ANXA1-derived peptide for targeting PD-L1 degradation inhibits tumor immune evasion in multiple cancers.

BACKGROUND: Immune checkpoint inhibitors (ICIs) therapy targeting programmed cell death 1 (PD-1)/programmed cell death ligand 1 (PD-L1) shows promising clinical benefits. However, the relatively low response rate highlights the need to develop an alternative strategy to target PD-1/PD-L1 immune checkpoint. Our study focuses on the role and mechanism of annexin A1 (ANXA1)-derived peptide A11 degrading PD-L1 and the effect of A11 on tumor immune evasion in multiple cancers. METHODS: Binding of A11 to PD-L1 was identified by biotin pull-down coupled with mass spectrometry analysis. USP7 as PD-L1's deubiquitinase was found by screening a human deubiquitinase cDNA library. The role and mechanism of A11 competing with USP7 to degrade PD-L1 were analyzed. The capability to enhance the T cell-mediated tumor cell killing activity and antitumor effect of A11 via suppressing tumor immune evasion were investigated. The synergistic antitumor effect of A11 and PD-L1 mAb (monoclonal antibody) via suppressing tumor immune evasion were also studied in mice. The expression and clinical significance of USP7 and PD-L1 in cancer tissues were evaluated by immunohistochemistry. RESULTS: A11 decreases PD-L1 protein stability and levels by ubiquitin proteasome pathway in breast cancer, lung cancer and melanoma cells. Mechanistically, A11 competes with PD-L1's deubiquitinase USP7 for binding PD-L1, and then degrades PD-L1 by inhibiting USP7-mediated PD-L1 deubiquitination. Functionally, A11 promotes T cell ability of killing cancer cells in vitro, inhibits tumor immune evasion in mice via increasing the population and activation of CD8+ T cells in tumor microenvironment, and A11 and PD-1 mAb possess synergistic antitumor effect in mice. Moreover, expression levels of both USP7 and PD-L1 are significantly higher in breast cancer, non-small cell lung cancer and skin melanoma tissues than those in their corresponding normal tissues and are positively correlated in cancer tissues, and both proteins for predicting efficacy of PD-1 mAb immunotherapy and patient prognosis are superior to individual protein. CONCLUSION: Our results reveal that A11 competes with USP7 to bind and degrade PD-L1 in cancer cells, A11 exhibits obvious antitumor effects and synergistic antitumor activity with PD-1 mAb via inhibiting tumor immune evasion and A11 can serve as an alternative strategy for ICIs therapy in multiple cancers.

Duck plague virus UL41 protein inhibits RIG-I/MDA5-mediated duck IFN-ß production via mRNA degradation activity.

Retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) are cytosolic pattern recognition receptors that initiate innate antiviral immunity. Recent reports found that duck RLRs significantly restrict duck plague virus (DPV) infection. However, the molecular mechanism by which DPV evades immune responses is unknown. In this study, we first found that the DPV UL41 protein inhibited duck interferon-ß (IFN-ß) production mediated by RIG-I and melanoma differentiation-associated gene 5 (MDA5) by broadly downregulating the mRNA levels of important adaptor molecules, such as RIG-I, MDA5, mitochondrial antiviral signalling protein (MAVS), stimulator of interferon gene (STING), TANK-binding kinase 1 (TBK1), and interferon regulatory factor (IRF) 7. The conserved sites of the UL41 protein, E229, D231, and D232, were responsible for this activity. Furthermore, the DPV CHv-BAC-ΔUL41 mutant virus induced more duck IFN-ß and IFN-stimulated genes (Mx, OASL) production in duck embryo fibroblasts (DEFs) than DPV CHv-BAC parent virus. Our findings provide insights into the molecular mechanism underlying DPV immune evasion.

Emergence of a novel pegivirus species in southwest China showing a high rate of coinfection with parvovirus and circovirus in geese.

