Lecture 13 PPT – Retroviruses & HIV Comprehensive Study Notes

Class VI – Retroviruses: Foundational Concepts

• Mature virion carries two capped & poly-A(+) RNA strands + two RT molecules
  • $\rightarrow$ RNA is not translated after uncoating
  • Converted in cytosol to $ss(-)\,DNA \xrightarrow{RT} dsDNA$
  • dsDNA enters nucleus and integrates into host genome (provirus)
  • Host $RNA\,Pol\,II$ transcribes proviral DNA $\Rightarrow (+)\,mRNA$ for proteins & new genomes
• Gene-expression regulation layers
  • Alternative splicing
  • mRNA nuclear export / cytosolic localisation
  • Translational control
  • Post-translational proteolysis, glycosylation, myristylation, etc.

Historical Perspective

• 1911 – Peyton Rous discovers RSV tumor virus in chickens.
• 1971 – Baltimore & Temin independently detect reverse transcriptase (RT).
• Following decade – proof that retroviral genomes integrate into host chromosomes $\Rightarrow$ oncogenesis.
• Howard Varmus: RSV oncogenesis via capture & mutation of a host proto-oncogene.
• Early 1980 s: Cluster of Pneumocystis pneumonia + Kaposi’s sarcoma $\Rightarrow$ led to discovery of HIV (Human Immunodeficiency Virus).

HIV Tropism & Immune-System Destruction

• gp120 (SU) surface glycoprotein binds CD4 – expressed on $T_H$, macrophages, dendritic cells.
• Co-receptors required:
  • $CCR5$ – macrophage infection (M-tropic)
  • $CXCR4$ – $T_H$ infection (T-tropic)
• Pathogenesis mechanism
  • gp120 is inserted into plasma membrane of infected cell $\to$ acts as viral envelope
  • Infected cell fuses with uninfected CD4$^{+}$ cell $\approx$ up to $500$ fusions/cell
  • Only $0.2\%$ infected cells suffice to wipe out entire susceptible population.
• Clinical course
  • Primary infection often asymptomatic; diagnosis by anti-HIV antibodies.
  • Viremia falls (clinical latency) while $T$ cells slowly decline.
  • Once $T$ cells cross critical threshold $\to$ opportunistic infections $\to$ AIDS.

Retroviral Genome Architecture

• Two identical $35S$ RNAs $\Rightarrow$ hybridize $\to 70S$ pseudo-diploid genome.
• Each RNA annealed to specific tRNA primer at pbs site:
  • Species-specific: $tRNA^{Pro}$, $tRNA^{Lys3}$, $tRNA^{Lys1,2}$ etc.
• Encapsidated with NC protein
  • Promotes RNA–RNA pairing, template switching, and high RT processivity.
• Long Terminal Repeats (LTRs) present at both ends; essential for integration & transcriptional control.

Reverse Transcriptase (RT) – Multifunctional Enzyme

• Active dimer (often heterodimer $p66/p51$ in HIV).
• Activities
  • $RDDP$ – RNA-dependent DNA Pol
  • $DDDP$ – DNA-dependent DNA Pol
  • Helicase/unwinding
  • RNase H – degrades RNA strand of RNA–DNA hybrids.

HIV Adhesin, Attachment & Uncoating

• Env glycoprotein trimer = (SU gp120) + (TM gp41)
  • TM spans membrane; SU non-covalently attached via disulfide (V1–V5 variable loops).
• Binding sequence
  1. gp120–CD4 interaction $\rightarrow$ conformational change exposes co-receptor site.
  2. gp120–CCR5/CXCR4 binding $\rightarrow$ triggers gp41 fusion peptide insertion and membrane fusion.
• Host chaperone cyclophilin A packaged in virion assists capsid uncoating.

