein
GAG— Integrase— RT— Protease

Protease is part of a large polypeptide, protease cleaves the polypeptide into many component parts, including RT, integrase, and viral core proteins and when this occurs the virus ‘immatures’

- if this does not occur, the virus cannot productively infect cells
- some of HIVs proteins are made as a large polyprotein, part of this has protease function and after synthesis protease cleaves polyprotein into its various subunits—> produce many proteins from the single precursor

12

Basic Retrovirus Lifecycle: Where do the 3 viral enzymes act

HIV is a lentivirus, a type of retrovirus that can infect non-dividing cells. Other retroviruses only infect dividing cells

Basic retrovirus lifecycle
Receptor binding
- receptor for HIV is CD4 protein—> viral Env protein then mediates membrane fusion with the host cell—> viral core is now in the cell - it must ‘uncoat’ and reverse transcription begins and is completed in the cytoplasm
- Viral DNA enters the nucleus due to nuclear localization signals on several of its associated proteins, integrates and then is transcribed
- since there is but a single promoter but multiple genes, RNA splicing events occur to make the needed mRNAs
- MRNAs exported from the nucleus and proteins made in the usual way—> virus assembles and buds at the plasma membrane and then the virus matures due to the action of the protease

Steps:
1. Receptor binding
2. Membrane fusion and entry
3. 3. Uncoating and reverse transcription - TARGETTED BY REVERSE TRANSCRIPTASE
4. Nuclear Uptake
5. Integration in nucleus - TARGETTED BY INTEGRASE
6. Transcription
7. RNA processing
8. Nuclear export
9. Translation
10. Assembly
11.Budding
12. Maturation - TARGETED BY PROTEASE

13

HIV epidemiology and definition of AIDS

GIEMSA STAIN slide 13

Pneumocystis pneumonia was rare until 1979 when 5 cases were found in LA

Over next year additional cases of this rare pneumonia were observed, patients all young, gay men

With giemsa stain at high magnification, the faint bluish dot like intracystic bodies of pneumocystis caring in lung are seen in this cytology preparation from a bronchoalveolar lovage

14

HIV and AIds
- infectious agent Kaposi’s sarcoma

Pic page 14

Before AIDS< kaposi’s sarcoma found in elderly Jewish men and immunocompromised, rare here

But cluster in SF and NY - young gay men

15

Morbidity and Mortality weekly report- summer of 1981

First description of AIDS, pneumocystis pneumonia and kaposi’s causes

16

AIDS described December 1981

By end of 1981 syndrome described- acquired immunodeficiency syndrome, 234 had died in US

17

HIV discovered, 1983

Cultured in vitro in 1984–> tests to screen blood but many ppl became infected with HIV following blood transfusions eg hemophiliacs screwed

2304 Americans died of aids in 1984

18

Definition of AIDS

HIV infected persons with CD4 < 200

HIV infected and have had an AIDS defining illness (opportunistic infection)

AIDS doesnt equate to HIV
- once a pt is diagnosed with AIDS they maintain this diagnosis even if CD4 counts increase above 200 due to effective therapy

19

HIV in US

45000 new HIV infx in US and 20,000 deaths due to aids per year—> total number of people in US HIV infected increasing —> 1.2 mill living with HIV in the US, 1/8 dont know they are infected

20

Important modes of HIV transmission

IV drug abuse (IVDA)

MSM

21

What parts ot eh country and sub pops have high HIV prevalence rates?

MSM = 70% new infx

African descent - nearly half of all new infx

Infx rate of women of African decent is 20 fold above that of whitte women and 5 fold above Hispanic women

Prevalence of HIV in DC is 2.5%

22

HIV has infected 70 million people; half have died

In 2016, there are 37 million people living with HIV infx—> 2.1 mill new infections and 1.1 mill death in 2016

23

Where is HIV particularly prevalent

Underdeveloped countries eg subsaharan Africa, Botswana, highest infection rates in the world

24

HAART therapy

Highly Active Antiretroviral Therapy

Reversed trend of increasing AIDS deaths in the US, greater prevalence because more people living with AIDS

25

Treatment in the developing world

Being expanded
New infections and deaths due to aids have fallen by 1/3 and people being treated has increased

26

When and Where did HIV come from?

Construct phylogenetic tree that shows genetic relationships between hundreds of virus strains isolated throughout the world

A and B have 98% sequence identity and are connected by a horizontal line, the length of which is proportional to genetic disease - 2% in this case

C and D are 96% similar etc

27

HIV family tree - what does it tell you?

Slide 28

HIV strains are grouped into ‘clades’, in the US almost all infections are caused by Claude B virus

This is why there is no AIDS vaccine— lots of diversity!

Star burst pattern- terminus of each line represents a virus strain and they are connected to eachother

Sequences cluster into groups termed clades and given letter designations

Virus strains within a clade are more similar to eachother than they are to viruses in other clades

In North America, virtually all HIV + ppl have a clade B virus, in other parts of the world, other clades predominate

**alll HIV strains arose from a common ancestor - single introduction of HIV into humans at some point in the past

28

Genetic Distance vs Year graph - what does it tell you?

Introduced into the population around 1910, cake from nonhuman primates

Year virus was isolated on the x axis and its genetic distance from the ancestor on the y axis—> linear regression—-> pandemic arose from single transmission event around 1910

29

What is HIV closely related to and what does this mean

SIV (Simian immunodeficiency virus)

Virtually all old world primates have their own strain of SIV - infected animals dont get sick- perfect host parasite relationship

SIV from African green monkey, when introduced into Asian rhesus macaques causes AIDS like illness bc the macaques do not have their own SIV

30

What is HIV1 most closely related to

What does this tell you about transmission

SIV from chimpanzees—> HIV1 arose by transmission of SIV from a chimp into a human

HIV2 arose via transmission of SIV from sooty mangabeys

HIV is an example of zoonosis