HIV Replication: Entry, Reverse Transcription, Integration, Expression, and Trafficking (Transcript-Based Notes)

Entry, uncoating, and genome features

HIV assembly starts with the viral envelope proteins engaging host receptors CD4 and CXCR4 (co-receptor). Binding induces a conformational change in the SU (surface) component of the envelope glycoprotein, enabling TM (transmembrane) to insert its fusion peptide into the host plasma membrane.
- Fusion occurs at neutral pH, delivering the viral capsid into the cytosol.
- Cyclophilin A (host chaperone) associates with the capsid and is essential for productive infection. Without cyclophilin A, the capsid cannot uncoat properly, and infection fails.
Two host components are highlighted (in blue in the visuals): cyclophilin A and a priming tRNA. These are host-derived, not viral, and are essential for initiating reverse transcription.
Priming tRNA for reverse transcription: initiation requires a primer from one of three host tRNAs (tRNA type involved in lysine incorporation). Any of these three can prime the reverse transcription process; the primer binds at the primer binding site (PBS) on the viral RNA.
Five-domain SU model: the SU component has five domains (domains 1–5) connected to receptor binding (CD4 and CXCR4) to initiate infection of CD4+ T cells. Interaction with both receptors triggers conformational changes enabling fusion.
After fusion, the capsid disassembles in the cytosol, exposing the retroviral genome for reverse transcription. The presence of cyclophilin A and primer tRNA is crucial for the infectious process.
In artificial lab conditions, HIV can be assembled without cyclophilin A, but in such conditions the capsid does not dissociate properly and infection cannot proceed.

Viral genome organization and initial reverse transcription

The genome has long terminal repeats (LTRs) at both ends; each end contains a U5 (5' untranslated region) at the 5' end and a U3 (3' untranslated region) at the 3' end.
The genome also contains a polypurine tract (PPT) important for reverse transcription; in the slide it’s referred to as a polycurine tract.
Primer binding site (PBS) is where the host tRNA primes reverse transcription.
The 5' end host tRNA-derived primer provides the hydroxyl group required for initiating reverse transcription. Reverse transcriptase (RT) then uses its RNA-dependent DNA polymerase (RDDP) activity to synthesize DNA from the RNA template.
RT has four activities, all utilized during reverse transcription in different steps:
- RNA-dependent DNA polymerase activity (synthesizes DNA from RNA template).
- DNA-dependent DNA polymerase activity (extends DNA using DNA as template).
- Helicase activity (facilitates strand separation during the process).
- Nuclease activity (degrades the RNA template as DNA is synthesized).
- The four activities are inserted at different stages of the reverse transcription process.
After priming and initiation, RT performs RNA-dependent DNAP synthesis to create the first DNA strand; the RNA template is degraded by RNase activity as needed, and the DNA strand becomes partially double-stranded.
Strand exchange occurs: a strand transfer event relocates the newly synthesized DNA to the end of the genome, enabling continued synthesis with the primer binding site and the R sequence (repeated region) acting as anchors.
The repeated region (R) and the U3/U5 regions are involved in later steps, including circularization and subsequent steps toward integration. The primer binding site (PBS) remains complementary to the tRNA primer during certain stages of reverse transcription.
The polypurine tract remains associated with cDNA to prevent complete degradation and to serve as primers for subsequent steps in reverse transcription.
At some point, reverse transcription may yield a linear dsDNA form; if helicase activity is present, a long linear dsDNA with repeated U3/R/U5 segments can be produced. If helicase activity is absent, a circular form may result (a dead end in this depiction).
Generally, reverse transcription concludes with generation of an almost complete double-stranded viral DNA that contains LTRs suitable for nuclear import and integration. The integrase enzyme remains associated with the viral genome throughout the process.

