Picornavirus 2C proteins: structure-function relationships and interactions with host factors.

Picornaviruses, which are positive-stranded, non-enveloped RNA viruses, are known to infect people and animals with a broad spectrum of diseases. Among the nonstructural proteins in picornaviruses, 2C proteins are highly conserved and exhibit multiple structural domains, including amphipathic α-helices, an ATPase structural domain, and a zinc finger structural domain. This review offers a comprehensive overview of the functional structures of picornaviruses' 2C protein. We summarize the mechanisms by which the 2C protein enhances viral replication. 2C protein interacts with various host factors to form the replication complex, ultimately promoting viral replication. We review the mechanisms through which picornaviruses' 2C proteins interact with the NF-κB, RIG-I, MDA5, NOD2, and IFN pathways, contributing to the evasion of the antiviral innate immune response. Additionally, we provide an overview of broad-spectrum antiviral drugs for treating various enterovirus infections, such as guanidine hydrochloride, fluoxetine, and dibucaine derivatives. These drugs may exert their inhibitory effects on viral infections by targeting interactions with 2C proteins. The review underscores the need for further research to elucidate the precise mechanisms of action of 2C proteins and to identify additional host factors for potential therapeutic intervention. Overall, this review contributes to a deeper understanding of picornaviruses and offers insights into the antiviral strategies against these significant viral pathogens.

Structure Prediction and Potential Inhibitors Docking of Enterovirus 2C Proteins.

Human enterovirus infections are mostly asymptomatic and occasionally could be severe and life-threatening. The conserved non-structural 2C from enteroviruses protein is a promising target in antiviral therapies against human enteroviruses. Understanding of 2C-drug interactions is crucial for developing the potential antiviral agents. While functions of enterovirus 2C proteins have been widely studied, three-dimensional structure information of 2C is limited. In this study, the structures of 2C proteins from 20 enteroviruses were simulated and reconstructed using I-TASSER programs. Subsequent docking studies of the known 22 antiviral inhibitors for 2C proteins were performed to uncover the inhibitor-binding characteristics of 2C. Among the potential inhibitors, the compound hydantoin exhibited the highest broad-spectrum antiviral activities with binding to 2C protein. The anti-enteroviral activity of GuaHCL, compound 19b, R523062, compound 12a, compound 12b, quinoline analogs 12a, compound 19d, N6-benzyladenosine, dibucaine derivatives 6i, TBZE-029, fluoxetine analogs 2b, dibucaine, 2-(α-hydroxybenzyl)-benzimidazole (HBB), metrifudil, pirlindole, MRL-1237, quinoline analogs 10a, zuclopenthixol, fluoxetine, fluoxetine HCl, and quinoline analogs 12c showed a trend of gradual decrease. In addition, the free energy with 22 compounds binding to EV 2C ranged from -0.35 to -88.18 kcal/mol. Our in silico studies will provide important information for the development of pan-enterovirus antiviral agents based on 2C.

Palmitoylation of SARS-CoV-2 S protein is critical for S-mediated syncytia formation and virus entry.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the ongoing coronavirus disease 2019 (COVID-19) pandemic. The S protein is the key viral protein for associating with ACE2, the receptor for SARS-CoV-2. There are many kinds of posttranslational modifications in S protein. However, the detailed mechanism of palmitoylation of SARS-CoV-2 S remains to be elucidated. In our current study, we characterized the palmitoylation of SARS-CoV-2 S. Both the C15 and cytoplasmic tail of SARS-CoV-2 S were palmitoylated. Fatty acid synthase inhibitor C75 and zinc finger DHHC domain-containing palmitoyltransferase (ZDHHC) inhibitor 2-BP reduced the palmitoylation of S. Interestingly, palmitoylation of SARS-CoV-2 S was not required for plasma membrane targeting of S but was critical for S-mediated syncytia formation and SARS-CoV-2 pseudovirus particle entry. Overexpression of ZDHHC2, ZDHHC3, ZDHHC4, ZDHHC5, ZDHHC8, ZDHHC9, ZDHHC11, ZDHHC14, ZDHHC16, ZDHHC19, and ZDHHC20 promoted the palmitoylation of S. Furthermore, those ZDHHCs were identified to associate with SARS-CoV-2 S. Our study not only reveals the mechanism of S palmitoylation but also will shed important light into the role of S palmitoylation in syncytia formation and virus entry.

