(45) Mouse hepatitis virus infectivity is reduced by host chaperone protein inhibitor MKT-077

Coronaviruses (CoV) are RNA viruses with high mutation rates that can lead to the accumulation of mutated viral proteins. Host chaperones, including heat shock protein 70 (Hsp70) family members, can buffer deleterious... [ view full abstract ]

Coronaviruses (CoV) are RNA viruses with high mutation rates that can lead to the accumulation of mutated viral proteins. Host chaperones, including heat shock protein 70 (Hsp70) family members, can buffer deleterious mutations by facilitating protein folding, stability, and function. In addition, Hsp70 family members can function as holdases and assist with virion disassembly, virion assembly, and protein transport.  To determine whether the members of the Hsp70 family of chaperones are required for CoV replication, we examined the sensitivity of mouse hepatitis virus (MHV, strain A59) to MKT-077, a small-molecule Hsp70 inhibitor that prevents substrate release. MKT-077 treatment of DBT-9 cells and BHK-R cells, which are engineered to express an MHVA59 receptor, resulted in a concentration-dependent reduction in MHV-A59 titer, with a maximal reduction of 2 log10.  To determine the target of Hsp70 in MHV replication, we assessed viral entry, transcription, and egress. Addition of MKT-077 at varying times during replication demonstrated that MKT-077 inhibits MHV post-entry step, and RT-qPCR analysis of genomic and subgenomic RNA levels demonstrated that MKT-077 did not impair viral transcription. Measurement the ratio of intracellular and extracellular virus titers showed that MKT-077 also does not prevent virus egress. To determine if MKT-077 could act independently of the cell, we measured MHV titer following incubation in solution with MKT-077. MKT-077 incubation resulted in a titer reduction comparable to treatment in DBT-9 cells. Our data indicate that MKT-077 reduces MHV-A59 infectivity independent of viral replication and may inhibit a viral protein, impair membrane integrity, or interact with Hsp70 packaged in the virion. The identification of the nature of MKT-077 inhibition will inform future efforts to identify a possible role for Hsp70 in CoV replication and stability.