Previously, we isolated a novel strain of goose pegivirus (GPgV) that infects geese and shows high levels of lymphotropism. This novel pegivirus strain is phylogenetically distinct from previously known Pegivirus species, Pegivirus A-K, and qualifies as a candidate new Pegivirus species, GPgV. GPgV is tentatively named Pegivirus M. Here, to better understand the epidemic of GPgV infection and the coinfection of this virus with other viruses in Southwest China, 25 geese in poor health from Sichuan Province and 24 geese in poor health from the municipality of Chongqing were collected. The geese were tested for 9 types of goose viruses (goose hemorrhagic polyomavirus, GPgV, astrovirus, parvovirus, circovirus, reovirus, coronavirus, paramyxovirus, and avian influenza virus) by RT-PCR or nested RT-PCR. GPgV RNA was detected in 2 out of 25 monoinfections and 8 out of 25 coinfections with other viruses on Sichuan farms and 2 out of 24 monoinfections and 10 out of 24 coinfections on Chongqing farms. Overall, 22 of the 49 (44.9%) geese were positive for GPgV, which indicated a high infection rate. To the best of our knowledge, this is the first report of GPgV coinfection with other epidemic viruses. This study enhances our understanding of the emergence and epidemiology of Pegivirus.

The role of SOCS proteins in the development of virus- induced hepatocellular carcinoma.

BACKGROUND: Liver cancer has become one of the most common cancers and has a high mortality rate. Hepatocellular carcinoma is one of the most common liver cancers, and its occurrence and development process are associated with chronic hepatitis B virus (HBV) and hepatitis C virus (HCV) infections. Main body The serious consequences of chronic hepatitis virus infections are related to the viral invasion strategy. Furthermore, the viral escape mechanism has evolved during long-term struggles with the host. Studies have increasingly shown that suppressor of cytokine signaling (SOCS) proteins participate in the viral escape process. SOCS proteins play an important role in regulating cytokine signaling, particularly the Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathway. Cytokines stimulate the expression of SOCS proteins, in turn, SOCS proteins inhibit cytokine signaling by blocking the JAK-STAT signaling pathway, thereby achieving homeostasis. By utilizing SOCS proteins, chronic hepatitis virus infection may destroy the host's antiviral responses to achieve persistent infection. CONCLUSIONS: This review provides recent knowledge regarding the role of SOCS proteins during chronic hepatitis virus infection and provides some new ideas for the future treatment of chronic hepatitis.

Duck hepatitis A virus 1 has lymphoid tissue tropism altering the organic immune responses of mature ducks.

Duck hepatitis A virus 1 (DHAV-1) is a highly prevalent pathogen within adult ducks causing acute as well as chronic hepatitis which closely emulates the progression of human hepatitis. However, the underlying mechanisms of DHAV-1 persistence and the pathogenesis of chronic liver disease are not well defined. The association between hematopoietic reservoirs of virus and persistent infection is increasingly concerning. Here, we explored the ability of lymphoid replication of DHAV-1 and the effect on immunity. We found that DHAV-1 was able to infect and replicate productively in the lymphoid organs of model ducks, persisting over 6 months. Moreover, a significant correlation of viral loads between these organs and blood was found, documenting a major contribution of lymphoid replication to DHAV-1 viraemia. Along with viral replication, the mRNA of PRRs and immune-related cytokines was up-regulated in these organs during the early phase of infection, showing tissue-dependent expression patterns but all inclining towards Th2 responses due to the consistently higher level of IL-4 than IL-2 and IFN-γ. Additionally, the expression of CCL19, CCL21, MHC-I and MHC-II, which are involved in T cell homing to the periphery and priming, was dysmodulated. Our data indicate that DHAV-1 possesses lymphoid tissue tropism, contributing to persistent infection and chronic hepatitis via altering the early endogenous transcription of immune-related genes and thereby perturbing organic immunity. These results may be useful to develop novel strategies to treat chronic viral hepatitis based on stimulation of the early innate system and regulation of T-cell trafficking.

