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. 2021 Jul:559:30-39.
doi: 10.1016/j.virol.2021.03.014. Epub 2021 Mar 26.

Chimeric Zika viruses containing structural protein genes of insect-specific flaviviruses cannot replicate in vertebrate cells due to entry and post-translational restrictions

Chandra S Tangudu  1 , Jermilia Charles  1 , Daniel Nunez-Avellaneda  1 , Alissa M Hargett  1 , Aaron C Brault  2 , Bradley J Blitvich  3
Affiliations

Chimeric Zika viruses containing structural protein genes of insect-specific flaviviruses cannot replicate in vertebrate cells due to entry and post-translational restrictions

Chandra S Tangudu et al. Virology. 2021 Jul.

Abstract

Long Pine Key virus (LPKV) and Lammi virus are insect-specific flaviviruses that phylogenetically affiliate with dual-host flaviviruses. The goal of this study was to provide insight into the genetic determinants that condition this host range restriction. Chimeras were initially created by replacing select regions of the Zika virus genome, including the premembrane and envelope protein (prM-E) genes, with the corresponding regions of the LPKV genome. Of the four chimeras produced, one (the prM-E swap) yielded virus that replicated in mosquito cells. Another chimeric virus with a mosquito replication-competent phenotype was created by inserting the prM-E genes of Lammi virus into a Zika virus genetic background. Vertebrate cells did not support the replication of either chimeric virus although trace to modest amounts of viral antigen were produced, consistent with suboptimal viral entry. These data suggest that dual-host affiliated insect-specific flaviviruses cannot replicate in vertebrate cells due to entry and post-translational restrictions.

Keywords: Chimeric; Entry; Flavivirus; Host range; Insect-specific.

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Conflict of interest statement

