Novel Approach for Insertion of Heterologous Sequences into Full-Length ZIKV Genome Results in Superior Level of Gene Expression and Insert Stability

Abstract

Zika virus (ZIKV) emerged in the Americas in 2015, presenting unique challenges to public health. Unlike other arboviruses of the Flaviviridae family, it is transmissible by sexual contact, which facilitates the spread of the virus into new geographic areas. Additionally, ZIKV can be transmitted from mother to fetus, causing microcephaly and other severe developmental abnormalities. Reliable and easy-to-work-with clones of ZIKV expressing heterologous genes will significantly facilitate studies aimed at understanding the virus pathogenesis and tissue tropism. Here, we developed and characterized two novel approaches for expression of heterologous genes of interest in the context of full-length ZIKV genome and compared them to two previously published strategies for ZIKV-mediated gene expression. We demonstrated that among the four tested viruses expressing nLuc gene, the virus constructed using a newly developed approach of partial capsid gene duplication (PCGD) attained the highest titer in Vero cells and resulted in the highest level of nLuc expression. Suitability of the PCGD approach for expression of different genes of interest was validated by replacing nLuc sequence with that of eGFP gene. The generated constructs were further characterized in cell culture. Potential applications of ZIKV clones stably expressing heterologous genes include development of detection assays, antivirals, therapeutics, live imaging systems, and vaccines.

Identifying optimal capsid duplication length for the stability of reporter flaviviruses

“…During this time, Volkova, et al, published a report on reporter ZIKV and the effect of capsid duplication size on replication. Their conclusion was that C50 is the optimal length for viral growth, with this being the shortest length that was not statistically attenuated compared to WT virus [ 18 ]. Based on this report, we also constructed a C50 ZIKV.…”

“…Two additional methods for stabilizing reporter flaviviruses have been newly developed, both focusing on blocking recombined reporter viruses from continued infection. Volkova and colleagues report a single-nucleotide insertion to the duplicated capsid portion of a reporter ZIKV that minimally perturbs critical RNA elements but causes a +1 frameshift [ 18 ]. If recombination occurs between the duplicated capsid sequences that flank the reporter gene, this frameshift mutation is incorporated into the viral polyprotein and causes mistranslation, effectively taking out recombined viruses from the population.…”

Construction of Stable Reporter Flaviviruses and Their Applications

“…Volkova et al developed a separate, elegant technique using ZIKV bearing NanoLuc as a model [ 39 ]. They built on the capsid duplication method, noting that, for ZIKV, the reported sizes used for the duplication ranged from 25 amino acids [ 28 ], to 33 amino acids [ 30 ], and even duplication of the full capsid [ 32 ].…”

“…Stability results from this construct were mixed, with two of five plaque-purified viral populations showing loss of reporter signal between passages five and ten in Vero cells by flow cytometry. This method has been used to design EGFP reporter LGTV [ 31 ], a tick-borne flavivirus, and Nanoluciferase (NanoLuc) and EGFP ZIKV constructs [ 39 ]. The E/NS1 NanoLuc ZIKV showed stability to ten passages but grew to lower titers than other reporter schemes.…”

“…The capsid duplication strategy has been successfully applied to many other flaviviruses, with slight variations giving different results in stability and utility, though addition of a reporter gene causes general attenuation in all cases [ 22 , 23 , 25 , 26 , 28 , 29 , 30 , 32 , 33 , 35 , 36 , 39 ]. As a rule, reporter viruses made without reducing the homology between duplicated capsids show marked instability, similar to the 3′ UTR reporter viruses [ 23 , 29 , 30 , 33 , 35 ].…”