Role of the host translational machinery in Zika virus replication

Abstract

The pandemic of ZIKV during 2013-2016 had alarmed the global world due to the rising microcephaly cases in newborn in Brazil. ZIKV genome encodes for a single open reading frame with multiple transmembrane domains that is cleaved into at least 3 structural and 7 non-structural proteins. To synthesize such a complex multi-pass protein, transmembrane domains have to be inserted correctly during de-novo synthesis. Flavivirus polyprotein is believed to be targeted to the translocon for insertion by the signal recognition particle (SRP) pathway, but experimental evidence is incomplete.

To determine which ER targeting pathways for nascent membrane protein insertion are necessary for ZIKV, components of the various targeting pathways were depleted in HeLa cells by DsiRNA transfection. Significantly lower viral titers were found in supernatants from Sec61α, SRP54 and EMC1 depleted cells after ZIKV infection, which are in line with the existing dogma that ZIKV utilizes SRP-dependent translocation as well as the EMC complex for replication.

Since SRP receptors (SR) are rarely found as hits in genome-wide screenings, the requirement of specific SRP components were further investigated. The 3 GTPases, SRα, SRβ and SRP54 were knocked down in HeLa, Vero and Huh7 cells and showed that synthesis of plasmid-encoded membrane proteins was inhibited in all the knocked down cells. In contrast, although significantly lower viral titers were detected in SRP54 depleted cells, this reduction was insignificant in SRα and SRβ depleted cells. In line with these results, viral protein amounts were attenuated in SRP54-depleted cells, while similar levels of viral proteins were detected in SRα and SRβ knockdown cells. These results showed that although SRP54 is necessary for ZIKV replication, SRα and SRβ are dispensable for ZIKV replication.

To test if SRα and SRβ independent replication is unique to ZIKV, SRα, SRβ and SRP54 knockdown A549 cells were infected with influenza. Significantly lower titers and HA proteins were found in all knockdown cells, indicating that unlike ZIKV, SRP54 and SR are necessary for influenza.

To quantify nascent protein synthesis in infected cells, ZIKV infected Vero cells were pulsed with a clickable methionine analog. Although nascent synthesis of host EGFR were depleted in all uninfected knockdown cells, it was comparable to the control in ZIKV infected SRα and SRβ knockdown cells, while modest increases of viral protein expression were observed in depleted cells, indicating that characteristics of protein translation is altered upon infection. To determine whether the architecture of the protein translocation machinery is altered in ZIKV-infected cells, we aimed to map the interactions of viral non-structural proteins with the translocation components. Interactions with host translocation machineries were sought by co-immunoprecipitation with FLAG-tagged viral NS proteins. Viral NS proteins interacted extensively with SRP, SR, translocon and ribosome components, hinting at the possibility that NS proteins bridge and strengthen interactions between the complex, and stabilize them on ER membrane for efficient viral replication.

In conclusion, despite ZIKV utilizing the SRP-dependent targeting, only knockdown of SRP54, but not SRα or SRβ inhibited viral replication, likely because of viral NS interactions with host translational machineries.