COMPETING INTERESTS

None of declare

Declaration of interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

FIGURE 1.
FIGURE 1.
A schematic of the viral chimeras produced in this study. Four chimeras were generated by replacing the prM-E, E, EDIII and NS5 regions of ZIKV with those of LPKV (denoted as cZIKV/LPKV-prME, cZIKV/LPKV-E, cZIKV/LPKV-EDIII and cZIKV/LPKVNS5) and an additional chimera was generated by replacing the prM-E region of ZIKV with those of LAMV (denoted as cZIKV/LAMV-prME). Each chimera name begins with a lowercase c to differentiate between viral chimeras and infectious chimeric viruses. In the schematic representing cZIKV/LPKV-EDIII, the location of EDIII within the E region is denoted, along with EDI, EDII, the stem region and transmembrane (TM) domain. The short hinge region that immediately precedes EDIII is not shown. Resulting amino acid chimeric sequences are denoted, with protease cleavage sites indicated by vertical lines. Sequences from the heterologous viruses (LPKV and LAMV) are underlined.
FIGURE 2.
FIGURE 2.
Western blot analysis of mosquito and vertebrate cells inoculated with ZIKV/LPKV-prME. The chimeric virus was passaged twice in C6/36 cells then sequentially passaged four more times in C6/36 cells (lanes 1–4, passage 3–6 respectively) or four times in Vero cells (lanes 8–11, Vero cell passage 1–4 respectively). C6/36 and Vero cells inoculated with ZIKV (lanes 5 and 12, respectively) and LPKV (lanes 6 and 13, respectively) were also included. Uninfected C6/36 and Vero cells were used as negative controls (lanes 7 and 14, respectively). Lysates were prepared at 4 or 5 days post-inoculation (Vero and C6/36 cells, respectively). Equal amounts of protein were resolved on 8–16% Tris-glycine gels and analyzed by Western blot using (A) anti-ZIKV NS1 polyclonal antibody, (B) anti-ZIKV prM polyclonal antibody or (C) anti-β-actin polyclonal antibody. The arrows show the expected migration positions of ZIKV prM and NS1 (molecular weights: 19 and 48 KDa, respectively) and cellular β-actin (molecular weight: 42 KDa).
FIGURE 3.
FIGURE 3.
Immunofluorescence analysis of mosquito cells inoculated with ZIKV/LPKV-prME and ZIKV/LAMV-prME. Subconfluent monolayers of C6/36 cells in six-well culture dishes were inoculated with recombinant ZIKV, ZIKV/LPKV-prME or ZIKV/LAMV-prME at a multiplicity of infection (MOI) of 1.0 or they were inoculated with media only (rows 1–4, respectively). Both chimeric viruses had undergone two passages in C6/36 cells prior to the experiment. At 5 days post-inoculation, cells were fixed with methanol and immunostained with anti-ZIKV capsid polyclonal antibody (column 2) or pan-flavivirus E protein monoclonal antibody (column 3), followed by Alexa 594-conjugated donkey anti-rabbit IgG. DAPI was used to visualize the nucleic (column 1). Merged images are also shown (column 4).
FIGURE 4.
FIGURE 4.
Monitoring first passage Vero cell cultures for ZIKV/LPKV-prME and ZIKV/LAMV-prME antigens by immunofluorescence assay. Subconfluent monolayers of Vero cells in six-well culture dishes were inoculated with recombinant ZIKV, ZIKV/LPKV-prME, ZIKV/LAMV-prME at an MOI of 1.0 or they were inoculated with media only (rows 1–4, respectively). Viruses had undergone two passages in C6/36 cells prior to the experiment. At 4 days post-inoculation, cells were fixed with methanol and immunostained with anti-ZIKV capsid polyclonal antibody (column 2) or pan-flavivirus E protein monoclonal antibody (column 3), followed by Alexa 594-conjugated donkey anti-rabbit IgG. DAPI was used to visualize the nucleic (column 1). Merged images are also shown (column 4).
FIGURE 5.
FIGURE 5.
Monitoring second passage Vero cell cultures for ZIKV/LPKV-prME and ZIKV/LAMV-prME antigens by immunofluorescence assay. Subconfluent monolayers of Vero cells in six-well culture dishes were inoculated with recombinant ZIKV, ZIKV/LPKV-prME, ZIKV/LAMV-prME or media only (rows 1–4, respectively). Viruses had undergone two passages in C6/36 cells followed by one passage in Vero cells prior to the experiment. At 4 days post-inoculation, cells were fixed with methanol and immunostained with anti-ZIKV capsid polyclonal antibody (column 2) or pan-flavivirus E protein monoclonal antibody (column 3), followed by Alexa 594-conjugated donkey anti-rabbit IgG. DAPI was used to visualize the nucleic (column 1). Merged images are also shown (column 4).
FIGURE 6.
FIGURE 6.
Morphology of vertebrate cells inoculated with the chimeric viruses. Subconfluent monolayers of Vero cells in six-well dishes were inoculated with (A) recombinant ZIKV, (B) wild-type LPKV, (C) media only as a negative control, (D) ZIKV/LPKV-prME previously passed twice in C6/36 cells (E) ZIKV/LPKV-prME previously passed twice in C6/36 cells followed by once in Vero cells and (F) ZIKV/LAMV-prME previously been passed twice in C6/36 cells. Images were taken at 4 days post-inoculation (ZIKV) or 7 days post-inoculation (all other cultures).
FIGURE 7.
FIGURE 7.
Western blot analysis of lysates harvested from mosquito and vertebrate cells inoculated with ZIKV/LAMV-prME. The chimeric virus was passaged twice in C6/36 cells then sequentially passaged two more times in C6/36 cells (lanes 1–2, passage 3–4 respectively) or two times in Vero cells (lanes 5–6, Vero cell passage 1–2 respectively). C6/36 and Vero cells inoculated with ZIKV (lanes 3 and 7, respectively) were also included. Uninfected C6/36 and Vero cells were used as negative controls (lanes 4 and 8, respectively). Lysates were prepared at 4 or 5 days post-inoculation (Vero and C6/36 cells, respectively). Equal amounts of protein were resolved on 8–16% Tris-glycine gels and analyzed by Western blot using (A) anti-ZIKV NS1 polyclonal antibody or (B) anti-β-actin polyclonal antibody. The arrows show the expected migration positions of ZIKV NS1 and cellular β-actin (molecular weights: 48 KDa and 42 KDa, respectively).
FIGURE 8.
FIGURE 8.
Alignment of the deduced amino acid sequences of the EDIII domains of select flaviviruses. The key beneath the alignment denotes sites where residues are strictly conserved across all sequences (*), sites with conservative replacements (:), sites with semi-conservative replacements (.) and sites with non-conservative replacements ( ). The alignment was performed using Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/). BinJV, Binjari virus; LAMV, Lammi virus; LPKV, Long Pine Key virus; ZIKV, Zika virus.
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