Genome-Wide Small Interfering RNA Screens Reveal VAMP3 as a Novel Host Factor Required for Uukuniemi Virus Late Penetration

Abstract

The Bunyaviridae constitute a large family of enveloped animal viruses, many of which are important emerging pathogens. How bunyaviruses enter and infect mammalian cells remains largely uncharacterized. We used two genome-wide silencing screens with distinct small interfering RNA (siRNA) libraries to investigate host proteins required during infection of human cells by the bunyavirus Uukuniemi virus (UUKV), a late-penetrating virus. Sequence analysis of the libraries revealed that many siRNAs in the screens inhibited infection by silencing not only the intended targets but additional genes in a microRNA (miRNA)-like manner. That the 7-nucleotide seed regions in the siRNAs can cause a perturbation in infection was confirmed by using synthetic miRNAs (miRs). One of the miRs tested, miR-142-3p, was shown to interfere with the intracellular trafficking of incoming viruses by regulating the v-SNARE VAMP3, a strong hit shared by both siRNA screens. Inactivation of VAMP3 by the tetanus toxin led to a block in infection. Using fluorescence-based techniques in fixed and live cells, we found that the viruses enter VAMP3 ؉ endosomal vesicles 5 min after internalization and that colocalization was maximal 15 min thereafter. At this time, LAMP1 was associated with the VAMP3 ؉ virus-containing endosomes. In cells depleted of VAMP3, viruses were mainly trapped in LAMP1-negative compartments. Together, our results indicated that UUKV relies on VAMP3 for penetration, providing an indication of added complexity in the trafficking of viruses through the endocytic network. IMPORTANCEBunyaviruses represent a growing threat to humans and livestock globally. Unfortunately, relatively little is known about these emerging pathogens. We report here the first human genome-wide siRNA screens for a bunyavirus. The screens resulted in the identification of 562 host cell factors with a potential role in cell entry and virus replication. To demonstrate the robustness of our approach, we confirmed and analyzed the role of the v-SNARE VAMP3 in Uukuniemi virus entry and infection. The information gained lays the basis for future research into the cell biology of bunyavirus infection and new antiviral strategies. In addition, by shedding light on serious caveats in large-scale siRNA screening, our experimental and bioinformatics procedures will be valuable in the comprehensive analysis of past and future high-content screening data.

MicroRNA miR-27 Inhibits Adenovirus Infection by Suppressing the Expression of SNAP25 and TXN2

“…Several previous reports demonstrated that the SNARE complex is involved in viral infection (46)(47)(48). Meier et al reported that siRNA-mediated knockdown of the SNARE protein VAMP3 resulted in the inhibition of endocytic intracellular trafficking of a bunyavirus, Uukuniemi virus, indicating that SNARE protein-mediated membrane fusion is needed for the efficient entry of Uukuniemi virus into cells (49). Ad infection was also inhibited by knockdown of SNAP25 at the step of viral entry (Fig.…”

Functional Genomic Strategies for Elucidating Human–Virus Interactions

“…While a consistently low number of exact genes overlap across related siRNA screens, it is nonetheless clear that similar screens find bioinformatically related genes, e.g., genes that cluster in common pathways and complexes like the nuclear pore complex (NPC) with HIV-1 and the vacuolar ATPase (V-ATPase) for IAV or HRV (Bushman et al, 2009;Hao et al, 2013;Perreira et al, 2015;Stertz & Shaw, 2011;Zhu et al, 2014). With closer study it became readily apparent that this low level of saturation within the dataset of each primary screen was due to a high level of false negatives (Hao et al, 2013;Meier et al, 2014;Zhu et al, 2014). False negatives with RNAi may come about for several reasons including difficulty in targeting a protein (prolonged protein half-life or sufficient remaining catalytic activity), nonspecific toxicity of siRNAs, and plate edge effects.…”

Stepwise Priming by Acidic pH and a High K + Concentration Is Required for Efficient Uncoating of Influenza A Virus Cores after Penetration

“…SFV-and VSV-infected cells were fixed at 5 h p.i., and UUKV-infected cells were fixed at 19 h p.i. SFV and VSV infection was detected on the basis of green fluorescent protein (GFP) expression, and UUKV infection was detected by immunostaining, as described previously (48).…”