Integration into the host genome

The viral DNA is transported toward the nucleus where integration occurs.
Integrase is depicted as a tetramer (four integrase molecules) bound to the ends of the viral genome. Its cofactor is a divalent metal ion (Mg2+ or Mn2+); no ATP is required for the integration reaction.
Before integration, integrase trims two nucleotides at the ends to expose the TG and CA sequences for integration (the classic HIV ends are typically described as TG at one end and CA at the other). The 5' and 3' ends are referred to as the ends to be integrated.
Integration results in a staggered cut of the host DNA, with the viral DNA integrated into the chromosome. The initial gaps are repaired by cellular machinery, producing a stable provirus.
In vitro, integration can appear random, but in vivo there are host factors that bias integration toward specific sequences or features (e.g., promoters in proximity to transcription factors like INI1). In HIV infection, integration is not completely random and often targets promoters via transcription factors that bind to enhancer elements in the long terminal repeats (LTRs).
Long terminal repeats (LTRs) contain several upstream activating sequences and transcription factor binding sites. Binding of factors such as GATA-3, NF-κB, ETS, USF, and other host transcription factors can influence transcriptional activation of the provirus and contribute to latency and reactivation.
If the provirus integrates near a host oncogene promoter within roughly 10 kilobases, transcription of that gene can be upregulated, contributing to oncogenesis in some scenarios.
Emergence from latency is driven by LTR enhancers; latency can persist for months to years, but viral transcription can spontaneously or upon activation proceed to produce viral RNAs and proteins.

From transcription to export: Tat, Rev, TAR, RRE, and export pathways

After integration, the provirus is driven by viral promoters; initial transcription is limited and most transcripts are short, with occasional production of full-length transcripts.
Transcription from the LTR yields a mix of RNAs: the majority are short, but a small proportion (~10%) of transcription produces full-length viral RNA. This small fraction is critical for producing all viral genes.
The full-length transcript includes a TAR element (Trans-Activation Response). Tat (Transactivator of Transcription) is produced from a short 5' transcript and is essential for robust full-length transcription.
TAR is an RNA stem-loop structure with extensive secondary structure and serves as the binding site for Tat. Tat recruits cellular transcription factors (notably Cyclin T and CDK9) and other components like TFIIH to hyperactivate transcription by phosphorylating the RNA polymerase II C-terminal domain (CTD).
Tat-TAR interactions recruit Cyclin T and CDK9, and phosphorylate RNA Pol II to enhance processivity and full-length transcription of the provirus.
Rev (Regulator of Expression of Virion Proteins) is produced from a short 10% transcript and binds the Rev Response Element (RRE) present in unspliced and partially spliced RNAs. Rev regulates export of these RNAs from the nucleus to the cytoplasm.
The export of unspliced (9 kb) and singly spliced (4 kb) RNAs requires the host export machinery and viral Rev: Rev interacts with RRE-containing RNAs and facilitates their export via the CRM1/exportin 1 pathway, in coordination with Ran-GTP. The transcript mentions export components: exportin, Ran, Ref proteins, TAP, and related factors, illustrating the export of these transcripts from the nucleus.
The fully spliced RNAs (2 kb) are exported via the standard cellular mRNA export pathway, aided by factors such as TAP (nuclear export factor) and Ran; the transcript names several specific factors (e.g., EIF5A, TFIIH) involved in downstream processes.
The Rev-responsive element (RRE) and TAR elements are essential for coordinating the expression of different viral proteins through regulated RNA processing, export, and translation.

Transcripts, export, and translation: roles of Rev, Tat, and export machinery

9 kb unspliced RNA serves as the primary mRNA for Gag and Gag-Pol precursor expression; 4 kb singly spliced RNAs encode Env (gp160 precursor) and other proteins; 2 kb fully spliced RNAs encode regulatory and accessory proteins (e.g., Tat, Rev, Nef).
An important balance is the ratio of unspliced to singly spliced RNAs. The optimal ratio is 2:1 (unspliced : singly spliced) for productive viral progeny production. If this ratio shifts, viral production drops or ceases.
Export of unspliced and singly spliced RNAs relies on Rev, RRE, and the cellular export machinery (exportin 1/CRM1, Ran-GTP). Export factors such as EIF5A, TAP, REF, and others are involved in the export and translation steps.
Translation of the 4 kb Env precursor (gp160) and 9 kb Gag/Pol precursor occurs in the cytosol after export; the Env precursor is processed through the secretory pathway, while Gag/Pol processing occurs later during assembly.
Rev and Tat function form a regulatory loop: Tat amplifies transcription; Rev ensures that unspliced and singly spliced RNAs are exported for production of structural proteins and enzymes.