Duck enteritis virus pUL47, as a late structural protein localized in the nucleus, mainly depends on residues 40 to 50 and 768 to 777 and inhibits IFN-ß signalling by interacting with STAT1.

Duck enteritis virus (DEV) is a member of the Alphaherpesvirinae subfamily. The characteristics of some DEV genes have been reported. However, information regarding the DEV UL47 gene is limited. In this study, we identified the DEV UL47 gene encoding a late structural protein located in the nucleus of infected cells. We further found that two domains of DEV pUL47, amino acids (aa) 40 to 50 and 768 to 777, could function as nuclear localization sequence (NLS) to guide the nuclear localization of pUL47 and nuclear translocation of heterologous proteins, including enhanced green fluorescent protein (EGFP) and beta-galactosidase (ß-Gal). Moreover, pUL47 significantly inhibited polyriboinosinic:polyribocytidylic acid [poly(I:C)]-induced interferon beta (IFN-ß) production and downregulated interferon-stimulated gene (ISG) expression, such as Mx and oligoadenylate synthetase-like (OASL), by interacting with signal transducer and activator of transcription-1 (STAT1).

Research Note: Duck plague virus glycoprotein I influences cell-cell spread and final envelope acquisition.

To determine the role of glycoprotein I (gI) in duck plague virus (DPV), a gI-deleted mutant (BAC-CHv-ΔgI) and a gI-revertant virus (BAC-CHv-ΔgI Rev) were constructed by using a markerless two-step Red recombination system implemented on the DPV genome cloned into a bacterial artificial chromosome (BAC). Mutants were characterized on duck embryo fibroblast (DEF) cells compared with wild-type virus. BAC-CHv-ΔgI produced viral plaques on DEF cells that were on average approximately 57.2% smaller than those produced by BAC-CHv-ΔgI Rev and wild-type virus. Electron microscopy confirmed that deleting of gI resulted in nucleocapsids accumulated around the cytoplasm vesicles and few of them could complete the final envelopment process. These results clearly indicated that DPV gI plays significant roles in viral cell-cell spread and viral final envelopment process.

Y772 phosphorylation of EphA2 is responsible for EphA2-dependent NPC nasopharyngeal carcinoma growth by Shp2/Erk-1/2 signaling pathway.

EphA2 is an important oncogenic protein and emerging drug target, but the oncogenic role and mechanism of ligand-independent phosphorylation of EphA2 at tyrosine 772 (pY772-EphA2) is unclear. In this study, we established nasopharyngeal carcinoma (NPC) cell lines with stable expression of exogenous EphA2 and EphA2-Y772A (phosphorylation inactivation) using endogenous EphA2-knockdown cells, and observed that pY772A EphA2 was responsible for EphA2-promoting NPC cell proliferation and anchorage-independent and in vivo growth in mice. Mechanistically, EphA2-Y772A mediated EphA2-activating Shp2/Erk-1/2 signaling pathway in the NPC cells, and Gab1 (Grb2-associated binder 1) and Grb2 (growth factor receptor-bound protein 2) were involved in pY772-EphA2 activating this signaling pathway. Our results further showed that Shp2/Erk-1/2 signaling mediated pY772-EphA2-promoting NPC cell proliferation and anchorage-independent growth. Moreover, we observed that EphA2 tyrosine kinase inhibitor ALW-II-41-27 inhibited pY772-EphA2 and EphA2-Y772A decreased the inhibitory effect of ALW-II-41-27 on NPC cell proliferation. Collectively, our results demonstrate that pY772-EphA2 is responsible for EphA2-dependent NPC cell growth in vitro and in vivo by activating Shp2/Erk-1/2 signaling pathway, and is a pharmacologic target of ALW-II-41-27, suggesting that pY772-EphA2 can serve as a therapeutic target in NPC and perhaps in other cancers.

Determinants of duck Tembusu virus NS2A/2B polyprotein procession attenuated viral replication and proliferation in vitro.

