The interaction of GRP78 and Zika virus E and NS1 proteins occurs in a chaperone-client manner.

Glucose regulated protein 78 (GRP78) is a chaperone protein that is a central mediator of the unfolded protein response, a key cellular stress response pathway. GRP78 has been shown to be critically required for infection and replication of a number of flaviviruses, and to interact with both non-structural (NS) and structural flavivirus proteins. However, the nature of the specific interaction between GRP78 and viral proteins remains largely unknown. This study aimed to characterize the binding domain and critical amino acid residues that mediate the interaction of GRP78 to ZIKV E and NS1 proteins. Recombinant EGFP fused GRP78 and individual subdomains (the nucleotide binding domain (NBD) and the substrate binding domain (SBD)) were used as a bait protein and co-expressed with full length or truncated ZIKV E and NS1 proteins in HEK293T/17 cells. Protein-protein interactions were determined by a co-immunoprecipitation assay. From the results, both the NBD and the SBD of GRP78 were crucial for an effective interaction. Single amino acid substitutions in the SBD showed that R492E and T518A mutants significantly reduced the binding affinity of GRP78 to ZIKV E and NS1 proteins. Notably, the interaction of GRP78 with ZIKV E was stably maintained against various single amino acid substitutions on ZIKV E domain III and with all truncated ZIKV E and NS1 proteins. Collectively, the results suggest that the principal binding between GRP78 and viral proteins is mainly a classic canonical chaperone protein-client interaction. The blocking of GRP78 chaperone function effectively inhibited ZIKV infection and replication in neuronal progenitor cells. Our findings reveal that GRP78 is a potential host target for anti-ZIKV therapeutics.

Comparative analysis of a Thai congenital-Zika-syndrome-associated virus with a Thai Zika-fever-associated virus.

In this study, we compared the characteristics of two strains of Zika virus (ZIKV) isolated in Thailand, one isolated from a febrile patient and one isolated from tissues of a fetus medically terminated due to congenital Zika syndrome (CZS). Replication profiles showed that the isolate from the fetal tissues replicated significantly more slowly than the fever-associated isolate in human lung A549 cells during the first 24 hours postinfection but showed a similar growth profile over longer-term infection. A much smaller difference was observed in Aedes albopictus C6/36 cells. In a quasispecies analysis, a high proportion (approximately 20%) of nonfunctional genomes was identified, caused by an adenine insertion in the prM gene. This insertion was found to be present in two Thai fever strains and as such may represent a common feature of Thai endemic ZIKV. Comparison between viral RNA copy number and viral titer showed that the isolate from fetal tissues was produced more efficiently than the fever-associated isolate. Together, these results suggest that different ZIKV isolates differ in their replication capacity, and this might contribute to the fetotropic potential of a particular strain.

Hsp90 interacts with multiple dengue virus 2 proteins.

Infections with the mosquito-borne dengue virus (DENV) remain a significant public health challenge. In the absence of a commercial therapeutic to treat DENV infection, a greater understanding of the processes of cellular replication is required. The abundant cellular chaperone protein heat shock protein 90 (Hsp90) has been shown to play a proviral role in the replication cycle of several viruses, predominantly through the stabilization of specific viral proteins. To investigate any potential role of Hsp90 in DENV infection the interaction between Hsp90 and DENV proteins was determined through co-immunoprecipitation experiments. Six DENV proteins namely envelope (E) and nonstructural (NS) proteins NS1, NS2B, NS3, NS4B and NS5 were shown to interact with Hsp90, and four of these proteins (E, NS1, NS3 and NS5) were shown to colocalize to a variable extent with Hsp90. Despite the extensive interactions between Hsp90 and DENV proteins, inhibition of the activity of Hsp90 had a relatively minor effect on DENV replication, with inhibition of Hsp90 resulting in a decrease of cellular E protein (but not nonstructural proteins) coupled with an increase of E protein in the medium and an increased virus titer. Collectively these results indicate that Hsp90 has a slight anti-viral effect in DENV infection.

Ubiquitin-Conjugating Enzyme E2 L3 is Downregulated by the Chikungunya Virus nsP2 Protease.

PURPOSE: Chikungunya virus (CHIKV) is a mosquito transmitted alphavirus that causes chikungunya fever in humans. The CHIKV non-structural protein 2 (nsP2) is a multifunctional protein that additionally modulates the host cell to dampen the innate immune response and inhibit other cellular processes. EXPERIMENTAL DESIGN: To further investigate the interactions of nsP2 with host cells, the protease domain of CHIKV nsP2 (nsP2-pro) is transfected into Hela cells, and differential protein expression is detected by 2D polyacrylamide gel electrophoresis. RESULTS: A total of 21 differentially regulated (six upregulated, 15 downregulated) spots are observed, of which five are identified by mass spectrometry. The downregulation of one of the identified proteins, ubiquitin-conjugating enzyme E2 L3 (UBE2L3) is confirmed by western blotting of both nsP2-pro transfection and CHIKV natural infection, and the downregulation of UBE2L3 is additionally shown to require an enzymatically active nsP2 protease domain. Transfection of full length UBE2L3 into HEK293T/17 cells prior to CHIKV infection reduce levels of infection and E protein expression but do not alter RNA genome levels. CONCLUSION: These results suggest that UBE2L3 is a cellular target of the CHIKV nsP2 protease, and this possibly mediates the pathogenesis of chikungunya fever.

Oleic acid Enhances Dengue Virus But Not Dengue Virus-Like Particle Production from Mammalian Cells.

Despite the recent introduction of a commercial vaccine, the mosquito-transmitted dengue virus is still a worldwide public health problem. Based on the live attenuated vaccine strategy, the commercial vaccine has a less than optimal protective profile. Virus-like particles (VLPs) offer an attractive alternate vaccination strategy due to the effectively native presentation of epitopes in the absence of any infectious genetic material. However, the production of amounts of VLP in a platform that can support commercial development remains a major obstacle. This study generated two DENV 2 VLPs [codon-optimized and chimeric DENV/Japanese encephalitis virus (JEV)] and directly compared yields of these constructs by western blotting and dot blot hybridization. The effect of oleic acid supplementation, a process known to increase DENV production in natural infection, was also investigated. Results showed that the chimeric construct gave a two- to threefold higher yield than the codon-optimized construct and that while oleic acid increased DENV virion production in natural infection, it inhibited VLP production. These results suggest that further optimization of DENV VLP expression is possible, but it will require more understanding of how native DENV infection remodels the host cell machinery.

Activity of andrographolide against dengue virus.

Dengue is the most prevalent arthropod-transmitted viral illness of humans, with an estimated 100 million symptomatic infections occurring each year and more than 2.5 billion people living at risk of infection. There are no approved antiviral agents against dengue virus, and there is only limited introduction of a dengue vaccine in some countries. Andrographolide is derived from Andrographis paniculata, a medicinal plant traditionally used to treat a number of conditions including infections. The antiviral activity of andrographolide against dengue virus (DENV) serotype 2 was evaluated in two cell lines (HepG2 and HeLa) while the activity against DENV 4 was evaluated in one cell line (HepG2). Results showed that andrographolide had significant anti-DENV activity in both cell lines, reducing both the levels of cellular infection and virus output, with 50% effective concentrations (EC50) for DENV 2 of 21.304 µM and 22.739 µM for HepG2 and HeLa respectively. Time of addition studies showed that the activity of andrographolide was confined to a post-infection stage. These results suggest that andrographolide has the potential for further development as an anti-viral agent for dengue virus infection.