Establishment of Tree Shrew Animal Model for Kaposi's Sarcoma-Associated Herpesvirus (HHV-8) Infection.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the most common cause of Kaposi's sarcoma (KS) and other malignant growths in humans. However, the lack of a KSHV-infected small animal model has hampered understanding of the mechanisms of KSHV infection, virus replication, pathogenesis, and persistence. This study was designed to explore the susceptibility of tree shrews as a possible KSHV-infected small animal model. A recombinant GFP (latent)/RFP (lytic)-positive rKSHV.219 strain was used to infect primary cells cultured from different tissues of tree shrews as an in vitro model and adult tree shrews as an in vivo model. KSHV latent nuclear antigen (LANA) and DNA were successfully detected in primary cells of tree shrews. Among them, tree shrew kidney epithelial cells (TSKEC) were the most susceptible cells to KSHV infection compared to other cells. KSHV genomic DNA, mRNA, and KSHV-specific proteins were readily detected in the TSKEC cultured up to 32 dpi. Moreover, KSHV DNA and mRNA transcription were also readily detected in the peripheral blood mononuclear cells (PBMCs) and various tissues of tree shrews infected with KSHV. Haematoxylin and eosin (HE) staining showed lymphocyte infiltration, lymphoid tissue focal aggregation, alveolar wall thickening, hepatocyte edema, hepatic necrosis in the spleen, lung, and liver of KSHV-infected animals. Additionally, immune-histochemical (IHC) staining showed that LANA or ORF62-positive cells were present in the spleen, lung, liver, and kidney of KSHV-infected tree shrews. Here, we have successfully established in vitro and in vivo KSHV latent infection in tree shrews. This small animal model is not only useful for studying the pathogenesis of KSHV in vivo but can also be a useful model to study transmission routes of viral infection and a useful platform to characterize the novel therapeutics against KSHV.

Recapitulating Zika Virus Infection in Vagina of Tree Shrew (Tupaia belangeri).

Sexual transmission of Zika Virus (ZIKV) elevates the risk of its dissemination in the female reproductive tract and causes a serious threat to the fetus. However, the available animal models are not appropriate to investigate sexual transmission, dynamics of ZIKV infection, replication, and shedding. The use of tree shrew as a small animal model of ZIKV vaginal infection was assessed in this study. A total of 23 sexually mature female tree shrews were infected with ZIKV GZ01 via the intravaginal route. There was no significant difference in change of body weight, and the temperature between ZIKV infected and control animals. Viral RNA loads were detected in blood, saliva, urine, and vaginal douching. ZIKV RNA was readily detected in vaginal lavage of 22 animals (95.65%, 22/23) at 1 dpi, and viral load ranged from 104.46 to 107.35 copies/ml, and the peak of viral load appeared at 1 dpi. The expression of key inflammatory genes, such as IL6, 8, CCL5, TNF-a, and CXCL9, was increased in the spleen of ZIKV infected animals. In the current study, female tree shrews have been successfully infected with ZIKV through the vaginal route for the first time. Interestingly, at first, ZIKV replicates at the local site of infection and then spreads throughout the host body to develop a robust systemic infection and mounted a protective immune response. This small animal model is not only valuable for exploring ZIKV sexual transmission and may also help to explain the cause of debilitating manifestations of the fetus in vivo.

Molecular docking of potential SARS-CoV-2 papain-like protease inhibitors.

SARS-CoV-2 papain-like protease is considered as an important potential target for anti-SARS-CoV-2 drug discovery due to its crucial roles in viral spread and innate immunity. Here, we have utilized an in silico molecular docking approach to identify the possible inhibitors of the SARS-CoV-2 papain-like protease, by screening 21 antiviral, antifungal and anticancer compounds. Among them, Neobavaisoflavone has the highest binding energy for SARS-CoV-2 papain-like protease. These molecules could bind near the SARS-CoV-2 papain-like protease crucial catalytic triad, ubiquitination and ISGylation residues: Trp106, Asn109, Cys111, Met208, Lys232, Pro247, Tyr268, Gln269, His272, Asp286 and Thr301. Because blocking the papain-like protease is an important strategy in fighting against viruses, these compounds might be promising candidates for therapeutic intervention against COVID-19.

Roles of telomeres and telomerase in age­related renal diseases (Review).

