The G285S mutation in nsP1 is sufficient to render Sindbis virus as a stable vector for gene delivery

Xiangwei Shi; Kangyixin Sun; You Hu; Qinghan Wang; Guoyang Liao; Li Li; Pengjie Wen; Leo E Wong; Fan Jia; Fuqiang Xu

doi:10.3389/fmicb.2023.1229506

The G285S mutation in nsP1 is sufficient to render Sindbis virus as a stable vector for gene delivery

Front Microbiol. 2023 Jul 20:14:1229506. doi: 10.3389/fmicb.2023.1229506. eCollection 2023.

Authors

Xiangwei Shi^{1

2

3

4}, Kangyixin Sun^{2

3

5}, You Hu^{2

3}, Qinghan Wang^{2

3}, Guoyang Liao⁶, Li Li^{2

3}, Pengjie Wen^{2

3}, Leo E Wong², Fan Jia^{2

3

4}, Fuqiang Xu^{1

2

3

4

5

7}

Affiliations

¹ State Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics, Key Laboratory of Magnetic Resonance in Biological Systems, Wuhan Center for Magnetic Resonance, Innovation Academy for Precision Measurement Science and Technology, Chinese Academy of Sciences, Wuhan, China.
² Shenzhen-Hong Kong Institute of Brain Science, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen, China.
³ NMPA Key Laboratory for Research and Evaluation of Viral Vector Technology in Cell and Gene Therapy Medicinal Products, Shenzhen Key Laboratory of Viral Vectors for Biomedicine, Key Laboratory of Quality Control Technology for Virus-Based Therapeutics, Guangdong Provincial Medical Products Administration, The Brain Cognition and Brain Disease Institute, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen, China.
⁴ University of Chinese Academy of Sciences, Beijing, China.
⁵ Wuhan National Laboratory for Optoelectronics, Huazhong University of Science and Technology, Wuhan, China.
⁶ College of Life Sciences, Wuhan University, Wuhan, China.
⁷ Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences, Shanghai, China.

Abstract

Neuroscience, gene therapy, and vaccine have all benefited from the increased use of viral vectors. Sindbis virus (SINV) is a notable candidate among these vectors. However, viral vectors commonly suffer from a loss of expression of the transgene, especially RNA viral vectors. In this study, we used a directed evolution approach by continuous passage of selection to identify adaptive mutations that help SINV to stably express exogenous genes. As a result, we found two adaptive mutations that are located at aa 285 (G to S) of nsP1 and aa 422 (D to G) of nsP2, respectively. Further study showed that G285S was sufficient for SINV to stabilize the expression of the inserted gene, while D422G was not. Combined with AlphaFold2 and sequence alignment with the genus Alphavirus, we found that G285S is conserved. Based on this mutation, we constructed a new vector for the applications in neural circuits mapping. Our results indicated that the mutant SINV maintained its anterograde transsynaptic transmission property. In addition, when the transgene was replaced by another gene, granulocyte-macrophage colony-stimulating factor (GM-CSF), the vector still showed stable expression of the inserted gene. Hence, using SINV as an example, we have demonstrated an efficient approach to greatly augment the gene delivery capacity of viral vectors, which will be useful to neuroscience and oncolytic therapy.

Keywords: Sindbis virus; adaptive mutation; directed evolution; gene delivery; neural circuits; stable vector.