Roles of ESCRT proteins (ALIX and CHIMP4A) and their interplay with ISG15 during tick-borne flavivirus infection

Flaviviruses are usually transmitted to humans via mosquito or tick bites. During infection, virus replication and assembly, whose cellular sites are relatively close, are controlled by virus proteins and a diverse range of host proteins. By siRNA-mediated gene silencing, we show that ALIX and CHMP4A, two members of the host endosomal sorting complex required for transport (ESCRT) protein machinery, are required for flavivirus infection. Using cell lines expressing subgenomic replicons and replicon virus-like particles, we demonstrate specific roles for ALIX and CHMP4A in viral replication and assembly, respectively. Employing biochemical methodology, we show that the ESCRT proteins are recruited by a putative specific late (L) domain motif LYXLA within the NS3 protein of tick-borne flaviviruses. Furthermore, to counteract the recruitment of ESCRT proteins, the host cells may elicit defense mechanisms. We found that ectopic expression of the interferon-stimulated gene 15 (ISG15) or the E3 ISG15-protein ligase (HERC5) reduced virus replication by suppressing the positive effects of ALIX and CHMP4A. Collectively, these results have provided new insights into flavivirus-host cell interactions that function as checkpoints, including the NS3 and the ESCRT proteins, the ISG15 and the ESCRT protein, at essential stages of the virus life cycle. IMPORTANCE Flaviviruses are important zoonotic viruses with high fatality rates worldwide. Here, we report that during infection the virus employs ESCRT protein members for virus replication and assembly. Among the ESCRT proteins, ALIX acts during virus replication, while CHMP4A is required during virus assembly. Other ESCRT protein members such as TSG101 are not required for virus production. The ESCRT, ALIX -CHMP4A complex, is recruited to NS3 through their interactions with the putative L domain motif of NS3, while CHMP4A is recruited to E. In addition, we demonstrate the antiviral mechanism of ISG15 and HERC5, which degrades ALIX and CHIMP4A, indirectly targets virus infection. In summary, we reveal host-dependency factors supporting flavivirus infection, but these factors may also be targeted by antiviral host effector mechanisms.

Humoral and cellular immunity in mice immunized with whole recombinant yeast expressing complex NS2B/NS3 protein of dengue serotype 3

A nonpathogenic edible yeast, Saccharomyces cerevisiae, has been identified as a vehicle to express many foreign antigens which elicit the immune response in mice. The complex NS2B/NS3 is a protease that represents a prime target for rational drug design for dengue infection. During infection, the NS3 protein is the main target for CD4+ and CD8+ T cell responses, which may be protective. However, no studies have been undertaken evaluating the use of recombinant yeast Saccharomyces cerevisiae INVSc1 expressing complex NSB/NS3 protease as a protective antigen against dengue infection. In the present study, we evaluated the humoral and cellular immune response elicited by recombinant yeast compared to wild-type yeast in the mouse model. Intraperitoneal (i.p.) administration of recombinant and wild-type yeast at 1 and 25 yeast units into BALB/c mice was used. These studies demonstrated that administration at a low concentration of recombinant yeast at 1 yeast units (YU) significantly elicits antibodies against DENV NS3 antigen. Furthermore, real-time PCR analysis revealed that NS2B/NS3-specific cytocines (TNF-a, IFN-©, IL-2) increased with moderate mode compared to wild-type yeast. The results in this study show the potential of recombinant yeast as an edible vaccine platform against dengue infection.

