Extend
Exchange
Cell model
NS1 truncated
Target verification
Summary
Reference
NA-boost IAV aims to transform the influenza virus through synthetic biological methods so that the neuraminidase (NA) can be better displayed on the surface of the influenza virus. This, in turn, improves the targeting of tumor cells with high TfR1 expression. This page will show the results obtained by our team in the competition cycle.
Rescuing and characterization of extended neuraminidase virus
Rescuing and characterization of extended neuraminidase viruses. By inserting amino acid segments from other influenza viruses into the stalk region of neuraminidase, we have achieved the purpose of extending neuraminidase and making it more prominent on the surface of the influenza virus. Referring to the method described by Castrucci MR et al.[1], we constructed four mutant influenza viruses with neuraminidase of different stalk lengths. In SD24, amino acids 38-61 of neuraminidase were deleted. In SD9, amino acids 43-51 were deleted. In SI14, 14 amino acids (43-56) from A/Tokyo/67(H2N2) were inserted at position 42. Building upon SI14, SI28 contains an additional 14 amino acids (positions 58-71) from A/Tern/Australia/G70C/75(H11N9).
By point mutation or homologous recombination, based on the A6 plasmid in the 12-plasmid rescue system of the influenza virus, we successfully constructed four mutant plasmids: A6-SD24, A6-SD9, A6-SI14, and A6-SI28. We also successfully rescued the mutant virus in HEK293T cells.
Initially, we used a 6-well plate for virus rescue. After harvesting the virus, we infected 293T cells with the virus, extracted the viral RNA, and reverse transcribed it into cDNA. The cDNA was then amplified by PCR and sent to a sequencing company for testing. The sequencing results confirmed the introduction of mutations at the gene level (Figure 1.2). Additionally, we observed cytopathic effects (CPE) in HEK293T cells 1-2 days after viral infection. The results demonstrated that all four mutant viruses effectively induced pathological changes in the cells (Figures 1.3 and 1.4).
Subsequently, we used a 10 cm dish to generate a large quantity of viruses. After harvesting the virus, we concentrated it through ultracentrifugation. The concentrated virus was subjected to testing using the hemagglutination assay of chicken red blood cells and Western blotting. The former test serves to further confirm the successful rescue of the virus, while the latter verifies the successful introduction of the mutation at the protein level. In the hemagglutination assay of chicken red blood cells, the SD9 and SI14 mutant viruses exhibited significant inhibition of chicken red blood cell agglutination, whereas SD24 and SI28 did not display such a phenomenon (Figure 1.5). This observation suggests that the SD24 and SI28 virus titer was too low, which was subsequently confirmed when we later quantified the virus. In the Western blotting experiment, we observed distinct NA proteins of varying lengths, including SD9, SI14 and the wild type. This observation provides conclusive evidence of the successful introduction of the mutation at the protein level (Figure 1.6).
The following figure shows the reverse view of the above figure, which is SI28, SI14, SD9, SD24 and WT from top to bottom.
In the process of preparing a large quantity of viruses, we discovered that although the SI28 virus was successfully rescued, its viral titer was unexpectedly low, which contradicted the findings reported in the original literature. Therefore, upon careful examination, we identified an error in the insertion of the base sequence of 14 amino acids from A/Tern/Australia/G70C/75(H11N9). Instead of copying amino acids 58-71, we mistakenly inserted amino acids 52-65. Subsequently, we redesigned the packaged plasmid and successfully validated it using a 6-well plate. Unfortunately, we were unable to complete the bulk virus preparation before the submission deadline for the wiki. However, we will continue this part of the experiment before the jamboree begins.
Rescuing and characterization of mutant virus with exchange packaging signal
By exchanging the packaging signals of the neuraminidase and hemagglutinin RNA segments, we reversed the expression levels of neuraminidase and hemagglutinin, thereby achieving the objective of better displaying neuraminidase on the surface of the influenza virus. Following the method outlined by Allen Zheng et al.[2], we performed an exchange of the packaging signals between the HA protein gene segment and NA protein gene segment of the WSN virus. Additionally, we mutated the initiation codon ATG within the 5'-end packaging signal to TTG.
