Week 5-Pathogenesis of human retroviruses: family Retroviridae

Retroviridae: virion structure

• Spherical, enveloped particles

• icosahedral or conical capsids

• contains reverse transcriptase intrase within capsid (required; needs to be packaged)

Retroviridae: classification

• infect wide range of organisms

• can cause AIDS and leukemia in humans

Retroviridae genome

• positive- sense ss-stranded RNA (7-10Kb)

• complexed with the nucleocapsid protein (NC)

• 2 identical copies of the (+)RNA in a dimer form are packaged into the virion

  • are identical sequences, not base pairs

• specific cellular tRNAs bound to genome RNAs

  • originate from tRNA from host cells

components of Retroviridae genome

• R = repeat sequences

• U5 and U3 = untranslated 5’ and 3’ sequences respectively

• PBS = primer binding site

  • where tRNA binds to act as a primer for reverse transcriptase

• ppt = polypyrimidine tract

  • if in middle of sequence can not be degraded

  • if att end of genomic sequence can be degraded

  • important during reverse transcriptase

Retroviridae: virion components

• envelope contains

  • a surface protein (SU) which is bound to the transmembrane protein (TM) that traverses the bilayer

• Viral matrix protein (MA)

  • coats the inner side of the membrane

• capsid protein (CA)

  • helps form the icosahedral or conical capsid

• core of the virus contains 3 main virally encoded enzymes

  • reverse transcriptase (RT)

  • integrase (IN)

  • protease (PR)

Retroviridae: cell entry

enter cells by the fusion pathway

• Viral SU protein interacts with cell surface receptors

• Envelope fuses with plasma membrane or is endocytosed followed by fusion within low pH endosomes

• Viral SU goes through conformational change upon receptor binding, exposing the hydrophobic region of TM protein, which inserts into the cell membrane

• Early phase includes entry, making a DNA copy of its RNA genome, and integrating it into the cellular genome

overview of Retroviridae: reverse transcription

• Viral RNA is converted into double-stranded DNA copy by reverse transcription

  • results in the production of proviral DNA

• RT is a dimer

  • has RNA-dependent (or DNA-dependent) DNA polymerase activity

  • has ribonuclease H activity

    • RNAse H selectively destroys the RNA in an RNA-DNA hybrid

  • lack of proofreading capability leads to high mutation rate

steps for Retroviridae: reverse transcription

1. Cellular tRNA bound to PBS used as primer → DNA copy of U5 & R

   • makes (-) DNA strand for begining region

   • DNA-RNA hybrid on 5’end of original genomic DNA

2. RNAse H digestion targets DNA-RNA hybrid; resulting in only the RNA region being degraded → leaves DNA copy of U5 &R and tRNA primer

3. first strand transfer. The 5’ R section compliments te 3’ R section. Thus the (-) U5 and R DNA segementt attach to the 3’ end with the tRNA

   • DNA copy anneals to 3’ end R sequence

4. reverse transcriptase makes a full-length (-) sense DNA genome copy

   • resuling U3-R-U5 sequence is a long terminal repeat = LTR

     • a pair of identical sequences of DNA, several hundred base pairs long, flank genes/pseudogenes/viral dna. forms a retrotransposon or an endogenous retrovirus or a retroviral provirus

     • Typically, an element flanked by a pair of LTRs will encode a reverse transcriptase and an integrase, allowing the element to be copied and inserted at a different location of the genome

5. RNAse H digestion removes almost all original RNA genome template, exceptt ppt (as it was in middle of sequence

6. Ppt serves as template for RT to synthesize a complementary strand to tthe 3’ end of the made (-)DNA genome

   • results in DNA RNA hybrid at the 3’ end (-) DNA stand

7. RNAse digest ppt & tRNA, leaving only U3-R-U5 PBS (+) RNA genome

8. Second strand transfer→ the PBS (+) moves and annealls to the PBS (-)

9. RT then extends both DNA strands

10. Linear dsDNA with LTRs on both ends are generated = proviral DNA

Retroviridae: integration

A copy of proviral DNA is integrated into the cellular genome at a random site

• some sites more likely to be integraded (if not methylated or compressed)

  • Carried out by the enzyme integrase (IN)

  • stably integrated into host genome

• Usually have to wait for nuclear envelope to break down for integration

Retroviridae: transcription & translation

• Host cell transcribes & translates virus genome

  • translation of the multiple mRNAs

  • processing enzymes cleave the polyproteins into single proteins

    • requires viral protease to cleave polyprotein

Retroviridae: budding & cell exit

• Late phase involves expression of viral RNA, viral protein synthesis and assembly of virions