Detailed Steps of Reverse Transcription (HIV Example)

• Primer jump 1
  • RT synthesises $\sim$18-nt strong-stop DNA across 5' LTR.
  • RNase H removes RNA at this region.
  • DNA relocates & anneals to complementary 3' LTR of second RNA strand.
• First full-length synthesis: RT extends to 5' end, degrading RNA template.
• RNase H leaves polypurine tract (PPT) intact $\to$ primer for second strand.
• Primer jump 2
  • Second strand synthesis begins at PPT, moves to 5' end, displaces tRNA primer.
  • Complementary sticky ends form; genome circularises temporarily.
• Final strand displacement + fill-in $\to$ linear dsDNA with U3–R–U5 LTRs at both ends.

Integration

• Viral Integrase (IN) generated by proteolytic cleavage of Gag-Pol polyprotein; packaged with RT (≈50$–$100 copies).
• Conditions & chemistry
  • Requires $Mg^{2+}$ or $Mn^{2+}$, no ATP.
  • Functions as tetramer; 2 active subunits + 2 structural.
• Mechanism
  1. IN trims 3' ends to expose conserved $\,TG$ dinucleotide $\Rightarrow$ pre-integration complex (PIC).
  2. PIC enters nucleus (not cell-cycle restricted for HIV).
  3. Staggered cut in host DNA (4–6 bp).
  4. 3'-OH viral ends attack phosphodiester bonds of host DNA $\rightarrow$ strand transfer.
  5. Host repair enzymes fill gaps, duplicating target sequence flanking provirus.
• Target DNA choice largely sequence-nonspecific in vitro; in vivo guided by host factors (e.g., Ini-1, RNA Pol III).

Transcriptional Initiation & Tat-Mediated Elongation

• LTR acts as promoter/enhancer; binds multiple host TFs: Gata-3, NF-IL6, LEF, USF, Ets-1, NF-κB, Sp1.
• RNAP II initiation efficient but early elongation stalls; only $\approx 10\%$ reach full length.
• Viral protein Tat (nuclear localisation & RNA-binding) rescues elongation.
  • Requires TAR RNA element (stem-loop at 5' end of nascent RNA).
  • Tat binds TAR RNA, recruits Cyclin T1 + CDK9 + TFIIH.
  • CDK9 phosphorylates RNAP II CTD $\Rightarrow$ processive elongation $\to$ abundant 9-kb transcripts.

Splicing, Polyadenylation & Balanced RNA Species

• Proviral transcript serves both as genomic RNA (9 kb) & mRNA.
• Protein coding correlation
  • Gag, Pol $\Leftarrow$ unspliced 9 kb RNA (also packaged).
  • Env $\Leftarrow$ singly spliced 4 kb RNA.
  • Regulatory (Tat, Rev, Nef, Vpr, Vif, Vpu) $\Leftarrow$ multiply spliced 2 kb RNAs.
• Splice-site efficiency dictates ratio; point mutations shifting balance toward full-length severely dampen virion output.
• Coupling with polyadenylation
  • Two identical polyA signals within LTRs; 5' signal silenced by $U1$ snRNP to avoid premature termination.
  • 3' splice site in terminal exon boosts 3' polyA cleavage.

mRNA Export – Cellular vs HIV Strategy

• Normal cellular route
  1. Completion of splicing $\rightarrow Uap56 \rightarrow Ref/Aly$
  2. Ref recruits Tap/p15; Tap exposes NES
  3. Tap–Xpo–Ran-GTP complex exports RNA.
• Problem: HIV needs to export unspliced & partially spliced RNAs.
• Rev protein solution
  • Domains: NES, NLS (binds Importin α/β), two dimerisation motifs.
  • Binds RRE (Rev-response element) in 3' region of 9 kb & 4 kb RNAs.
  • Process
  1. Early phase: only Tat & small RNAs produced (RRE removed).
  2. Rev translated, imported to nucleus.
  3. Rev dimer binds RRE $\Rightarrow$ recruits eIF5A $\Rightarrow$ Exportin 1(Xpo) + Ran-GTP + Sam68.
  4. Complex docks at nuclear pore (Can/Nup214, Nup98) $\Rightarrow$ cytosol.
  5. Ran-GAP GTP hydrolysis releases RNP; Rev recycled to nucleus.
• Leptomycin B (Xpo inhibitor) blocks both cellular NES-dependent export & HIV RNA export.

