DNAJC14 (also designated DRIP78, Jiv and HDJ3) is a member of the Hsp40 family of protein chaperones 
. Proteins in this family contain a 70 amino acid motif, designated the J-domain, which recruits Hsp70 family members and stimulates the ATP hydrolysis step of the chaperone process. J-domain containing proteins are involved in diverse cellular processes. The human DNAJC family has 23 members with the presence of the J domain being the single common feature. Although not extensively studied, involvement of these proteins in mitochondrial import, translation, endocytosis and exocytosis has been noted 
. DNAJC14 has previously been implicated in the life cycle of a member of the Flaviviridae.
The bovine homolog of this factor, Jiv, is essential for the polyprotein cleavage and replication of the pestivirus BVDV. Jiv acts as a required co-factor for the viral NS2 autoprotease, influencing its cleavage from NS3 and modulating RNA replication, virus production and cytopathogenicity of this pestivirus 
. In contrast to our findings with YFV, increased expression of Jiv results in higher levels of BVDV RNA replication and virus-induced cell death. Interestingly, some cytopathic biotypes of BVDV are naturally occurring recombinant viruses, which have insertions of DNAJC14 in the NS2-3 coding region. A 90 amino acid domain common to all of the Jiv-containing cytopathic BVDV isolates is designated Jiv90 (see ). This sequence is distinct from the J-domain and contains two conserved CXXCXXXH motifs.
DNAJ proteins regulate the ATPase cycle of Hsp70 via their J domain, with the HPD motif critical in accelerating the Hsp70 ATPase activity, while the substrate-binding domain loads the substrate onto Hsp70 
. Our studies with mutant H471Q suggest that the critical HPD motif within the J domain is required for DNAJC14 antiviral function, suggesting that ATP-driven Hsp70 chaperone activity may be involved in the process of RNA replication and its inhibition. Since Hsp70 chaperone activity occurs via a stoichiometric mechanism, with a single Hsp70 monomer per substrate 
it seems likely that DNAJC14/Hsp70 chaperone activity is required for YFV replication complex assembly and that overexpression of DNAJC14 disrupts the chaperone/substrate complex. Dimerization of some Hsp40 family members is evolutionarily conserved and required for their function 
. The CT1 mutant lacks the ability to multimerize () and fails to inhibit YFV () as well as HCV (data not shown). Thus multimerization is likely critical for DNAJC14's antiviral function.
In our studies, we demonstrated that DNAJC14 noninhibitory mutants are found in YFV replication complexes, as measured by colocalization and coprecipitation with NS3 (). Moreover, endogenous DNAJC14 rearranges upon YFV infection and is found colocalized with active replication complexes, as determined by the presence of dsRNA (). This suggests that YFV replication complexes assemble at a specific ER membrane site where DNAJC14 is located, and that DNAJC14 (and likely Hsp70) is specifically recruited to facilitate formation of the viral replication complex. DNAJC14 overexpression would then result in disrupted chaperone/substrate stoichiometry and inhibit replication complex assembly. Alternatively, YFV may hijack DNAJC14-containing membranes for its replication complex assembly, and overexpression may inhibit the distribution and recruitment of other host factors localized to this membrane microdomain and required for replication complex formation.
We realized that the inhibitory effect of DNAJC14 on YFV was diminished at later time points post infection and that inhibition was dose dependent, with higher levels of DNAJC14 resulting in lower levels of virus replication (). One possible explanation is that at early time points, overexpressed DNAJC14 is in vast excess to its substrates (viral proteins) and this inappropriate stoichiometry results in inhibition of replication complex formation. Since DNAJC14 does not inhibit virus genome translation (), nor polyprotein processing (), viral protein would be predicted to accumulate with time. At some point, the level of viral protein(s) would result in an appropriate DNAJC14 to substrate ratio to allow the chaperone process to occur and thus overcome DNAJC14's inhibitory effect. This is not dissimilar to the scenario occurring with BVDV, in which the DNAJC14 Jiv90 domain interacts with BVDV NS2-3 at a ratio of 1
1. This stoichiometric mechanism might be a common requirement for normal DNAJC14 cellular function, as either overexpression or sequestration of DNAJC14 inhibits dopamine D1 receptor transport 
We found that multiple Flaviviridae
were inhibited under conditions of DNAJC14 overexpression and wondered whether viruses from other families might be similarly affected. We tested DNAJC14's effects on Sindbis virus, a positive strand RNA virus from the Alphavirus
genus. In contrast to the flaviviruses, we found that DNAJC14 overexpression had no effect on viral replication, as measured by expression of a fluorescent reporter from the viral subgenomic RNA (data not shown). Thus replication complex formation for Sindbis virus is not likely affected by DNAJC14 overexpression. Interestingly, however, Sindbis virion production was reduced by DNAJC14 overexpression (data not shown). Thus a step in Sindbis virus assembly, such as glycoprotein maturation and transport from the ER-Golgi to the plasma membrane, where Sindbis budding occurs, may require DNAJC14-containing membrane microdomains and chaperone function. In addition, overexpression of DNAJC14 reduced VSV virion production (data not shown), suggesting an effect on VSV glycoprotein ER-golgi transport. Since it has been reported that DNAJC14 is involved in dopamine D1 receptor transport 
, it is likely that DNAJC14 facilitates specific membrane processes including vesicle transport and viral replication complex assembly. It is possible that many virus families have specific requirements for chaperone processes at various steps in their life cycle. Understanding these requirements and identifying the chaperones and proteins undergoing the chaperone process may lead to insights into similarities and differences between different virus families in these critical life cycle steps.
DNAJC14 can both facilitate and inhibit YFV replication. Based on all of our findings, we propose the following model (): Translation of the incoming YFV RNA and subsequent polyprotein processing generates the viral proteins necessary for the viral RNA replication process. DNAJC14 functions as a chaperone system, most likely with involvement of Hsp70, to facilitate a step in the YFV membrane-associated multiprotein complex assembly that is critical for the formation of replication complexes. The TM domains within DNAJC14 target the protein to a specific subcellular ER membrane location, wherein substrate selection and YFV replication complex formation occurs. Multimerization of DNAJC14 via its C-terminus is likely required for assembly of the chaperone/substrate complex. It is possible that each DNAJC14 monomer binds a substrate and together they promote the proper folding and interaction of the substrate pair, which might be different sites on the same protein, two different YFV proteins, or a viral protein and host protein necessary for viral replication. Newly generated viral RNA is produced, which after translation generates new substrate for the chaperone process, and the formation of additional replication complexes. Overexpression of DNAJC14 mutants that fail to multimerize (CT1, CT2, CT3, CT4 or CT5), or contain mutations in the critical J domain (L466P, H471Q, ) has no effect on virus replication; these mutants fail to interact with and disrupt the normal chaperone components and therefore exhibit no antiviral activity. Expression of full-length (wildtype) DNAJC14 results in an excess of DNAJC14 relative to the substrate, and complexes with an inappropriate stoichiometric ratio are formed, disrupting the chaperone process (). The N-terminal truncation mutants, which contain the C terminal multimerization motif and an intact J domain, also interact with the chaperone components, disrupting the proper chaperone/substrate stoichiometry. With time, continued translation of the incoming viral genome (or genome generated by very low levels of viral replication) results in the accumulation of viral proteins. Once the optimal substrate/chaperone ratio is established, the restored chaperone process results in replication complex formation and viral RNA replication. Further studies are required to address the viral and cellular substrate(s) for DNAJC14 and to determine if other host factors (for example, Hsp70) participate in this important chaperone process.