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.

A 2D-proteomic analysis identifies proteins differentially regulated by two different dengue virus serotypes.

The mosquito transmitted dengue virus (DENV) is a major public health problem in many tropical and sub-tropical countries around the world. Both vaccine development and drug development are complex as the species Dengue virus consist of four distinct viruses (DENV 1 to DENV 4) each of which is composed of multiple lineages and strains. To understand the interaction of DENV with the host cell machinery, several studies have undertaken in vitro proteomic analysis of different cell lines infected with DENV. Invariably, these studies have utilized DENV 2. In this study we sought to define proteins that are differentially regulated by two different DENVs, DENV 2 and DENV 4. A 2-dimensional proteomic analysis identified some 300 protein spots, of which only 11 showed differential expression by both DENVs. Of these, only six were coordinately regulated. One protein, prohibitin 1 (PHB1) was downregulated by infection with both DENVs. Overexpression of PHB1 increased DENV protein expression, level of infection and genome copy number. DENV E protein colocalized with PHB, and there was a direct interaction between DENV 2 E protein and PHB1, but not between DENV 4 E protein and PHB1. The low number of proteins showing coordinate regulation after infection by different DENVs is a cause for concern, particularly in determining new druggable targets, and suggests that studies should routinely investigate multiple DENVs.

Glycolysis is reduced in dengue virus 2 infected liver cells.

Infections with dengue virus (DENV) remain a worldwide public health problem. A number of bona fide cellular targets of DENV have been identified including liver cells. Despite the many lines of evidence confirming the involvement of hepatocytes during DENV infection, only a few studies have used proteomic analysis to understand the modulation of the cellular proteome occurring upon DENV infection. We utilized a 2D-gel electrophoresis analysis to identify proteins that were differentially regulated by DENV 2 infection of liver (Hep3B) cells at 12 h post infection (hpi) and at 48 hpi. The analysis identifies 4 proteins differentially expressed at 12 hpi, and 14 differentially regulated at 48 hpi. One candidate protein identified as downregulated at 48 hpi in the proteomic analysis (GAPDH) was validated in western blotting in Hep3B cells, and subsequently in induced pluripotent stem cell (iPSC) derived human hepatocytes. The reduced expression of GAPDH was coupled with an increase in NADH, and a significantly reduced NAD + /NADH ratio, strongly suggesting that glycolysis is down regulated in response to DENV 2 infection. Metformin, a well characterized drug used in the treatment of diabetes mellitus, is an inhibitor of hepatic gluconeogenesis was shown to reduce the level of DENV 2 infection and new virus production. Collectively these results show that although glycolysis is reduced, glucose is still required, possibly for use by the pentose phosphate pathway to generate nucleosides required for viral replication.

Strain Variation Can Significantly Modulate the miRNA Response to Zika Virus Infection.

Zika virus (ZIKV) is a mosquito-transmitted virus that has emerged as a major public health concern due to its association with neurological disorders in humans, including microcephaly in fetuses. ZIKV infection has been shown to alter the miRNA profile in host cells, and these changes can contain elements that are proviral, while others can be antiviral in action. In this study, the expression of 22 miRNAs in human A549 cells infected with two different ZIKV isolates was investigated. All of the investigated miRNAs showed significant changes in expression at at least one time point examined. Markedly, 18 of the miRNAs examined showed statistically significant differences in expression between the two strains examined. Four miRNAs (miR-21, miR-34a, miR-128 and miR-155) were subsequently selected for further investigation. These four miRNAs were shown to modulate antiviral effects against ZIKV, as downregulation of their expression through anti-miRNA oligonucleotides resulted in increased virus production, whereas their overexpression through miRNA mimics reduced virus production. However, statistically significant changes were again seen when comparing the two strains investigated. Lastly, candidate targets of the miRNAs miR-34a and miR-128 were examined at the level of the mRNA and protein. HSP70 was identified as a target of miR-34a, but, again, the effects were strain type-specific. The two ZIKV strains used in this study differ by only nine amino acids, and the results highlight that consideration must be given to strain type variation when examining the roles of miRNAs in ZIKV, and probably other virus infections.