Duck Tembusu virus (DTMUV), a mosquito-borne Flavivirus, has caused serious economic losses for the Chinese poultry industry. The genome is translated into a polyprotein that is cleaved to mature protein by host and viral proteases in the host cell, and this proteolytic process is important for the viral life cycle. However, the cleavage mechanism of DTMUV polyprotein is still unclear. In this study, we identified that several amino acids (P1-R, P1'-G, P2-R, P3-T, and P4-V) were vital for NS2A/2B cleavage. Meanwhile, both NS2A and NS2B were essential in cis for polyprotein NS2A/2B intramolecular cleavage. Subsequently, a DTMUV replicon and an infectious clone showed that the P1 site is essential to viral replication, while a mutation in P1' could boost viral RNA replication. Furthermore, a recombinant virus with P1 and P1' site mutations named rDTMUV-NS2A/2B-P1P1'(AA) was rescued from transfected BHK21 cells. The maximum viral titers and viral genome copies of rDTMUV-NS2A/2B-P1P1'(AA) were much lower than those of rDTMUV-WT both in the intracellular and extracellular samples of transfected and infected BHK21 cells. Taken together, the NS2A/2B cleavage sites processed by the NS2B3 protease are vital for DTMUV proliferation and virulence.

Duck IFIT5 differentially regulates Tembusu virus replication and inhibits virus-triggered innate immune response.

Mammalian interferon-induced protein with tetratricopeptide repeats family proteins (IFITs) play important roles in host innate immune response to viruses. Recently, studies have shown that IFIT from poultry also plays a crucial part in antiviral function. This study first reports the regulation of duck Tembusu virus (DTMUV) replication by IFIT5 and the effect of duck IFIT5 (duIFIT5) on the innate immune response after DTMUV infection. Firstly, duIFIT5 was obviously increased in duck embryo fibroblast cells (DEFs) infected with DTMUV. Compared to the negative control, we found that in the duIFIT5-overexpressing group, the DTMUV titer at 24 h post infection (hpi) was significantly reduced, but the viral titer was strikingly increased at 48 hpi. Moreover, overexpression of duIFIT5 could significantly inhibit IFN-ß transcription and IFN-ß promoter activation at indicated time points after DTMUV infection. Further, in DTMUV-infected or poly(I:C)-stimulated DEFs, overexpression of duIFIT5 also significantly inhibited the activation of NF-κB and IRF7 promoters, as well as the activation of downstream IFN induced the interferon-stimulated response element (ISRE) promoter. Meanwhile, the transcription level of antiviral protein Mx, but not OASL, was obviously decreased at various time points. The opposite results were obtained by knockdown of duIFIT5 in DTMUV-infected or poly(I:C)-stimulated DEFs. Compared to the negative control, knockdown of duIFIT5 promoted DTMUV titer and DTMUV envelope (E) protein expression at 24 hpi, but DTMUV titer and E protein expression was markedly decreased at 48 hpi. Additionally, the promoters of IFN-ß, NF-κB, IRF7 and ISRE were significantly activated in the duIFIT5 knockdown group. Collectively, duIFIT5 differentially regulates DTMUV replication and inhibits virus-triggered innate immune response.

Duck Tembusu Virus Utilizes miR-221-3p Expression to Facilitate Viral Replication via Targeting of Suppressor of Cytokine Signaling 5.

