HIV-1 Infection Cell Models for Research

HIV-1 remains a major global health issue. According to the World Health Organization (WHO), approximately 39 million people were living with HIV at the end of 2022, with 1.3 million new infections and 630,000 AIDS-related deaths in that year. The disease burden is highest in sub-Saharan Africa, but it affects all regions. Antiretroviral therapy (ART) has transformed HIV from a fatal disease to a manageable chronic condition, but there is no cure and lifelong treatment is required. Key risk factors include unprotected sex, injection drug use, and mother-to-child transmission. Without treatment, HIV progresses to AIDS, with a median survival of about 3 years after AIDS onset. However, with effective ART, life expectancy can approach that of the general population. The National Cancer Institute (NCI) notes that HIV-infected individuals have an increased risk of certain cancers, such as Kaposi sarcoma, non-Hodgkin lymphoma, and cervical cancer, due to immunosuppression.

HIV-1 is an ideal model for studying viral pathogenesis, host-virus interactions, and immune evasion. Its complex life cycle involves multiple host factors, making it a rich area for mechanistic studies. Public datasets, such as those from the HIV Sequence Database and the Los Alamos HIV Databases, provide extensive genetic and clinical data. Open questions include the mechanisms of viral latency, the role of host restriction factors, and the development of a cure or vaccine. Gene-edited cell models are crucial for dissecting these mechanisms and for drug discovery.

HIV-1 infection involves several key steps:

1. Entry: The viral envelope glycoprotein gp120 binds to the CD4 receptor and a co-receptor (CCR5 or CXCR4) on the host cell, triggering fusion and entry.

2. Reverse Transcription: The viral RNA is reverse-transcribed into DNA by the viral enzyme reverse transcriptase.

3. Integration: The viral DNA integrates into the host genome by the viral integrase enzyme.

4. Transcription and Translation: The integrated provirus is transcribed and translated to produce viral proteins.

5. Assembly and Budding: New viral particles assemble at the cell membrane and bud off, releasing mature virions.

Each step involves host factors that can be targeted by gene editing to study their function or to create resistance models.

While HIV-1 does not cause mutations in host genes, it exploits host genetic variants that affect susceptibility and progression. Key host genes include:

Data from population studies and the NCBI dbSNP database.

HIV-1 modulates multiple host signaling pathways to facilitate replication and evade immune responses. Key networks include:

• • NF-κB pathway: Activated by viral proteins (e.g., Tat) to promote viral transcription.
• • PI3K/AKT pathway: Enhanced by HIV-1 to promote cell survival and viral replication.
• • JAK/STAT pathway: Modulated by viral proteins to interfere with interferon signaling.
• • Wnt/β-catenin pathway: Dysregulated in HIV-associated cancers.

These pathways are potential targets for therapeutic intervention and can be studied using gene-edited cell models.

Commonly used cell lines for HIV-1 research include:

Organoids, such as tonsil or gut organoids, are emerging as more physiologically relevant models to study HIV-1 transmission and latency.

Animal models are essential for studying HIV-1 pathogenesis and testing therapies. Key models include:

• • Humanized mice: Immunodeficient mice engrafted with human immune cells (e.g., NSG mice) support HIV-1 infection.
• • Simian immunodeficiency virus (SIV) models: Used in non-human primates to study AIDS pathogenesis.
• • Feline immunodeficiency virus (FIV): Used in cats as a model for HIV.

These models are limited by cost, availability, and differences from human infection.

CRISPR-based gene editing has revolutionized HIV-1 research by enabling the creation of isogenic cell lines with specific gene knockouts or knock-ins. For example:

• • CD4 knockout cell lines: Used to study the role of CD4 in viral entry and to generate resistant cells.
• • CCR5 knockout cell lines: Mimic the CCR5-Δ32 mutation, providing resistance to CCR5-tropic HIV-1.
• • CXCR4 knockout cell lines: Used to study CXCR4-tropic HIV-1 entry.
• • Reporter cell lines: Engineered to express fluorescent or luminescent proteins upon HIV-1 infection, enabling real-time monitoring.

These gene-edited models are commercially available from various sources and are sequence-verified to ensure accuracy. They are essential for functional genomics, drug screening, and understanding viral-host interactions.

Gene-edited cell lines are used to validate host factors involved in HIV-1 replication. For example, knocking out the host gene SAMHD1 in macrophages increases HIV-1 replication, confirming its role as a restriction factor. Similarly, knocking out LEDGF/p75 reduces viral integration, demonstrating its importance. These models allow researchers to dissect the function of specific genes in the viral life cycle.

Isogenic cell lines with specific mutations (e.g., CCR5 knockout) are used to screen antiviral drugs. For example, CCR5 knockout cells are resistant to CCR5-tropic HIV-1, making them useful for testing entry inhibitors. Additionally, gene-edited cells can be used to generate drug-resistant viral variants by passaging in the presence of antiviral drugs, enabling the study of resistance mechanisms.

CRISPR screens using gene-edited cells can identify host factors that are essential for HIV-1 replication, which can serve as potential drug targets. For example, a genome-wide CRISPR screen in T cells identified novel host factors required for HIV-1 infection, such as TPST2 and SLC35B2. These findings provide new avenues for therapeutic intervention.