Env trafficking, folding, and maturation (gp160 to gp120/gp41)

Env is translated as gp160 and enters the secretory pathway via the SRP (signal recognition particle) targeting to the ER, where the ribosome-nascent chain complex is routed to the ER membrane and the translocon.
In the ER, folding and processing occur with the help of chaperones and folding enzymes:
- Glucosidases I and II trim glucose residues on the nascent glycoprotein, allowing calnexin/calreticulin chaperone binding (via a glycoprotein folding cycle).
- ERp57 and protein disulfide isomerase (PDI) catalyze disulfide bond formation and proper folding.
- If misfolding occurs, reglucosylation by UGGT reinitiates the cycle; trimming and reglucosylation cycles continue until proper folding is achieved.
- If folding fails repeatedly, the protein may be exported to the cytosol and degraded by the proteasome; otherwise, properly folded Env proceeds to trafficking.
Properly folded gp160 is trafficked to the Golgi where furin protease cleaves it into gp120 (SU) and gp41 (TM), generating the mature envelope glycoproteins.
The Env complex then traffics through the Golgi to the plasma membrane, aided by secretory vesicles and the secretory pathway, including COP II vesicles and SNARE/fusion machinery.
Glycosylation and processing along the secretory pathway:
- Initial N-linked glycosylation occurs on nascent gp160 with high-mannose glycans as it traffics through the ER.
- Golgi processing trims mannose residues and adds N-acetylglucosamine, galactose, and sialic acid in successive Golgi compartments (cis, medial, trans).
- The protein traverses the Golgi, acquiring a mature glycan profile suitable for surface expression.
The mature gp120/gp41 complex is trafficked to the plasma membrane, often via lipid rafts (cholesterol- and sphingolipid-rich microdomains) that help target the complex to the correct membrane regions.
Lipid raft association and secretory vesicle trafficking ensure gp120/gp41 are presented on the cell surface as a functional holoenzyme complex capable of engaging CD4 and coreceptors on new target cells.
A furin protease step in the medial Golgi finalizes maturation by cleaving gp160 into gp120 and gp41; gp120/gp41 remain associated as a functional complex anchored in the viral membrane.
The mature envelope dictates receptor specificity and fusion potential for new target cells, enabling entry for subsequent infection.

Vpu (BPU) and immune evasion during virion assembly and release

A viral protein, referred to as BPU in the transcript (commonly known as Vpu), is involved in two key immune-evasion roles:
- Enhances export of CD4 from the endoplasmic reticulum (ER) and promotes degradation of surface CD4, reducing CD4 accumulation and blocking superinfection and interference with envelope expression.
- Contributes to downregulation of MHC class I molecules (via targeting components of the antigen presentation pathway), reducing recognition by cytotoxic T cells.
By preventing CD4 accumulation and downregulating MHC I, HIV reduces immune detection and enhances survival of infected cells.
HIV Vpu also participates in enabling efficient release of virions by antagonizing tetherin (not elaborated in detail in this transcript, but generally part of Vpu’s function in immune evasion).