Through homologous recombination and point mutation, we successfully constructed the A6-HAps plasmid and A4-NAps plasmid. However, upon replacing the A6 plasmid and A4 plasmid within the 12-plasmid system with these two plasmids, we were unable to effectively rescue the mutant virus.
We suspected that we were unable to effectively rescue the virus due to the presence of residual packaging signals in the coding regions of the HA protein and NA protein. Therefore, we introduced synonymous mutations in this region to avoid the influence of the remaining packaging signal. Through homologous recombination, we successfully constructed the A6-HAps(mut) plasmid and A4-NAps(mut) plasmid. However, we still did not succeed in rescuing the PS mutant virus in the 6-well plate virus rescue assay.
We carefully examined the A6-HAps(mut) plasmid and A4-NAps(mut) plasmid and found that there were two initiation codons (ATG) remaining within the packaging signal at the 5' end of the A4-NAps(mut) plasmid, which might be the cause of this issue. Therefore, we made point mutations in the A4-NAps(mut) plasmid and successfully rectified the plasmid. However, due to a lack of time, we did not complete the virus rescue before the wiki submission deadline. We will continue this part of the experiment before the jamboree begins.
Construction and characterization of a cell model with high TfR1 expression
The original intention of packaging the NA boost influenza virus was to target tumor cells with high TfR1 expression. Therefore, using Chinese hamster ovary (CHO) cells as a basis, we constructed cell models with different levels of TfR1 expression through plasmid transfection in order to evaluate the infectivity of the mutant virus. CHO cells are not susceptible to influenza virus, making them suitable for comparing differences in virus infection ability.
We used a 12-well plate to construct the model, transfected 1ug of pCMV3-TfR1 plasmid into each well, and changed the solution after transfection for 6 hours. Two days later, we extracted total protein from the cells and performed a Western blotting experiment to determine the level of TfR1 expression. The results of the Western blotting experiment indicated successful construction of CHO cells with high expression of TfR1 (Figure 3.1).
Rescuing the NS1-truncated Mutant Virus
In the 'human practice' aspect of our project, experts in the field raised concerns about the safety of oncolytic viruses. They suggested that we should consider the safety issue while improving the virus's targeting ability towards tumors. Through literature research, we discovered that oncolytic viruses based on the influenza virus primarily focus on the NS1 protein. Adolfo García-Sastre et al. found that an influenza virus lacking the NS1 protein can only replicate and survive in cells lacking interferon[3]. Subsequently, numerous studies have emerged on virus oncolysis through cutting NS1 protein and arming cytokines[4].
We consider that truncating the NS1 protein of the influenza virus prevents its replication in normal cells, ensuring the safety of the virus. Furthermore, the truncation of NS1 does not conflict with our modification of the surface proteins of the influenza virus in theory. This led us to the idea of attempting to rescue the NS1-truncated influenza virus. Using the A8 plasmid in the 12-plasmid system, we successfully deleted the NS1 protein and retained only the NS2 protein. Unfortunately, despite several attempts, we were unable to rescue the NS1-deleted influenza virus in HEK293T cells. Due to time constraints, we could not continue further experimentation in this area. Our plan is to wait for positive results from our original design before exploring suitable conditions for rescuing the NS1-deleted influenza virus.
Target verification of mutant virus
After rescuing the NA-boosted influenza virus and constructing the corresponding cell model with high TfR1 expression, our aim is to verify the targeting ability of the mutant virus.
First, we need to quantify the virus accurately. Eight hours after infecting CHO cells with the virus, we extracted the viral RNA and reverse transcribed it into cDNA. Using the A7 plasmid (containing transcribed M1 protein RNA) in the twelve-plasmid system, we prepared a series of concentration gradients and obtained a standard curve through real-time fluorescence quantitative PCR. Next, we calculated the virus titers based on the standard curve and the Cq value of the virus cDNA. We measured the virus titers of SD24, SD9, Wild Type, SI14, and SI28 (Figure 5.1). The results showed that the virus titer of the wild-type influenza virus was the highest. The titers of SI14 and SD9 viruses were lower than that of the wild-type, while the titers of SD24 and SI28 were significantly lower than that of the wild-type.
