Development of Zika Virus E Variants for Pseudotyping Retroviral Vectors Targeting Glioblastoma Cells.

A fundamental idea for targeting glioblastoma cells is to exploit the neurotropic properties of Zika virus (ZIKV) through its two outer envelope proteins, prM and E. This study aimed to develop envelope glycoproteins for pseudotyping retroviral vectors that can be used for efficient tumor cell infection. Firstly, the retroviral vector pNLlucAM was packaged using wild-type ZIKV E to generate an E-HIVluc pseudotype. E-HIVluc infection rates for tumor cells were higher than those of normal prME pseudotyped particles and the traditionally used vesicular stomatitis virus G (VSV-G) pseudotypes, indicating that protein E alone was sufficient for the formation of infectious pseudotyped particles. Secondly, two envelope chimeras, E41.1 and E41.2, with the E wild-type transmembrane domain replaced by the gp41 transmembrane and cytoplasmic domains, were constructed; pNLlucAM or pNLgfpAM packaged with E41.1 or E41.2 constructs showed infectivity for tumor cells, with the highest rates observed for E41.2. This envelope construct can be used not only as a tool to further develop oncolytic pseudotyped viruses for therapy, but also as a new research tool to study changes in tumor cells after the transfer of genes that might have therapeutic potential.

Transduction Efficiency of Zika Virus E Protein Pseudotyped HIV-1gfp and Its Oncolytic Activity Tested in Primary Glioblastoma Cell Cultures.

The development of new tools against glioblastoma multiforme (GBM), the most aggressive and common cancer originating in the brain, remains of utmost importance. Lentiviral vectors (LVs) are among the tools of future concepts, and pseudotyping offers the possibility of tailoring LVs to efficiently transduce and inactivate GBM tumor cells. Zika virus (ZIKV) has a specificity for GBM cells, leaving healthy brain cells unharmed, which makes it a prime candidate for the development of LVs with a ZIKV coat. Here, primary GBM cell cultures were transduced with different LVs encased with ZIKV envelope variants. LVs were generated by using the pNLgfpAM plasmid, which produces the lentiviral, HIV-1-based, core particle with GFP (green fluorescent protein) as a reporter (HIVgfp). Using five different GBM primary cell cultures and three laboratory-adapted GBM cell lines, we showed that ZIKV/HIVgfp achieved a 4-6 times higher transduction efficiency compared to the commonly used VSV/HIVgfp. Transduced GBM cell cultures were monitored over a period of 9 days to identify GFP+ cells to study the oncolytic effect due to ZIKV/HIVgfp entry. Tests of GBM tumor specificity by transduction of GBM tumor and normal brain cells showed a high specificity for GBM cells.

Generation of Viral Particles with Brain Cell-Specific Tropism by Pseudotyping HIV-1 with the Zika Virus E Protein.

Flaviviruses are a family of RNA viruses that includes many known pathogens, such as Zika virus (ZIKV), West Nile virus (WNV), dengue virus (DENV), and yellow fever virus (YFV). A pseudotype is an artificial virus particle created in vitro by incorporating the flavivirus envelope proteins into the structure of, for example, a retrovirus such as human immunodeficiency virus type-1 (HIV-1). They can be a useful tool in virology for understanding the biology of flaviviruses, evaluating immune responses, developing antiviral strategies but can also be used as vectors for gene transfer experiments. This protocol describes the generation of a ZIKV/HIV-1 pseudotype developed as a new tool for infecting cells derived from a highly malignant brain tumor: glioblastoma multiforme grade 4.