Gag/Pol processing, genome packaging, and virion assembly

The nine kilobase (kb) transcript encodes the Gag and Pol polyproteins in a single precursor, which are transported to cellular membranes where assembly occurs.
The Gag-Pol precursor includes protease (PR), reverse transcriptase (RT), and integrase (IN) domains, along with structural Gag components (MA, CA, NC).
Gag and Gag-Pol assemble on cellular membranes, and the viral protease cleaves the polyproteins into mature products:
- Gag is processed into MA (matrix), CA (capsid), and NC (nucleocapsid).
- Pol is processed to yield RT, IN, and PR (protease).
During assembly, the NC, MA, and CA proteins organize into the mature virion structure, and the RNA genome is packaged into the assembling virion.
The envelope glycoproteins (gp120/gp41) are incorporated into the budding virion through interactions with Gag and the assembling viral particle, coordinating assembly with envelope acquisition.

Viral export, maturation, and budding (secretory pathway to plasma membrane)

The translation products from 4 kb and 9 kb RNAs feed into the secretory and translation pathways to produce Env and other structural proteins that will be incorporated into new virions.
The Env glycoproteins (gp120/gp41) are trafficked to the plasma membrane, where budding occurs.
The assembly and budding process leverages the secretory vesicle trafficking system:
- Secretory vesicles bud from the Golgi and are guided to the plasma membrane via Rab GTPases and SNARE proteins (V-SNAREs and T-SNAREs in the target membrane).
- Vesicle docking/fusion involves SNARE complexes, NSF, and SNAPs to mediate fusion with the plasma membrane and release of virions.
The glycoproteins and other envelope components are retained on the viral surface as the virion buds from the host cell, acquiring a complete envelope and functional attachment glycoproteins for subsequent infection of new cells.

Post-entry considerations and pathogenesis implications

The HIV genome includes regulatory features that influence latency, reactivation, and pathogenesis:
- LTRs contain enhancers and promoter elements that control transcription of the provirus, contributing to latency and reactivation potential.
- Transcriptional activators (Tat) and regulatory elements (TAR, RRE) coordinate transcription, RNA processing, and export.
- The HIV genome can remain latent for varying periods; reactivation can occur upon cellular activation and transcription factor engagement.
Integration site biases and oncogenic potential: integration near host gene promoters can alter transcriptional activity of host genes, potentially contributing to oncogenesis in some contexts. The presence of LTR enhancer elements further modulates host gene transcription near the integration site.
The balance of viral RNA species (unspliced 9 kb, singly spliced 4 kb, and fully spliced 2 kb) regulates the production of structural versus regulatory proteins, with the Tat/TAR and Rev/RRE axis controlling expression and export.
The downstream consequences include production of viral structural proteins (Gag, Pol, Env) and accessory proteins, virion assembly, release, and spread, with immune evasion strategies (e.g., Vpu’s effects on CD4 downregulation and MHC I degradation) contributing to persistence.

Key terms and concepts for quick review

Cyclophilin A: host chaperone essential for HIV infection during uncoating.
Priming tRNA: host tRNA used as a primer for reverse transcription at the PBS; any of three tRNAs capable of priming.
Primer Binding Site (PBS): region where host tRNA binds to initiate reverse transcription.
PPT (polypurine tract): RNA sequence necessary for reverse transcription priming; often called a polypurine tract in this transcript.
LTR (Long Terminal Repeat): regulatory regions at both ends of the HIV genome; contain U5 and U3 regions and repeat (R) sequences.
U5 and U3: untranslated regions at 5' and 3' ends of the viral genome.
R (repeat region): sequences within LTRs involved in reverse transcription and integration.
TAR (Trans-Activation Response element): RNA element within the transcript that binds Tat and recruits transcriptional machinery.
RRE (Rev Response Element): RNA element that binds Rev to facilitate export of unspliced and singly spliced RNAs.
Rev: regulatory protein that exports unspliced and singly spliced RNAs via the CRM1/exportin 1 pathway (Ran-GTP dependent).
Tat: transcriptional activator that enhances processivity of RNA polymerase II on the provirus via TAR recruitment and recruitment of Cyclin T and CDK9.
Gag: structural polyprotein; processed into MA (matrix), CA (capsid), NC (nucleocapsid).
Pol: polyprotein containing protease (PR), reverse transcriptase (RT), and integrase (IN).
Env: envelope precursor gp160 processed into gp120 (SU) and gp41 (TM).
gp120: surface glycoprotein that binds CD4 and coreceptors.
gp41: transmembrane glycoprotein that mediates membrane fusion.
Vpu (BPU in this transcript): viral protein that promotes CD4 degradation/export, and aids MHC I downregulation to evade immune detection.
Integrase: enzyme that catalyzes integration of viral DNA into host genome; described as a tetramer; requires a divalent metal cofactor (Mg2+ or Mn2+).
Secretory pathway and trafficking: SRP, translocon, ER chaperones (calnexin, calreticulin, ERp57), ER glucosidases I/II, UGGT; COP II vesicles; SNAREs (V-SNAREs and T-SNAREs); Rab GTPases; NSF and SNAPs; lipid rafts.
Golgi processing: trimming of mannose residues, addition of N-acetylglucosamine, galactose, and sialic acid; furin cleavage to yield gp120/gp41; final assembly at the plasma membrane.
Embedding practical implications: understanding these steps highlights targets for antiretroviral therapy (e.g., RT inhibitors, protease inhibitors, integrase inhibitors) and helps explain latency and immune evasion mechanisms in HIV.