Effects of hepatitis C virus NS3 protein on expression of heat shock protein 70 and Glypican3 as the markers of hepatocellular carcinoma

Background and Aims: Hepatitis C virus (HCV) infection is an important risk factor for the development of liver cancer. The HCV NS3 protein plays a key role in the virus life cycle and can affect normal cellular activities, such as cell proliferation, cell death, and cell signaling pathways. Moreover, it may influence malignancy development. Two cellular genes, heat shock protein 70 (HSP70) and Glypican3 (GPC3), that are assessed in this study, play important roles in the regulation of the cell signaling pathways, including cell proliferation. This study aimed to evaluate the effects of HCV NS3 protein on the expressions of these two genes in the Balb/C mouse model. Materials and Methods: This study was performed on three groups of male mice of Balb/C (n=8). The first group received NS3 plasmid, the second group received hepatitis B virus HBx plasmid, and the negative control group received distilled water. Two injections were administered via the tail vein, and after the last injection, RNA was extracted from the liver tissue. Next, the cDNA synthesis and real-time polymerase chain reaction for relevant genes were performed. Results: Findings revealed that the relative expression of the selected genes in the NS3 group was significant in comparison with the negative control group (P=0.0229 for GPC3 and 0.0020 for HSP70). However, there was no significant difference between the NS3 group and the HBx group (P=0.4516 for GPC3 and 0.6740 for HSP70). Conclusion: Results showed that NS3 protein may affect the increasing expression of the mentioned genes. Nevertheless, for more precise understanding, much more studies should be performed, such as evaluation of the effect of NS3 on other involved proteins in cell signaling pathways, studying other domains of NS3, performance of pathological and histological tests, usage of various experimental methods, assessment of the role of NS4A as a cofactor for NS3, and usage of vectors with more stability.

ZBTB38 in Porcine Alveolar Macrophages Positively Regulates the Proliferation of Japanese Encephalitis Virus

Background Japanese encephalitis (JE) is an important zoonotic disease caused by Japanese encephalitis virus (JEV), and pigs are intermediate host of this disease. Previous studies have confirmed that JEV can proliferate in the respiratory tract of mice and spread through it. Therefore, this study aimed to screen the proteins interacting with JEV on porcine alveolar macrophage cell and verify its role in the proliferation of JEV.Methods and results Porcine alveolar macrophages cell line 3D4/21 were infected with JEV, and obvious cytopathic effect (CPE) was observed. Zinc finger and BTB domain containing 38 (ZBTB38) was screened out as an interacting protein using co-immunoprecipitation assay and validated through knockout and overexpression of ZBTB38 in 3D4/21 cells. The results demonstrated that loss of ZBTB38 function basically had no effect on the attachment and entry processes of JEV, while the transcription level of JEV envelope gene, the expression level of NS3 protein and the number of virions were all significantly down-regulated in the subsequent infection stage. Conclusion Overall, one core conclusion was drawn in this paper that ZBTB38 promotes the proliferation of JEV especially in the middle and late stages of infection. This study provides new information for understanding the pathogenic mechanism of JEV, especially the respiratory transmission caused by JEV infection.

Construction and Characterization of the NS3 Protein of Bluetongue Virus as A Bait for Application in Yeast Two-hybrid System

Background: Bluetongue is an arthropod transmitted viral disease causing range of clinical manifestations in small ruminants. The causative agent, Bluetongue virus (BTV) possesses several structural (SPs) and non-structural proteins (NSPs) associated with its morphogenesis and virulence. NS3, a multifunctional protein of BTV execute a decisive role in virus pathogenesis and release. In the present study, the NS3 protein of BTV was cloned into a pGBKT7-BD vector as bait and characterized for its expression. The suitability of NS3 as a bait to screen interactions with host proteins was also assessed. Methods: The NS3 gene was cloned in the pGBKT7-BD vector to generate the bait plasmid. The recombinant pGBKT7-NS3 bait was sequence confirmed and characterized for auto-activation, toxicity and expression. Thereafter, the bait was mated with yeast two-hybrid (Y2H) cDNA library and screened for the host-BTV protein-protein interactions (PPI). Conclusion: The constructed bait was found suitable for mating, however, no protein hits appeared on forward library screening. The sequence analysis revealed the presence of the transmembrane domains (TM) within the NS3 sequence that may have resulted in the failure of protein interaction studies in Y2H.

