Part:BBa_K5142031
Vaccinia virus A27L
This part includes the coding sequence (cds) of A27L, a membrane protein of vaccinia virus (VACV). VACV is a member of the genus Orthopoxvirus in the family Poxviridae, which is one of the most intensively studied poxviruses. Because of its low virulence and pathogenicity, VACV has been extensively engineered for various applications, such as vaccines, gene vectors, and drug carriers. VACV has two morphologically distinct infectious virions: intracellular mature virus (IMV), which resides in the cytoplasm, and extracellular enveloped virus (EEV), which is released to the extracellular environment for viral dissemination (1). The A27L protein consists of 110 amino acids and contains several functional domains, including an N-terminal heparin-binding sequence (HBS), a disulfide-linked domain (DLD), a C-terminal leucine zipper domain (LZD), flexible linker regions, and an α-helical coiled-coil domain (CCD). The N-terminal HBS promotes virus attachment to the cell membrane through specific binding to heparin molecules on the cell surface. The C-terminal LZD anchors the protein to the viral membrane through interactions with the A17L protein. A27L is important for the formation of EEV but not IMV. Deficiency of A27L leads to a dramatic reduction of EEV and thus impairs viral dissemination (2). Considering this function of A27L, scientists usually generate a non-efficiently spreading viral mutant by deleting the A27L gene before further genetic engineering, in order to insert exogenous DNA into the viral genome without using other genetic markers and reduce biosafety risk (3). When the viral engineering is complete, A27L deficiency can retained for safety or viral ability to spread can be restored by reintroducing the A27L gene if necessary. In this project, we incorporated click-reaction sites to A27L directly, so that the workload of viral engineering could be reduced. Briefly, we employed the techniques of DNA recombination to convert the codons of three phenylalanines at the N-terminus of A27L into amber suppressor codons (UAG), creating the mutant A27L-3stop. Then we introduced a specific artificial translation system including an amber suppressor tRNA and benzoylphenylalanine-specific tyrosyl-tRNA synthetase as well as 4-azido-L-phenylalanine, to generate the modified VACV with A27L containing 4-azido-L-phenylalanines which could be served as click-reaction sites.
Reference 1.Vanderplasschen A, Hollinshead M, Smith GL. Intracellular and extracellular vaccinia virions enter cells by different mechanisms. J Gen Virol. 1998;79 ( Pt 4):877-87. 2.Vazquez MI, Esteban M. Identification of functional domains in the 14-kilodalton envelope protein (A27L) of vaccinia virus. J Virol. 1999;73(11):9098-109. 3.Lorenzo MM, Sanchez-Puig JM, Blasco R. Genes A27L and F13L as Genetic Markers for the Isolation of Recombinant Vaccinia Virus. Sci Rep. 2019;9(1):15684.
The cds of A27L was cloned by PCR amplification from the genome of vaccinia virus (GenBank ID: JX489136.1).
The A27L protein is a crucial viral protein that plays essential roles in virus attachment, membrane fusion, intracellular transport of the virus within the host cell, and the formation of enveloped virion particles. The protein encoded by the A27L gene is necessary for the cell-to-cell transmission of viral particles. Deleting the A27L gene significantly impairs the virus's ability to spread between cells, resulting in substantially reduced plaque size.
Therefore, we chose the A27L protein as the target for our experiments. Without an artificial translation system, the modified virus cannot synthesize complete A27L, leading to a drastic reduction in EEV formation, which greatly decreases the infectiousness and pathogenicity of the modified virus. Viruses unable to synthesize complete A27L mostly remain at the IMV stage, unable to spread over long distances, thereby ensuring the safety of the experiment and its applications. Only when an artificial translation system and unnatural amino acids are added can the modified virus synthesize complete A27L.
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Illegal SpeI site found at 355
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Illegal NotI site found at 7
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Illegal XbaI site found at 16
Illegal SpeI site found at 355
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