Connections to broader principles and real-world relevance

The HIV life cycle exemplifies a complex, multi-step replication strategy that leverages host cell machinery at nearly every stage (entry, reverse transcription, nuclear import, transcriptional regulation, RNA export, protein processing, trafficking, assembly, and egress).
The balance between different RNA species (unspliced vs. spliced) and the regulatory roles of Tat, Rev, TAR, and RRE illustrate how viruses fine-tune gene expression to optimize replication while evading host defenses.
Integration into the host genome creates a reservoir of latent provirus with the potential for reactivation, a central challenge in HIV treatment and eradication strategies.
Viral proteins such as Vpu (BPU) and the interplay with MHC I trafficking demonstrate sophisticated immune evasion that complicates host immune responses and informs vaccine and therapeutic design.
The detailed recycling of host quality-control and secretory pathway components (ER chaperones, calnexin/calreticulin cycle, Golgi processing, SNAREs, Rab GTPases) highlights how viruses co-opt normal cellular processes for infectious particle production.

Ethical and practical considerations for research and therapy

Understanding these mechanisms informs antiviral drug development targeting RT, integrase, and protease; it also underscores the importance of combination therapies to prevent resistance.
Latency and viral reservoir dynamics underscore the ethical and clinical need for strategies addressing long-term persistence of HIV, including cure research and long-term treatment considerations.
In research settings, handling such material requires rigorous biosafety practices to prevent exposure and environmental release; the knowledge also emphasizes the importance of responsible communication given the potential misuse of detailed replication information.

Summary takeaways

HIV entry, uncoating, and reverse transcription rely on host factors (cyclophilin A and host tRNA primers) and the viral genome features (PBS, PPT, LTRs, R, TAR, RRE).
RT performs four activities during reverse transcription; end-processing and strand transfers culminate in a dsDNA provirus.
Integrase integrates the proviral DNA into the host genome, with a staggered cut mechanism and potential biases toward transcription factor-rich sites.
Transcription from the integrated provirus is controlled by Tat, TAR, Rev, RRE, and host transcription/export machinery, producing a regulated set of RNAs (9 kb unspliced, 4 kb singly spliced, 2 kb fully spliced).
Env biosynthesis, processing (gp160 -> gp120/gp41), glycosylation, and trafficking through the secretory pathway generate functional envelope proteins that are presented on the cell surface.
Vpu aids in immune evasion and viral egress, including CD4 downregulation and MHC I interference.
Gag/Pol processing yields the structural and enzymatic components necessary for virion assembly and maturation; final budding and release generate infectious virions capable of initiating new rounds of infection.
The described steps collectively illuminate why HIV persists and challenges therapeutic intervention, and they connect to foundational cell biology principles (secretory pathway, SNARE-mediated fusion, transcriptional regulation, and RNA processing).