Expression of dengue virus and Zika virus NS2B-NS3pro constructs alter cellular fatty acids, but co-expression with a Zika virus virus-like particle is detrimental to virus-like particle expression.

OBJECTIVE: Studies have shown that Flavivirus infection remodels the host cell to favour viral replication. In particular, the host cell lipid profile is altered, and it has been proposed that this process alters membrane fluidity to allow wrapping of the outer structural proteins around the viral nucleocapsid. We investigated whether expression of the Zika virus (ZIKV) and dengue virus (DENV) protease induced alterations in the cellular lipid profile, and subsequently whether co-expression of these proteases with VLP constructs was able to improve VLP yield. RESULTS: Our results showed that both ZIKV and DENV proteases induced alterations in the lipid profile, but that both active and inactive proteases induced many of the same changes. Neither co-transfection of protease and VLP constructs nor bicistronic vectors allowing expression of both protease and VLP separated by a cell cleavable linker improved VLP yield, and indeed many of the constructs showed significantly reduced VLP production. Further work in developing improved VLP expression platforms is required.

Production of Zika Virus Virus-Like Particles.

Zika virus (ZIKV) is a mosquito-transmitted virus that has caused major outbreaks of disease around the world over the last few years. The infectious ZIKV consists of a structural protein outer shell surrounding a nucleocapsid. Virus-like particles (VLP) consist of the outer structural protein shell, but without the nucleocapsid, and are hence noninfectious. VLP, however, are structurally equivalent to the native virus and thus present a similar antigenic profile. These properties make them good candidates for vaccine development. ZIKV VLP can be generated on a laboratory scale by cloning the relevant structural proteins into a eukaryotic expression vector and transfecting the construct into mammalian cells. The secreted VLP can be harvested from the culture medium and purified by sucrose cushion ultracentrifugation. Validation of the VLP is achieved through western blotting and electron microscopy.

Analysis of the virus propagation profile of 14 dengue virus isolates in Aedes albopictus C6/36 cells.

OBJECTIVE: The mosquito transmitted RNA virus dengue virus (DENV) shows significant variation as a consequence of the lack of proofreading activity of the RNA-dependent RNA polymerase that synthesizes new virus genomes. How this variation affects DENV replication, and how this in turn impacts drug development remains largely unknown. Given the technical limitations in working with large numbers of isolates few studies have sought to investigate this area. This study used a panel of 14 DENV isolates of different serotypes and origins to determine how much virus replication in Aedes albopictus C6/36 cells was affected by DENV variability. RESULTS: The results showed that there was considerable variation, with peak titers ranging from 6Log10 to 8Log10, and maximum titer being reached from day 3 to day 9 post infection. While strains from DENV 1 and 4 serotypes showed considerable uniformity, DENV 2 and 3 strains showed much greater variation. Overall, these results show that serotype specific strain variation can have a significant impact on DENV replication, suggesting that studies either investigating DENV pathogenesis or developing drug therapeutics should consider the contribution of DENV variability.

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.

High correlation between Zika virus NS1 antibodies and neutralizing antibodies in selected serum samples from normal healthy Thais.

Despite the widespread presence of the mosquito transmitted Zika virus (ZIKV) over much of Southeast Asia, the number of reported cases remains low. One possibility is that residents in Southeast Asia are immunologically protected, although the nature of any such protection remains unclear. This study sought to investigate the presence of antibodies directed to ZIKV NS1 protein in a selected sub-set of samples from a well characterized cohort of serum samples from normal, healthy Thais that had been previously characterized for the presence of neutralizing antibodies to ZIKV, DENV 1-4, and JEV. Because of similarities in molecular weight between the flavivirus E and NS1 proteins, an immunoblot system was established in which the NS1 antigen was not denatured, allowing detection of the dimer form of NS1, distinctly clear from the migration position of the E and NS1 monomer proteins. The results showed that antibodies to ZIKV NS1 protein were only detected in samples with ZIKV neutralizing antibodies (27/30 samples), and no sample (0/30) with a ZIKV plaque reduction neutralization test (PRNT)90 < 20 showed evidence of anti-ZIKV NS1 antibodies. The high correlation between the presence of ZIKV NS1 antibodies and ZIKV PRNT suggests that immunological protection against ZIKV infection in Thailand arises from prior exposure to ZIKV, and not through cross neutralization.