ADP-ribosyltransferase PARP11 suppresses Zika virus in synergy with PARP12

Background Zika virus (ZIKV) infection and ZIKV epidemic have been continuously spreading silently throughout the world and its associated microcephaly and other serious congenital neurological complications poses a significant global threat to public health. Type I interferon response to ZIKV infection in host cells suppresses viral replication by inducing the expression of interferon-stimulated genes (ISGs). Methods The study aims to demonstrate the anti-ZIKV mechanism of PARP11. PARP11 knock out and overexpressing A549 cell lines were constructed to evaluate the anti-ZIKV function of PARP11. PARP11−/−, PARP12−/− and PARP11−/−PARP12−/− HEK293T cell lines were constructed to explain the synergistic effect of PARP11 and PARP12 on NS1 and NS3 protein degradation. Western blotting, immunofluorescence and immunoprecipitation assay were performed to illustrate the interaction between PARP11 and PARP12. Results Both mRNA and protein levels of PARP11 were induced in WT but not IFNAR1−/− cells in response to IFNα or IFNβ stimulation and ZIKV infection. ZIKV replication was suppressed in cells expressed PARP11 but was enhanced in PARP11−/− cells. PARP11 suppressed ZIKV independently on itself PARP enzyme activity. PARP11 interacted with PARP12 and promoted PARP12-mediated ZIKV NS1 and NS3 protein degradation. Conclusion We identified ADP-ribosyltransferase PARP11 as an anti-ZIKV ISG and found that it cooperated with PARP12 to enhance ZIKV NS1 and NS3 protein degradation. Our findings have broadened the understanding of the anti-viral function of ADP-ribosyltransferase family members, and provided potential therapeutic targets against viral ZIKV infection.

ADP-Ribosyltransferase PARP11 Suppresses Zika Virus in Synergy with PARP12

Zika virus (ZIKV) infection and ZIKV epidemic have been continuously spreading silently throughout the world and its associated microcephaly and other serious congenital neurological complications poses a significant global threat to public health. ZIKV infection stimulates type I interferon response in host cells which suppresses viral replication by inducing the expression of interferon-stimulated genes (ISGs). Here, we identified ADP-ribosyltransferase PARP11 as an anti-ZIKV ISG and found that PARP11 suppressed ZIKV independently on itself PARP enzyme activity. Furthermore, PARP11 interacted with PARP12 and promoted PARP12-mediating ZIKV NS1 and NS3 protein degradation. Homo family PARP11 and PARP12 cooperated with each other on ZIKV suppression and the anti-ZIKV function of PARP11 mostly dependent on the existence of PARP12. Our findings have broadened the understanding of the anti-viral function of PARP11, and more importantly suggest a potential therapeutics target against ZIKV infection.

HCV Core/NS3 Protein Immunization with “N-Terminal Heat Shock gp96 Protein (rNT (gp96))” Induced Strong and Sustained Th1-Type Cytokines in Immunized Mice

Feeble cellular responses induced by T cell-based vaccines are a major challenge for the development of an effective vaccine against Hepatitis C virus (HCV) infection. To address this challenge, the potential of N-terminal fragment of gp96 heat shock protein (rNT (gp96) as an adjuvant was evaluated and compared to that of the CpG (as a recognized Th1-type adjuvant) in the formulation of HCV core/NS3 antigens in three immunization strategies of protein/protein, DNA/DNA, and DNA/protein. Immunized mice were evaluated for elicited immune responses in week 3 (W3) and 11 post-immunizations. Our results demonstrated that the protein (subunit) vaccine formulated with rNT (gp96) in protein/protein strategy (core/NS3 + gp96) was significantly more efficient than CpG oligodeoxynucleotides (CpG ODN) formulation and all other immunization strategies in the induction of Th1-type cytokines. This group of mice (core/NS3 + gp96) also elicited a high level of anti-Core-NS3 total immunoglobulin G (IgG) with dominant IgG2a isotype at W3. Thus, the co-administration of recombinant NT (gp96) protein with rHCV proteins might be a promising approach in the formulation of HCV subunit vaccine candidates for induction of high levels of Th1 cytokines and humoral responses.