  • Envelope proteins go through the host secretory pathway= expressed on PM

  • budding occurs at sites where envelope proteins cluster

Retroviridae antiviral

• NRTI & NNRTI: inhibits reverse transcription

• HIV-P1: stops viral maturation

  • HIV buds off then comples virion maturation

Retroviridae: Lentiviruses

• for slow progression of disease

• results Human immunodeficiency virus types 1 and 2 (HIV-1, HIV-2)

Human Immunodeficiency Virus Type I genome structure

• Spherical enveloped particle

• 100nm

• conical capsid

• 9.3Kb ssRNA positive sense genome (2 RNAs/virion)

• Lysine tRNA bound to genome RNAs

is a complex retrovirus

Human Immunodeficiency Virus Type I genome structure

• complex retrovirus (encodes 25 mRNAs)

  • simpler retroviruses make 2 mRNAs

• Uses alternative splicing leads to several mRNAs; makes

  • structural proteins

  • regulatory proteins

Human Immunodeficiency Virus Type I encodes

• three capsid proteins

  • matrix (MA)

  • capsid (CA)

  • nucleocapsid (NC)

• Three enzymes

  • protease (PR)

  • reverse transcriptase (RT)

  • integrase (IN)

• Two envelope proteins

  • surface (SU)

  • transmembrane (TM)

• Five regulatory proteins (6 non-structural)

  • Vif (viral infectivity factor)

  • Vpu (virion protein unique to HIV-1)

  • Tat (transactivator of transcription)

  • Rev (regulator of expression of virion proteins)

  • Nef (negative effector)

  • Vpr (virion protein R)

HIV-1 cell entry

• targets cells of the immune system by recognizing CD4 antigens and chemokine receptors

  • CD4 is found on both T lymphocytes and macrophages

  • A co-receptor of either CCR5 or CXCR4 is also required

  • CXCR4 is expressed on T cells

  • CCR5 is expressed on macrophages

    • T-cell tropic or macrophage tropic virus strains

• example of Model of HIV-1 entry:

1. gp120 interacts with CD4

2. conformational change exposes region on gp120 that interacts with chemokine receptor

3. fusion domain of gp41 now becomes exposed

4. close proximity of viral and cell membrane induces fusion = viral nucleocapsid enters cell cytosoll

Unlike other retroviruses, HIV-1 directs transport of proviral DNA into the cell nucleus

• allows for productive infection in non-dividing cells

• many regulatory proteins aid in this process

oncogenic retroviruses

• Acute transforming retroviruses express mutated forms of cellular growth signaling proteins

  • Acute transforming retroviruses express mutated forms of cellular growth signaling proteins

  • growth factors, receptors, transcription factors etc.

  • bring in genes simillar to host, which is unregulated (like growth factor), result in rapidly dviding cells cancer

example

• Viral Src is nearly identical to cellular c-src

  • codes for a protein tyrosine kinase

    • under the control of a viral promoter src is unregulated=uncontrolled cell growth = tumours

HIV & AIDS

• Acquired immune deficiency syndrome (AIDS) was first described in 1981

• HIV replicates in and kills lymphocytes and macrophages

  • due tto infectious immune cell

  • -Infection results in depletion of CD4+ T cells, resulting immune-incompetent

  • Opportunistic infections by other pathogens are often fatal

• HIV is transmitted through sexual contact and blood exchange

  • sharing needles

still a global HIV epidemic, but reduced numbers significantly (good health promotions, trying to reduce it more)

Stages of AIDS

• Acute infection

  • inital infection of HIV virion

  • High viral RNA, slight CD4+ T cells

    • some immune response and thus flu like symtoms

    • spike in CD4+ cells

  • virions begin to infect/enter immune cells

• Clinical latency

  • virus RNA levels consistent (virus load setpoint)

  • steady decline of CD4+ cells

• AIDS

  • once CD4+ deacrease pass a threshold result in AIDS

  • lack of immune cells, increase viral RNA (nothing stopping them, and will multiply)

HIV & treatment

• No vaccine developed

  • Imbokodo vaccine study:

    • “Mosaic” vaccine that combines epitopes from many different HIV strains, making a truly global vaccine

    • Enrolled women in sub-Saharan Africa – 2600 participants vaccinated

      • induced neutralizing antibodies in animals (but not shown in humans, thus was canceled).

        • Used Alum adjuvants

• Antiviral drugs can control HIV-1 infection and prevent disease progression

  • Treatment involves a combination of drugs targeted at various steps in the viral life cycle (e.g. reverse transcription, fusion, protease processing, integration etc.) = anti-retroviral therapy

  • e.x

    • PrEP (pre-exposure prophylaxis)

      • for after exposure