Age­related renal diseases, which account for various progressive renal disorders associated with cellular and organismal senescence, are becoming a substantial public health burden. However, their aetiologies are complicated and their pathogeneses remain poorly understood. Telomeres and telomerase are known to be essential for maintaining the integrity and stability of eukaryotic genomes and serve crucial roles in numerous related signalling pathways that activate renal functions, such as repair and regeneration. Previous studies have reported that telomere dysfunction served a role in various types of age­related kidney disease through various different molecular pathways. The present review aimed to summarise the current knowledge of the association between telomeres and ageing­related kidney diseases and explored the contribution of dysfunctional telomeres to these diseases. The findings may help to provide novel strategies for treating patients with renal disease.

Genetic diversity and drug resistance of Mycobacterium tuberculosis in Yunnan, China.

BACKGROUND: China is a country with high burden of tuberculosis (TB), especially drug-resistant TB (DR-TB), which is still a serious health problem in Yunnan Province. Mycobacterium tuberculosis (MTB) is the pathogenic microorganism of TB. The epidemiological characteristics of MTB strains in local areas need to be described. METHODS: A total of 430 clinical MTB isolates were collected from Yunnan Province and genotyped through the method of 24-locus mycobacterial interspersed repetitive unit-variable number tandem DNA repeats (MIRU-VNTR). RESULTS: The genotypes of the 24 loci showed abundantly genetic diversity, and allelic diversity index (h) of these loci varied from 0.012 to 0.817. Among the 430 strains, 30 clusters and 370 unique genotypes were identified. Beijing family was the predominant lineage (70.47%) in Yunnan MTB strains, and the other lineages contained T family (5.81%), MANU2 (0.70%), LAM (3.26%), CAS (0.23%), New-1 (8.37%), and some unknown clades (11.16%). A total of 74 TB strains were identified as drug resistance through drug susceptibility testing (DST), including 38 multidrug-resistant TB (MDR-TB) and 36 single-drug-resistant TB (SDR-TB). The frequency of MDR-TB strains was significantly higher in Beijing family (10.89%) than that in non-Beijing family (3.94%, P = 0.032). CONCLUSIONS: Although MTB strains showed high genetic diversity in Yunnan, China, the Beijing family was still the dominant strain. A high frequency of MDR-TB strains was recorded in the Beijing family.

Infectivity of Zika virus on primary cells support tree shrew as animal model.

Zika virus (ZIKV) is a mosquito-borne flavivirus that caused the public health emergency. Recently, we have proved a novel small animal tree shrew was susceptive to ZIKV infection and presented the most common rash symptoms as ZIKV patients. Here we further cultured the primary cells from different tissues of this animal to determine the tissue tropism of ZIKV infection in vitro. The results showed that the primary cells from tree shrew kidney, lung, liver, skin and aorta were permissive to ZIKV infection and could support viral replication by the detection of viral specific RNA intra- and extra-cells. In comparing, the skin fibroblast and vascular endothelial cells were highly permissive to ZIKV infection with high releasing of active virus particles in supernatants proved by its infectivity in established neonatal mouse model. The expressions of ZIKV envelop and nonstructural protein-1, and the effects and strong immune response of primary tree shrew cells were also detected followed by ZIKV infection. These findings provide powerful in vitro cell-level evidence to support tree shrew as animal model of ZIKV infection and may help to explain the rash manifestations in vivo.

Screening mutations in drug-resistant Mycobacterium tuberculosis strains in Yunnan, China.

Drug-resistant tuberculosis (DR-TB), especially multidrug-resistant tuberculosis (MDR-TB), is a serious medical and societal problem in China. The purpose of this study was to evaluate the mutation characteristics of drug-resistant Mycobacterium tuberculosis (M. tuberculosis) isolates in Yunnan, China. Drug susceptibility testing (DST) was performed in 523 clinical M. tuberculosis isolates. Six drug resistance genes (katG, inhA, rpoB, rpsL, embB, and pncA) were selected to screen for mutations. In total, 54 clinical M. tuberculosis strains were identified as drug-resistant by DST, including 18 single drug-resistant (SDR) strains and 36 multidrug-resistant (MDR) strains. Twenty-four types of mutations in five genes (excluding the inhA gene) were screened in forty-one strains. Six novel mutations were identified in this study, including three missense mutations (p.S302R in katG, p.D78G in embB, and p.M1I in pncA), two frameshift mutations (408 ins A and 538-580 del in pncA), and one mutation in a control region (-6 C>T located upstream of rpsL). The mutation frequencies in the hotspot mutation regions in the katG, rpoB, rpsL, embB, and pncA genes were 92.5%, 44.4%, 54.2%, 52.6%, and 37.5%, respectively. The mutation spectra and frequencies seemed somewhat unique in the Yunnan DR-TB strains.