Duck Tembusu virus (DTMUV), a member of Flaviviridae family, causes acute egg-drop syndrome in ducks. MicroRNAs (miRNAs) have been found to be involved in various biological processes, including tumor genesis, viral infection, and immune response. However, the functional effect of miRNAs on DTMUV replication remains largely unclear. This study aimed to elucidate the role of host microRNA-221-3p (miR-221-3p) in regulating DTMUV replication. Here, we indicated that the expression of miR-221-3p was significantly upregulated in duck embryo fibroblasts (DEFs) during DTMUV infection. Transfection of miR-221-3p mimic significantly reduced interferon (IFN) ß production, whereas transfection of miR-221-3p inhibitor conversely significantly increased the expression of IFN-ß in DTMUV-infected DEF. Moreover, we found that viral RNA copies, viral E protein expression level, and virus titer, which represent the replication and proliferation of virus, were all enhanced when transfecting the miR-221-3p mimic into DEF; reverse results were also observed by transfecting the miR-221-3p inhibitor. We also found that the expression of suppressor of cytokine signaling 5 (SOCS5) was downregulated in DEF infected with DTMUV. Besides, we further proved that SOCS5 is a target of miR-221-3p and that miR-221-3p could negatively modulate SOCS5 expression at both mRNA and protein levels. Finally, our results showed that overexpression of SOCS5 inhibited DTMUV replication and knockdown of SOCS5 enhanced DTMUV replication. Thus, our findings reveal a novel host evasion mechanism adopted by DTMUV via miR-221-3p, which may hew out novel strategies for designing miRNA-based vaccines and therapies.

Heterologous prime-boost: an important candidate immunization strategy against Tembusu virus.

BACKGROUND: Tembusu virus (TMUV), a newly emerging pathogenic flavivirus, spreads rapidly between ducks, causing massive economic losses in the Chinese duck industry. Vaccination is the most effective method to prevent TMUV. Therefore, it is urgent to look for an effective vaccine strategy against TMUV. Heterologous prime-boost regimens priming with vaccines and boosting with recombinant adenovirus vaccines have been proven to be successful strategies for protecting against viruses in experimental animal models. METHODS: In this study, heterologous and homologous prime-boost strategies using an attenuated salmonella vaccine and a recombinant adenovirus vaccine expressing prM-E or the E gene of TMUV were evaluated to protect ducks against TMUV infection for the first time, including priming and boosting with the attenuated salmonella vaccine, priming and boosting with the recombinant adenovirus vaccine, and priming with the attenuated salmonella vaccine and boosting with the recombinant adenovirus vaccine. Humoral and cellular immune responses were detected and evaluated. We then challenged the ducks with TMUV at 12 days after boosting to assay for clinical symptoms, mortality, viral loads and histopathological lesions after these different strategies. RESULTS: Compared with the homologous prime-boost strategies, the heterologous prime-boost regimen produced higher levels of neutralizing antibodies and IgG antibodies against TMUV. Additionally, it could induce higher levels of IFN-γ than homologous prime-boost strategies in the later stage. Interestingly, the heterologous prime-boost strategy induced higher levels of IL-4 in the early stage, but the IL-4 levels gradually decreased and were even lower than those induced by the homologous prime-boost strategy in the later stage. Moreover, the heterologous prime-boost strategy could efficiently protect ducks, with low viral titres, no clinical symptoms and histopathological lesions in this experiment after challenge with TMUV, while slight clinical symptoms and histopathological lesions were observed with the homologous prime-boost strategies. CONCLUSIONS: Our results indicated that the heterologous prime-boost strategy induced higher levels of humoral and cellular immune responses and better protection against TMUV infection in ducks than the homologous prime-boost strategies, suggesting that the heterologous prime-boost strategy is an important candidate for the design of a novel vaccine strategy against TMUV.

Duck plague virus gE serves essential functions during the virion final envelopment through influence capsids budding into the cytoplasmic vesicles.