We constructed three cell models to assess the infection efficiency of the virus. One model was transformed with the pCMV3-TfR1 plasmid, another with the pCMV3 plasmid, and the third model was not transfected with any plasmid. Two days after transfecting CHO cells with the respective plasmids, we conducted the virus attack experiment. The amount of virus used for the attack was determined through absolute quantification, ensuring consistent titers across different virus types.
After an 8-hour virus challenge, we extracted virus RNA from the cells and reverse transcribed it into cDNA. We then evaluated the virus infection ability using real-time fluorescence quantitative PCR, with GAPDH selected as the internal reference protein. Due to the low titer of the SD24 virus, we were unable to ensure consistent added amounts with the other viruses, even if we used up all the available virus. Therefore, we did not perform a targeted evaluation of SD24, but focused on evaluating the other three mutant viruses and wild types.
The results (Figure 5.2) demonstrate that the infection efficiency of the wild-type influenza virus with high TfR1 expression is doubled compared to the blank plasmid group, confirming the crucial role of TfR1 in influenza virus infection.
The insertion of 14 amino acids moderately improved the virus's infection efficiency, as described in the literature. However, it did not enhance the targeting of TfR1 high-expression cells. This suggests that the insertion of 14 amino acids may not effectively display NA on the surface of the influenza virus.
The deletion of nine amino acids reduced the virus's infection efficiency to some extent, consistent with the literature. Furthermore, the infection efficiency of the SD9 mutant influenza virus with high TfR1 expression was only 20% higher than that of the blank plasmid group. This finding confirms the important role of neuraminidase in the interaction between the influenza virus and TfR1, providing validation for the concept of our project from a different perspective.
The insertion of 28 amino acids significantly weakened the virus's infection efficiency, which contradicts the literature. Upon sequence comparison, we discovered that the reason for this discrepancy was an incorrect insertion of the extended sequence. We plan to conduct a new round of evaluation after expressing the correct SI28 to rectify this issue.
Summary
In this iGEM project, we attempted to rescue eight mutant influenza viruses, namely S15, S30, SD24, SD9, SI24, SI28, SI28-new, and PS, in order to modify the display state of neuraminidase on the surface of influenza viruses. We successfully rescued six mutant viruses (S15, SD24, SD9, SI24, SI28, SI28-new) and evaluated the TfR1 targeting of three mutant viruses (SD9, SI24, SI28). Unfortunately, due to time constraints, we were unable to rescue the PS mutant virus before the wiki submission. However, after three rounds of engineering cycles, we have identified the reason for the failure and plan to make another attempt in the future.
Additionally, we were unable to complete the targeted evaluation of the SI28-new mutant viruses before the wiki submission. Furthermore, we did not evaluate the targeting of SD24 due to its low viral titer. Nevertheless, the results obtained for the wild-type virus and SD9 mutant virus are promising. Based on these findings, we have demonstrated that cells with high TfR1 expression are more susceptible to influenza virus infection, and the length of the neuraminidase protein plays a crucial role in determining this susceptibility. In our future work, we aim to rescue more influenza viruses with different neuraminidase display states and conduct targeted evaluations to identify influenza viruses that exhibit enhanced targeting towards cells with high TfR1 expression.
Reference
[1]Castrucci MR, Kawaoka Y. Biologic importance of neuraminidase stalk length in influenza A virus. J Virol. 1993;67(2):759-764. doi:10.1128/JVI.67.2.759-764.1993
[2]Zheng A, Sun W, Xiong X, et al. Enhancing Neuraminidase Immunogenicity of Influenza A Viruses by Rewiring RNA Packaging Signals. J Virol. 2020;94(16):e00742-20. Published 2020 Jul 30. doi:10.1128/JVI.00742-20
[3]García-Sastre A, Egorov A, Matassov D, et al. Influenza A virus lacking the NS1 gene replicates in interferon-deficient systems. Virology. 1998;252(2):324-330. doi:10.1006/viro.1998.9508
[4]Kabiljo J, Laengle J, Bergmann M. From threat to cure: understanding of virus-induced cell death leads to highly immunogenic oncolytic influenza viruses. Cell Death Discov. 2020;6:48. Published 2020 Jun 11. doi:10.1038/s41420-020-0284-1