Viruses in the Flavivirus genus of the Flaviviridae family are 
arthropod-transmitted and contribute to staggering numbers of human infections 
and significant deaths annually across the globe. To identify cellular factors 
with antiviral activity against flaviviruses, we screened a cDNA library using 
an iterative approach. We identified a mammalian Hsp40 chaperone protein 
(DNAJC14) that when overexpressed was able to mediate protection from yellow 
fever virus (YFV)-induced cell death. Further studies revealed that DNAJC14 
inhibits YFV at the step of viral RNA replication. Since replication of bovine 
viral diarrhea virus (BVDV), a member of the related Pestivirus genus, is also 
known to be modulated by DNAJC14, we tested the effect of this host factor on 
diverse Flaviviridae family members. Flaviviruses, including the pathogenic 
Asibi strain of YFV, Kunjin, and tick-borne Langat virus, as well as a 
Hepacivirus, hepatitis C virus (HCV), all were inhibited by overexpression of 
DNAJC14. Mutagenesis showed that both the J-domain and the C-terminal domain, 
which mediates self-interaction, are required for anti-YFV activity. We found 
that DNAJC14 does not block YFV nor HCV NS2-3 cleavage, and using non-inhibitory 
mutants demonstrate that DNAJC14 is recruited to YFV replication complexes. 
Immunofluorescence analysis demonstrated that endogenous DNAJC14 rearranges 
during infection and is found in replication complexes identified by dsRNA 
staining. Interestingly, silencing of endogenous DNAJC14 results in impaired YFV 
replication suggesting a requirement for DNAJC14 in YFV replication complex 
assembly. Finally, the antiviral activity of overexpressed DNAJC14 occurs in a 
time- and dose-dependent manner. DNAJC14 overexpression may disrupt the proper 
stoichiometry resulting in inhibition, which can be overcome upon restoration of 
the optimal ratios due to the accumulation of viral nonstructural proteins. Our 
findings, together with previously published work, suggest that the members of 
the Flaviviridae family have evolved in unique and important ways to interact 
with this host Hsp40 chaperone molecule.