Duck plague virus (DPV), a member of the alphaherpesviruses subfamily, causes massive ducks death and results in a devastating hit to duck industries in China. It is of great significance for us to analyze the functions of DPV genes for controlling the outbreak of duck plague. Thus, glycoproteins E (gE) of DPV, which requires viral cell-to-cell spreading and the final envelopment in herpes simplex virus 1 (HSV-1) and pseudorabies virus (PRV), was chosen herein. The gE mutant virus BAC-CHv-ΔgE was constructed by using a markerless two-step Red recombination system implemented on the DPV genome cloned into a bacterial artificial chromosome (BAC). Viral plaques on duck embryo fibroblast (DEF) cells of BAC-CHv-ΔgE were on average approximately 60% smaller than those produced by BAC-CHv virus. Viral replication kinetics showed that BAC-CHv-ΔgE grew to lower titers than BAC-CHv virus did in DEF cells. Electron microscopy confirmed that deleting of DPV gE resulted in a large number of capsids accumulating around vesicles and very few of them could bud into vesicles. The drastic inhibition of virion formation in the absence of the DPV gE indicated that it played an important role in virion morphogenesis before the final envelopment of intracytoplasmic nucleocapsids.

Binding of Duck Tembusu Virus Nonstructural Protein 2A to Duck STING Disrupts Induction of Its Signal Transduction Cascade To Inhibit Beta Interferon Induction.

Duck Tembusu virus (DTMUV), which is similar to other mosquito-borne flaviviruses that replicate well in most mammalian cells, is an emerging pathogenic flavivirus that has caused epidemics in egg-laying and breeding waterfowl. Immune organ defects and neurological dysfunction are the main clinical symptoms of DTMUV infection. Preinfection with DTMUV makes the virus impervious to later interferon (IFN) treatment, revealing that DTMUV has evolved some strategies to defend against host IFN-dependent antiviral responses. Immune inhibition was further confirmed by screening for DTMUV-encoded proteins, which suggested that NS2A significantly inhibited IFN-ß and IFN-stimulated response element (ISRE) promoter activity in a dose-dependent manner and facilitated reinfection with duck plague virus (DPV). DTMUV NS2A was able to inhibit duck retinoic acid-inducible gene-I (RIG-I)-, and melanoma differentiation-associated gene 5 (MDA5)-, mitochondrial-localized adaptor molecules (MAVS)-, stimulator of interferon genes (STING)-, and TANK-binding kinase 1 (TBK1)-induced IFN-ß transcription, but not duck TBK1- and interferon regulatory factor 7 (IRF7)-mediated effective phases of IFN response. Furthermore, we found that NS2A competed with duTBK1 in binding to duck STING (duSTING), impaired duSTING-duSTING binding, and reduced duTBK1 phosphorylation, leading to the subsequent inhibition of IFN production. Importantly, we first identified that the W164A, Y167A, and S361A mutations in duSTING significantly impaired the NS2A-duSTING interaction, which is important for NS2A-induced IFN-ß inhibition. Hence, our data demonstrated that DTMUV NS2A disrupts duSTING-dependent antiviral cellular defenses by binding with duSTING, which provides a novel mechanism by which DTMUV subverts host innate immune responses. The potential interaction sites between NS2A and duSTING may be the targets of future novel antiviral therapies and vaccine development.IMPORTANCE Flavivirus infections are transmitted through mosquitos or ticks and lead to significant morbidity and mortality worldwide with a spectrum of manifestations. Infection with an emerging flavivirus, DTMUV, manifests with clinical symptoms that include lesions of the immune organs and neurological dysfunction, leading to heavy egg drop and causing serious harm to the duck industry in China, Thailand, Malaysia, and other Southeast Asian countries. Mosquito cells, bird cells, and mammalian cell lines are all susceptible to DTMUV infection. An in vivo study revealed that BALB/c mice and Kunming mice were susceptible to DTMUV after intracerebral inoculation. Moreover, there are no reports about DTMUV-related human disease, but antibodies against DTMUV and viral RNA were detected in serum samples of duck industry workers. This information implies that DTMUV has expanded its host range and may pose a threat to mammalian health. However, the pathogenesis of DTMUV is largely unclear. Our results show that NS2A strongly blocks the STING-induced signal transduction cascade by binding with STING, which subsequently blocks STING-STING binding and TBK1 phosphorylation. More importantly, the W164, Y167, or S361 residues in duSTING were identified as important interaction sites between STING and NS2A that are vital for NS2A-induced IFN production and effective phases of IFN response. Uncovering the mechanism by which DTMUV NS2A inhibits IFN in the cells of its natural hosts, ducks, will help us understand the role of NS2A in DTMUV pathogenicity.

