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.
IMPORTANCE: Bunyaviruses 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.