Protein Processing, Assembly & Maturation

• Env (gp160) synthesis
  • Co-translational ER entry; signal peptidase cleaves leader.
  • PDI forms SU–TM disulfide bond; extensive N-linked glycosylation.
  • Golgi protease Furin cleaves gp160 $\to$ mature gp120 (SU) + gp41 (TM).
• Vpu functions
  • Binds CD4 in ER, escorts to cytosol for ubiquitin-proteasome degradation.
  • Also retro-transports MHC-I to avoid CTL recognition.
• Gag polyprotein
  • Myristoylation signal (aa 15–31) anchors Gag to plasma membrane.
  • Viral protease (PR) cleaves Gag $\to$ MA, CA, NC; later cleaves Pol to liberate PR, RT, IN.
• Assembly requirements
  • 5' $\psi$ region – SL1 & SL3 stem-loops mediate RNA dimerisation & NC binding.
  • Structural proteins gather at inner leaflet of membrane enriched in gp41 cytosolic tails.
  • Two RNA genomes + tRNA primer + enzymatic proteins incorporated.
  • Budding $\rightarrow$ immature virion; PR activity during/after release yields mature infectious particle.

Immune Evasion: Error-Prone Replication

• RT lacks 3'$\rightarrow$5' exonuclease proofreading; error rate $\approx 10^{-4}$ per nt.
  • $\approx$1 mutation per gene per replication cycle.
• Consequences
  • Rapid antigenic drift of gp120 $\Rightarrow$ antibodies from early infection ineffective against progeny.
  • Vaccine design complicated; targets mutate before immune memory useful.

Therapeutic Strategies & Challenges

• AZT (3'-azido-3'-deoxythymidine) – nucleoside analog chain terminator; RT selects AZT-resistant mutants quickly.
• Protease inhibitors (PI) – block PR cleavage; mutants emerge.
• HAART / cART – combination of NRTI + NNRTI + PI + integrase/entry inhibitors lowers viral load; long-term efficacy still under study.

Natural Resistance & Experimental Vaccines

• Some individuals naturally resistant
  • CCR5-Δ32 homozygotes – truncated, non-functional receptor $\Rightarrow$ prevents R5 strain entry.
  • Robust cytotoxic T-cell responses clearing infected cells.
• Novel vaccination ideas
  • DNA vaccines encoding conserved catalytic motifs (e.g., RT active-site $\,DDX$ triad) trigger cell-mediated immunity not subject to antigenic drift.
  • Conjugation to Bacillus anthracis PA antigen or use of attenuated rabies vectors to enhance delivery & immunogenicity.
• Ongoing question: will multi-target vaccines / combination therapies shift virus impossibly? Too early to conclude.

Key Numerical / Statistical Points

• Hybridised retroviral RNA sedimentation: $70S$.
• Fusion potential: 1 infected cell may fuse with ≈500 CD4$^{+}$ cells.
• Population sterilised if ≥0.2\% cells infected.
• In absence of Tat, only ≈10\% of initiated transcripts read through whole genome.
• Staggered host‐DNA cut length during integration: 4–6 nt duplication.

Ethical, Practical & Philosophical Implications

• High mutability questions feasibility of classic prophylactic vaccines.
• Socio-economic burden: life-long drug regimens, access inequality.
• Potential for oncogenic insertion events (retroviral gene-therapy vectors must be engineered for integration specificity).
• Understanding natural resistance (CCR5-Δ32) guides gene-editing prospects (CRISPR-Cas9 therapies).

Integrative Connections to Prior Knowledge

• Reverse transcription mechanistically parallels telomerase activity (RNA template, DNA polymerisation).
• Integration reminiscent of transposon cut-and-paste; HIV IN shares catalytic D,D(35)E motif with transposases.
• Regulation of nuclear export via Rev mimics CRM1-dependent NES seen in many cellular & viral proteins.