Molecular Engineering of Virus Tropism

Abstract

Engineered viral vectors designed to deliver genetic material to specific targets offer significant potential for disease treatment, safer vaccine development, and the creation of novel biochemical research tools. Viral tropism, the specificity of a virus for infecting a particular host, is often modified in recombinant viruses to achieve precise delivery, minimize off-target effects, enhance transduction efficiency, and improve safety. Key factors influencing tropism include surface protein interactions between the virus and host-cell, the availability of host-cell machinery for viral replication, and the host immune response. This review explores current strategies for modifying the tropism of recombinant viruses by altering their surface proteins. We provide an overview of recent advancements in targeting non-enveloped viruses (adenovirus and adeno-associated virus) and enveloped viruses (retro/lentivirus, Rabies, Vesicular Stomatitis Virus, and Herpesvirus) to specific cell types. Additionally, we discuss approaches, such as rational design, directed evolution, and in silico and machine learning-based methods, for generating novel AAV variants with the desired tropism and the use of chimeric envelope proteins for pseudotyping enveloped viruses. Finally, we highlight the applications of these advancements and discuss the challenges and future directions in engineering viral tropism.

Keywords: AAV; AAV serotype; AAV variant; HSV; capsid; g-deleted rabies virus; lentivirus; pseudotyping virus; retrovirus; viral application; viral vectors; virus envelope; virus envelope chimera.

Figure 1

Cultured mouse brain slices, prepared as described previously [11], were transduced with baculovirus, delivering a green fluorescence protein (GFP) pseudotyped with Vesicular Stomatitis Virus (VSV) glycoprotein (BacMam technology, Thermo Fisher Scientific). (a) After a period of 24 h following transduction with the engineered baculovirus, a GFP is expressed in the vicinity of the infection area (20× objective, zoom of 1, single tile). (b) Images of the mouse brain slice, depicting the scale of gene expression with fluorescence only, visible light, and the overlap of both fluorescence and visible light (20× objective, zoom of 1, assembled tile scans). (c) Robust GFP expression in brain slices are evident 48 h post-infection—images depicting fluorescence only, visible light, and the overlap of both fluorescence and visible light. Black boxes in (c) are regions where no data was acquired by tile scan (20× objective, zoom of 1, assembled tile scans).

Figure 2

Recombinant lentiviruses pseudotyped with SARS-CoV-2 Spike delivering a Red Fluorescent Protein (Lenti-RFP-Spike). (a) Cryo-EM image of the purified Lenti-RFP-Spike imaged on Talos Arctica 200kV equipped with Gatan K2 Summit detector at a magnification of 36,000×. (b) HEK293T stably expressing human Angiotensin-Converting Enzyme 2 (HEK293T-hACE2) infected with a Lenti-RFP-Spike, fixed with 1% formaldehyde and stained with anti-spike antibodies conjugated to GFP (20× objective, zoom of 2, 400× magnification). (c) Lenti-RFP-Spike binds to the hACE2 receptor on the surface of the HEK293T-hACE2 cells to enter and express RFP in infected cells (20× objective, 40× magnification).