Activated ADP-ribosylation factor 4 (ARF4) is required for ZIKA virus (ZIKV) infectious cycle

Identification: Li, Mingyuan


Description

 

Activated ADP-ribosylation factor 4 (ARF4) is required for ZIKA virus (ZIKV) infectious cycle
 
MY Li1, YL Siu1, T Naik1, X Ji2, CF, Qin2, R Bruzzone1 and S Sanyal1
1HKU-Pasteur Research Pole and Center of Influenza Research, School of Public Health;
2Beijing Institute of Microbiology and Epidemiology
      
The life cycle of flaviviruses, such as ZIKV, is a complex process relying on specific interactions with host cellular factors which, in turn, provide the basis to devise optimal prophylactic and therapeutic treatment options. To date, the viral-host interactions during the ZIKV life cycle are largely unknown. Our previous results revealed that ARF4 and ARF5, which are GTP-binding proteins regulating vesicular traffic, facilitated the egress of dengue virus. Our current studies using CRISPR-Cas9-mediated ARF4/5 knock out (ARF4/5KO) cells demonstrate that ZIKV production was drastically suppressed in ARF4KO cells, whereas had no changes in ARF5KO cells. We found by real-time RT-PCR that the viral genome was significantly reduced in ARF4KO cells at 1h post-infection, which indicate that the entry of ZIKV was regulated by ARF4. Similar amount of viral particles was detected on the surface of both WT and ARF4KO cells when endocytosis-induced ZIKV internalization was blocked at 4°, suggesting that deletion of ARF4 did not affect ZIKV attachment. Immunofluorescence (IF) and flow cytometry analyses with a fluorescently-labeled ZIKV shows that fusion of virus particles in late endosomes was drastically reduced in ARF4KO cells, implying that ARF4 might be vital in regulating ZIKV internalization to endosomal compartments and subsequent viral fusion. Besides, several reports corroborate our hypothesis that ARF4 is involved in distinct stages of ZIKV infection. Immunoprecipitation assay shows a direct interaction between ARF4 and ZIKV NS2B protein. IF shows a positive correlation between subcellular distribution of ARF4 with the intracellular transport of newly synthesized ZIKV E protein. Release of ZIKA recombinant subviral particles from ARF4KO cells was greatly reduced. Moreover, ZIKV infection was rescued in ARF4KO cells that stably re-express ARF4 WT or a constitutively active mutant, which is defective in GTP hydrolysis. In contrast, production of ZIKV was still blocked in cells stably transduced with a constitutively inactive, GDP-bound form of ARF4. Together these observations reveal a novel function of ARF4 in assisting ZIKV to complete its infectious cycle.
 

 

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