DEF Cell-Derived Exosomal miR-148a-5p Promotes DTMUV Replication by Negative Regulating TLR3 Expression.

Duck tembusu virus (DTMUV) is a single-stranded, positive-polarity RNA flavivirus that has caused considerable economic losses in China in recent years. Innate immunity represents the first line of defense against invading pathogens and serves as an important role in resisting viral infections. In this study, we found that the infection of ducks by DTMUV triggers Toll-like receptors (TLRs) and (RIG-I)-like receptors (RLRs) signaling pathways and inducing abundant of pro-inflammatory factors and type I interferons (IFNs), in which melanoma differentiation-associated gene 5 (MDA5) and Toll-like receptor 3 (TLR3) play important immunity roles, they can inhibit the replication process of DTMUV via inducing type I IFNs. Moreover, we demonstrated that type I IFNs can inhibit the DTMUV replication process in a time- and dose-dependent manner. Exosomes are small membrane vesicles that have important roles in intercellular communication. MicroRNAs (miRNAs) are small non-coding RNAs that can modulate gene expression and are common substances in exosomes. In our experiment, we successfully isolated DEF cells derived exosome for the first time and explored its function. Firstly, we found the expression of miR-148a-5p is significantly decreased following DTMUV infect. Then we found miR-148a-5p can target TLR3 and down-regulate the expression of TLR3, serving as a negative factor in innate immunity. Unfortunately, we cannot find miRNAs with different expression changes that can target MDA5. Lastly, our experimental results showed that TLR3 was one of the causes of miR-148a-5p reduction, suggesting that the high level of TLR3 after DTMUV infect can both trigger innate immunity and suppress miR-148a-5p to resist DTMUV.

Innate Immune Evasion of Alphaherpesvirus Tegument Proteins.

Alphaherpesviruses are a large family of highly successful human and animal DNA viruses that can establish lifelong latent infection in neurons. All alphaherpesviruses have a protein-rich layer called the tegument that, connects the DNA-containing capsid to the envelope. Tegument proteins have a variety of functions, playing roles in viral entry, secondary envelopment, viral capsid nuclear transportation during infection, and immune evasion. Recently, many studies have made substantial breakthroughs in characterizing the innate immune evasion of tegument proteins. A wide range of antiviral tegument protein factors that control incoming infectious pathogens are induced by the type I interferon (IFN) signaling pathway and other innate immune responses. In this review, we discuss the immune evasion of tegument proteins with a focus on herpes simplex virus type I.

Therapeutic effects of duck Tembusu virus capsid protein fused with staphylococcal nuclease protein to target Tembusu infection in vitro.

Tembusu virus (TMUV), a member of the genus flavivirus, primarily causes egg-drop syndrome in ducks and is associated with low disease mortality but high morbidity. The commercially available live vaccines for treating TMUV currently include the main WF100, HB, and FX2010-180P strains, and efficient treatment and/or preventative measures are still urgently needed. Capsid-targeted viral inactivation (CTVI) is a conceptually powerful new antiviral strategy that is based on two proteins from the capsid protein of a virus and a crucial effector molecule. The effector molecule can destroy the viral DNA/RNA or interfere with the proper folding of key viral proteins, while the capsid protein mainly plays a role in viral integration and assembly; the fusion proteins are incorporated into virions during packaging. This study aimed to explore the potential use of this strategy in duck TMUV. Our results revealed that these fusion proteins can be expressed in susceptible BHK21 cells without cytotoxicity and possess excellent Ca2+-dependent nuclease activity, and their expression is also detectable in DF-1 cells. Compared to those in the negative controls (BHK21 and BHK21/pcDNA3.1(+) cells), the numbers of viral RNA copies in TMUV-infected BHK21/Cap-SNase and BHK21/Cap-Linker-SNase cells were reduced by 48 h, and the effect of Cap-Linker-SNase was superior to that of Cap-SNase. As anticipated, these results suggest that these fusion proteins contribute to viral resistance to treatment. Thus, CTVI might be applicable for TMUV inhibition as a novel antiviral therapeutic candidate during viral infection.

