HIV-1 Gene-Edited Cell Models for Drug Discovery

According to the World Health Organization (WHO), approximately 39.0 million people were living with HIV globally at the end of 2022, with 1.3 million new infections and 630,000 AIDS-related deaths that year. Sub-Saharan Africa accounts for the majority of cases. Antiretroviral therapy (ART) has transformed HIV-1 infection from a fatal disease to a manageable chronic condition, but it does not eradicate the virus. Latent reservoirs remain a major barrier to a cure. Key risk factors include unprotected sex, injection drug use, and mother-to-child transmission. Without treatment, HIV-1 infection progresses to AIDS, with a median survival of approximately 11 years from seroconversion (NCI).

• • HIV-1 is an ideal model for studying viral-host interactions, immune evasion, and latency. Key research areas include:
• • Mechanisms of viral entry via CD4 and co-receptors (CCR5, CXCR4).
• • Reverse transcription and integration into the host genome.
• • Establishment and maintenance of latent reservoirs.
• • Immune escape and viral evolution.
• • Development of curative strategies (shock and kill, block and lock).
• • Public datasets from the Los Alamos HIV Sequence Database, NCBI GenBank, and clinical trials provide extensive genomic and clinical data. Open questions include the molecular basis of latency reversal and the role of host restriction factors.

• • HIV-1 replication involves several key steps:

1. Attachment and entry: gp120 binds CD4 and a co-receptor (CCR5 or CXCR4).

2. Fusion and uncoating: viral envelope fuses with host membrane; capsid releases viral RNA.

3. Reverse transcription: viral reverse transcriptase converts RNA into double-stranded DNA.

4. Integration: integrase inserts viral DNA into the host genome.

5. Transcription and translation: host machinery produces viral proteins.

6. Assembly and budding: new virions assemble and exit the cell.

7. Maturation: protease cleaves polyproteins to form infectious particles.

HIV-1 does not cause somatic mutations in host cells, but host genetic variants influence susceptibility and disease progression. Key host factors include:

Data from NCBI dbSNP, ClinVar, and genome-wide association studies (GWAS).

• • HIV-1 hijacks multiple host signaling pathways to facilitate replication and evade immune responses:
• • NF-kB pathway: viral proteins (Tat, Nef) activate NF-kB, promoting viral transcription and inflammation.
• • PI3K/AKT pathway: Nef activates PI3K/AKT to enhance cell survival and viral production.
• • MAPK/ERK pathway: Tat and gp120 activate MAPK, modulating gene expression and apoptosis.
• • Interferon signaling: HIV-1 counteracts type I interferon responses via Vif and Vpu.
• • CD4 and co-receptor signaling: gp120 binding triggers downstream cascades affecting T-cell activation.

Commonly used cell lines for HIV-1 research:

Organoid models: tonsil, thymus, and gut organoids support HIV-1 infection and recapitulate tissue-specific immune responses. They are valuable for studying mucosal transmission and latency.

• • HIV-1 does not infect rodents, so humanized mouse models are essential:
• • Humanized NSG mice (NOD-scid-IL2Rgamma-null) engrafted with human CD34+ hematopoietic stem cells: support HIV-1 infection, latency, and immune responses.
• • BLT (bone marrow-liver-thymus) mice: reconstitute a human immune system; used for transmission and latency studies.
• • Non-human primates: SIV and SHIV models in macaques are used for pathogenesis and vaccine studies.
• • Transgenic rats: express human CD4 and CCR5; support limited HIV-1 replication.

• • CRISPR/Cas9 gene editing enables precise modification of host and viral genes in cell lines. Examples include:
• • CD4 knockout cell lines: used to study alternative entry pathways.
• • CCR5 knockout cell lines: model natural resistance; used for testing CCR5 inhibitors.
• • CXCR4 knockout cell lines: study X4-tropic HIV-1 entry.
• • APOBEC3G knockout cell lines: investigate host restriction factors.
• • HIV-1 LTR reporter cell lines: contain fluorescent or luciferase reporters under the HIV-1 promoter for monitoring latency and reactivation.
• • Commercially available, sequence-verified isogenic cell lines accelerate research by providing consistent, defined genetic backgrounds. These models are essential for functional validation of host factors and drug targets.

• • CRISPR knockout and knock-in lines are used to validate host factors essential for HIV-1 replication. For example:
• • Knockout of CCR5 in CD4+ T cells confirms its role as a co-receptor for R5-tropic HIV-1.
• • Knockout of LEDGF/p75 (PSIP1) impairs HIV-1 integration, validating it as a drug target.
• • Knock-in of HIV-1 restriction factors (e.g., TRIM5alpha) in permissive cells reveals mechanisms of species-specific restriction.

• • Isogenic cell pairs (wild-type vs. knockout) are used to screen for antiviral compounds and study resistance:
• • CCR5 knockout cells are resistant to R5-tropic HIV-1; used to test CCR5 antagonists (e.g., maraviroc).
• • Cells with mutations in reverse transcriptase or protease are used to assess drug resistance profiles.
• • Latency models (e.g., J-Lat cells) with integrated HIV-1 LTR reporters are used to screen latency-reversing agents.

• • CRISPR screens identify host factors that modulate HIV-1 infection and latency:
• • Genome-wide knockout screens in CD4+ T cells have identified novel restriction factors (e.g., SERINC5, MxB).
• • Synthetic lethality screens in HIV-1-infected cells identify host pathways required for viral replication, revealing potential drug targets.
• • CRISPR activation (CRISPRa) screens identify factors that reactivate latent HIV-1, informing shock-and-kill strategies.