Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus that causes the most prevalent viral encephalitis in Asia. The NS5 protein of JEV is a key component of the viral replicase complex, which plays a crucial role in viral pathogenesis. In this study, tandem affinity purification (TAP) followed by mass spectrometry analysis was performed to identify novel host proteins that interact with NS5. Heat shock protein 70 (Hsp70), eukaryotic elongation factor 1-alpha (eEF-1α) and ras-related nuclear protein (Ran) were demonstrated to interact with NS5. In addition to NS5, Hsp70 was also found to interact with NS3 which is another important member of the replicase complex. It was observed that the cytoplasmic Hsp70 partially colocalizes with the components of viral replicase complex including NS3, NS5 and viral dsRNA during JEV infection. Knockdown of Hsp70 resulted in a significantly reduced JEV genome replication. Further analysis reveals that Hsp70 enhances the stability of viral proteins in JEV replicase complex. These results suggest an important role for Hsp70 in regulating JEV replication, which provides a potential target for the development of anti-JEV therapies.

Pseudorabies virus (PRV) has been widely used as a live trans‐synaptic tracer for mapping neuronal circuits. Systematically identifying mature PRV virion proteomes and defining co‐purified host proteins are necessary to fully understand the detailed mechanism underlying PRV transmission processes. Here, a PRV virion purification strategy based on sorting with flow cytometry is developed and the mature extracellular and intracellular PRV virion proteomes using LC coupled with MS/MS are characterized. In addition to viral proteins, a large number of host proteins are also identified, including proteins related to actin cytoskeletal dynamics and membrane protrusion. How many of these host proteins are true virion components are unknown and the majority of these may not be. Through functional analysis, it is found that IRSp53 and fascin are critical for the egress process and play a role in direct cell–cell transmission. Moreover, it is shown that CDC42 and Rac1 are also involved in the production of mature extracellular virions. The results suggest that the formation of the filopodia‐like cytoskeleton and the rearrangement of the membrane, which are both associated with IRSp53 and fascin, may be important for the transmission of viruses used in neuronal tracing.

Mapping the neural circuits facilitates understanding the brain’s working mechanism. Pseudorabies virus (PRV; Bartha stain) as a tracer can infect neurons and retrogradely transport in neural circuits. To illuminate the network, tracers expressing reporter genes at a high level are needed. In this study, we optimized the expression level of reporter genes and constructed two new retrograde trans-multisynaptic tracers PRV531 and PRV724, which separately express more robust green and red fluorescent proteins than the existing retrograde tracers PRV152 and PRV614. PRV531 and PRV724 can be used for mapping the neural circuit of the central nervous system (CNS) and the peripheral nervous system (PNS). Overall, our work adds two valuable tracers to the toolbox for mapping neural circuits.