Biochemical characterization of recombinant Avihepatovirus 3C protease and its localization.

BACKGROUND: The picornaviral 3C protease mediates viral polyprotein maturation and multiple cleavages of host proteins to modulate viral translation and transcription. The 3C protease has been regarded as a valid target due to its structural similarity among different picornaviruses and minimal sequence similarity with host proteins; therefore, the development of potent inhibitors against the 3C protease as an antiviral drug is ongoing. Duck hepatitis A virus (DHAV) belongs to the Picornavidea family and is a major threat to the poultry industry. To date, little is known about the roles of the DHAV 3C protease plays during infection. METHODS: In this study, we compared the full-length DHAV 3C protein sequence with other 3C sequences to obtain an alignment for the construction of a phylogenetic tree. Then, we expressed and purified recombinant DHAV 3C protease in the BL21 expression system using nickel-NTA affinity chromatography. The optimization of the cleavage assay conditions and the kinetic analysis for DHAV 3C protease were done by in vitro cleavage assays with a fluorogenic peptide respectively. The inhibitory activity of rupintrivir against the DHAV 3C protease was further evaluated. The localization of the 3C protease in infected and transfected cells was determined using immunofluorescence and confocal microscopy. RESULTS: Under different expression conditions, the 3C protease was found to be highly expressed after induction with 1 mM IPTG at 16 °C for 10 h. We synthesized a fluorogenic peptide derived from the cleavage site of the DHAV polyprotein and evaluated the protease activity of the DHAV 3C protease for the first time. We used fluorimetric based kinetic analysis to determine kinetic parameters, and Vmax and Km values were determined to be 16.52 nmol/min and 50.78 µM, respectively. Rupintrivir was found to exhibit inhibitory activity against the DHAV 3C protease. Using polyclonal antibody and an indirect immunofluorescence microscopy assay (IFA), it was determined that the DHAV 3C protease was found in the nucleus during infection. In addition, the DHAV 3C protease can enter into the nucleus without the cooperation of viral proteins. CONCLUSIONS: This is the first study to examine the activity of the DHAV 3C protease, and the activity of the DHAV 3C protease is temperature-, pH- and NaCl concentration- dependent. The DHAV 3C protease localizes throughout DHAV-infected cells and can enter into the nucleus in the absence of other viral proteins. The kinetic analysis was calculated, and the Vmax and Km values were 16.52 nmol/min and 50.78 µM, respectively, using the Lineweaver-Burk plot.

Alpha-Herpesvirus Thymidine Kinase Genes Mediate Viral Virulence and Are Potential Therapeutic Targets.

Alpha-herpesvirus thymidine kinase (TK) genes are virulence-related genes and are nonessential for viral replication; they are often preferred target genes for the construction of gene-deleted attenuated vaccines and genetically engineered vectors for inserting and expressing foreign genes. The enzymes encoded by TK genes are key kinases in the nucleoside salvage pathway and have significant substrate diversity, especially the herpes simplex virus 1 (HSV-1) TK enzyme, which phosphorylates four nucleosides and various nucleoside analogues. Hence, the HSV-1 TK gene is exploited for the treatment of viral infections, as a suicide gene in antitumor therapy, and even for the regulation of stem cell transplantation and treatment of parasitic infection. This review introduces the effects of α-herpesvirus TK genes on viral virulence and infection in the host and classifies and summarizes the current main application domains and potential uses of these genes. In particular, mechanisms of action, clinical limitations, and antiviral and antitumor therapy development strategies are discussed.