Retroviruses - HIV-1

genome of HIV-1?

• positive sense ssRNA

• two identical copies of RNA genome covered with NC

• cellular tRNA attached to RNA

capsid of HIV-1?

conical capsid made of CA (capsid proteins)

is HIV-1 enveloped?

• enveloped, derived from PM

• contains SU and TM

  • SU = surface protein

  • TM = transmembrane protein

How does HIV-1 enter the cell? Be able to describe actions of SU (gp120) and TM (gp41) in virus entry.

1. Gp120 on HIV binds CD4 on T cell/macrophage → Gp120 conformational change

2. exposes CCR5 or CXCR4 co-receptor binding site

3. Co-receptor binding to gp120 -> gp120 conformational change

4. Triggers gp41 to mediate fusion of viral and cytoplasmic membranes

What are the 2 activities of the RT? Why are both of these activities important?

RNA and DNA-dependent DNA pol and RNase H activity

1. RNase H activity destroys the RNA part of an RNA-DNA hybrid

2. these two activities are required for the conversion of viral RNA → retroviral DNA

viral RNA → retroviral DNA?

(hints: U3, R, U5, ppt, 1 and 2nd transfers, )

1. DNA copy of R and U5

2. removal of RNA from DNA-RNA hybrid

3. (1st transfer) DNA copy of Rseq hybridizes with 3’ R seq in genomic RNA

4. full length DNA copy of genome is generated, generating a U3-R-U5 sequence at the right end of DNA copy (long terminal repeat LTR)

5. template RNA is removed by RNase H except for polypurine tract (ppt is digestion resistant)

6. synthesis is initiated by ppt RNA primer, extending through the LTR and 18 nucleotides of tRNA hybridized to PBC

7. removal of ppt sequence and tRNA by RNase H activity

8. (2nd transfer) exposed PBS can hybridize with PBS at other end of strand

9. both (-) and (+) DNA strands are extended by RT (DNA-dep, DNA pol)

- binds primer binding sequence (PBS), acts as primer for RT

Cellular tRNA

Describe the proviral DNA. How is it different from the retrovirus genome?

1. Proviral DNA remains associated w virus capsid = preintegration complex

2. It is too big to enter through nuclear pores

How does proviral DNA get access to cellular chromosomal DNA in a lentivirus? IN a retrovirus that isnt a lentivirus?

Lentivirus 

1. Can mediate transport of viral DNA complex through nuclear pore

2. MA, Vpr, integrase 

3. Viral integrase mediates the integration of proviral DNA into the host cell DNA = cleavage-ligation reaction

Other retroviruses

1. Wait for breakdown of nuclear envelope, usually during cell division 

2. Can’t infect terminally differentiated cells 

3. Must infect cells that undergo mitosis

What catalyzes integration of proviral DNA?

Viral integrase 

What are the 2 outcomes after HIV-1 provirus integration?

Active infection

1. Viral mRNAs transcribed

2. Production of progeny virus 

3. Occurs only in activated T cells

Latent infection 

1. Viral RNA is not expressed but its maintained for long periods in genome

When theres a latent infection, theres a barrier to - of virus by - because immune system can’t - that cell is infected

full elimination, immune system/drug therapy, recognize

What is the function of the retroviral LTR in initiation mRNA synthesis?

1. Retroviral LTR = promoter/enhancer

2. LTR binds NFKB and NFAT (TFs)

3. LTR must bind these TFs to activate transcription

   1. TCR binding/exposure to various cytokines

   2. TFs move to nucleus/become activated

How do you get production of mRNA that encodes the Env protein?

splicing of primary transcript

full length RNA yields?

singly spliced?

doubly spliced?

• synthesis of Gag and Gag/Pol polyproteins

• synthesis of Env, Vpr, Vif, and Vpu

• synthesis of Tat, Rev, Nef

How is the Gag/Pol polyprotein produced?

(hint: mention RNA structure, stop codon, ribosomal shifting, reading frame)

• unspliced, full length RNA is used to synthesize Gag and Gag/Pol polyproteins

• there is a stop codon between Gag and Gag/Pol regions and they’re often in different reading frames

• RNA stem-loop causes stalling at the heptamer sequence and the shift of the ribosome back 1 nucleotide, resuming translation in a different reading frame, resulting in the stop codon not being recognized, resulting in synthesis of Gag/Pol

Tat protein?

what if there is no Tat?

• transactivator of transcription

• increases amount of viral RNA produced by increasing elongation efficiency of cellular RNA pol II

no tat?

• transcripts are short

• HIV-1 genes aren’t highly expressed

Rev protein?

• regulator of expression of viral proteins

• mediates nuclear export of unspliced and singly spliced HIV-1 mRNAs

• Rev has NLS and NES that mediates its entry into and exit out of the nucleus

• Rev binds RRE (Rev response element) in unspliced and singly-spliced RNA, dragging them out of the nucleus for translation

• Rev returns to nucleus to shuttle more HIV-1 mRNAs

Describe the process of assembly of a new retroviral particle?

1. - protein travels through golgi + PM

2. Env cleaved → - and -

3. - and - modify PM that will bud off to be viral envelope

4. - and - released into - after translation and they associate with each other

5. capsid assembly

   1. genome RNA binds Gag via -

   2. - incorporated into assembling virus particle

   3. Gag targeted to PM by - tag added to polyprotein after translation, bringing assembling particle to membrane

6. virus particle buds from PM

1. Env

2. SU and TM

3. SU + TM

4. cytosol

5. Gag, Gag/Pol

6. packaging signal

7. tRNA primer

8. myristate

When are the viral proteases activated?

1. - becomes activated

2. Cleaves - and - into structural and enzymatic proteins

3. Virus rearranges into its mature form, becomes infectious

when virus particle buds from PM

1. Viral protease PR

2. Gag, Gag/Pol polyproteins

What are the 3 phases of HIV-1 infection? Be able to describe what is happening in terms of CD4+ T cells and viral load at each phase

1. Acute infection phase

   1. Viral load is high

   2. Virus replicates in GALT killing CD4 T cells

2. asymptomatic phase (clinical latency)

   1. Viral load stabilizes at viral set point 

   2. CD4 T cells levels drop 

3. AIDS (acquired immunodeficiency syndrome)

   1. Viral load increases

   2. CD4 T cells levels drop below 200 uL/ul

What stages of HIV-1 life cycle are targeted for treatment?

1. RT

2. protease

3. virus entry/fusion

4. integrase

what drugs target RT?

nucleoside and non-nucleoside RT inhibitors

1. AZT = azidothymidine - nucleoside analog

   1. has an altered sugar group

   2. when taken up and phosphorylated by the cell, the AZT triphosphate incorporates into the growing DNA strand, causing termination due to the missing OH group

2. Nevirapine, tenofovir, efavirenz = non-nucleoside analog, binds to RT at site close to active site for DNA pol activity and slows rate of DNA pol

what drugs target protease?

- = protease inhibitor

• a peptide that binds - but it can’t be cleaved, taking up the space so that the protease can’t cleave other polyproteins

ritonavir

• HIV-1 protease

whats the difference between nucleoside and non-nucleoside RT inhibitors

• nucleoside RT inhibitors act as competitive substrates for the enzyme, mimicking natural nucleotides

• non-nucleoside RT inhibitors bind to a different site on the enzyme, inducing a conformational change that disrupts its activity

Why are HIV-1 antiretroviral drugs always used in combo?

HIV-1 antiretroviral drugs are almost always used in combination because HIV can rapidly develop resistance to single drug treatments