Analysis of the Zika and Japanese Encephalitis Virus NS5 Interactomes.

Mosquito-borne flaviviruses, including dengue virus (DENV), Japanese encephalitis virus (JEV), and Zika virus (ZIKV), are major human pathogens. Among the flaviviral proteins, the nonstructural protein 5 (NS5) is the largest, most conserved, and major enzymatic component of the viral replication complex. Disruption of the common key NS5-host protein-protein interactions critical for viral replication could aid in the development of broad-spectrum antiflaviviral therapeutics. Hundreds of NS5 interactors have been identified, but these are mostly DENV-NS5 interactors. To this end, we sought to investigate the JEV- and ZIKV-NS5 interactomes using EGFP immunoprecipitation with label-free quantitative mass spectrometry analysis. We report here a total of 137 NS5 interactors with a significant enrichment of spliceosomal and spliceosomal-associated proteins. The transcription complex Paf1C and phosphatase 6 were identified as common NS5-associated complexes. PAF1 was shown to play opposite roles in JEV and ZIKV infections. Additionally, we validated several NS5 targets and proposed their possible roles in infection. These include lipid-shuttling proteins OSBPL9 and OSBPL11, component of RNAP3 transcription factor TFIIIC, minichromosome maintenance, and cochaperone PAQosome. Mining this data set, our study expands the current interaction landscape of NS5 and uncovers several NS5 targets that are new to flavivirus biology.

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.

Involvement of fatty acid synthase in dengue virus infection.

BACKGROUND: The mosquito transmitted Dengue virus (DENV) remains a significant public health problem in many tropical and subtropical countries. Increasing evidence has suggested that during the infection process cellular lipids play important roles at several stages of the replication cycle. This study sought to characterize the changes in lipid metabolism gene expression and investigated the role of one enzyme, fatty acid synthase, in DENV infection. METHODS: Transcriptional profiles of genes associated with lipid metabolism were evaluated by real-time PCR after infection of different cell lines (HepG2 and HEK293T/17) and with different DENVs (laboratory adapted and low passage). Expression profiles of genes were evaluated by western blotting. A critical lipid metabolism protein, fatty acid synthase was down-regulated through siRNA and inhibited with orlistat and the effect on DENV infection determined by flow cytometry, plaque assay, western blotting and confocal microscopy. RESULTS: The results showed alterations of gene transcription and expression were seen in genes variously associated with lipogenesis, lipolysis and fatty acid ß-oxidation during DENV infection. Interference of fatty acid synthase with either siRNA or orlistat had marked effects on virus production, with orlistat having an EC50 value of 10.07 µM at 24 h post infection. However, non-structural protein expression was largely unaffected. CONCLUSIONS: While drug treatment reduced virus titer by up to 3Log10, no significant effect on DENV non-structural protein expression was observed, suggesting that fatty acid synthase acts through an effect on virion formation.

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.

Activity of andrographolide against chikungunya virus infection.

Chikungunya virus (CHIKV) is a re-emerging mosquito-borne alphavirus that has recently engendered large epidemics around the world. There is no specific antiviral for treatment of patients infected with CHIKV, and development of compounds with significant anti-CHIKV activity that can be further developed to a practical therapy is urgently required. Andrographolide is derived from Andrographis paniculata, a herb traditionally used to treat a number of conditions including infections. This study sought to determine the potential of andrographolide as an inhibitor of CHIKV infection. Andrographolide showed good inhibition of CHIKV infection and reduced virus production by approximately 3log10 with a 50% effective concentration (EC50) of 77 µM without cytotoxicity. Time-of-addition and RNA transfection studies showed that andrographolide affected CHIKV replication and the activity of andrographolide was shown to be cell type independent. This study suggests that andrographolide has the potential to be developed further as an anti-CHIKV therapeutic agent.