Characterising Non-Structural Protein NS4 of African Horse Sickness Virus

African horse sickness is a serious equid disease caused by the orbivirus African horse sickness virus (AHSV). The virus has ten double-stranded RNA genome segments encoding seven structural and three non-structural proteins. Recently, an additional protein was predicted to be encoded by genome segment 9 (Seg-9), which also encodes VP6, of most orbiviruses. This has since been confirmed in bluetongue virus and Great Island virus, and the non-structural protein was named NS4. In this study, in silico analysis of AHSV Seg-9 sequences revealed the existence of two main types of AHSV NS4, designated NS4-I and NS4-II, with different lengths and amino acid sequences. The AHSV NS4 coding sequences were in the +1 reading frame relative to that of VP6. Both types of AHSV NS4 were expressed in cultured mammalian cells, with sizes close to the predicted 17–20 kDa. Fluorescence microscopy of these cells revealed a dual cytoplasmic and nuclear, but not nucleolar, distribution that was very similar for NS4-I and NS4-II. Immunohistochemistry on heart, spleen, and lung tissues from AHSV-infected horses showed that NS4 occurs in microvascular endothelial cells and mononuclear phagocytes in all of these tissues, localising to the both the cytoplasm and the nucleus. Interestingly, NS4 was also detected in stellate-shaped dendritic macrophage-like cells with long cytoplasmic processes in the red pulp of the spleen. Finally, nucleic acid protection assays using bacterially expressed recombinant AHSV NS4 showed that both types of AHSV NS4 bind dsDNA, but not dsRNA. Further studies will be required to determine the exact function of AHSV NS4 during viral replication.     

Viroporin Activity of the Foot-and-Mouth Disease Virus Non-Structural 2B Protein

Viroporins are a family of low-molecular-weight hydrophobic transmembrane proteins that are encoded by various animal viruses. Viroporins form transmembrane pores in host cells via oligomerization, thereby destroying cellular homeostasis and inducing cytopathy for virus replication and virion release. Among the Picornaviridae family of viruses, the 2B protein encoded by enteroviruses is well understood, whereas the viroporin activity of the 2B protein encoded by the foot-and-mouth disease virus (FMDV) has not yet been described. An analysis of the FMDV 2B protein domains by computer-aided programs conducted in this study revealed that this protein may contain two transmembrane regions. Further biochemical, biophysical and functional studies revealed that the protein possesses a number of features typical of a viroporin when it is overexpressed in bacterial and mammalian cells as well as in FMDV-infected cells. The protein was found to be mainly localized in the endoplasmic reticulum (ER), with both the N- and C-terminal domains stretched into the cytosol. It exhibited cytotoxicity in Escherichia coli, which attenuated 2B protein expression. The release of virions from cells infected with FMDV was inhibited by amantadine, a viroporin inhibitor. The 2B protein monomers interacted with each other to form both intracellular and extracellular oligomers. The Ca²⁺ concentration in the cells increased, and the integrity of the cytoplasmic membrane was disrupted in cells that expressed the 2B protein. Moreover, the 2B protein induced intense autophagy in host cells. All of the results of this study demonstrate that the FMDV 2B protein has properties that are also found in other viroporins and may be involved in the infection mechanism of FMDV.     

利用植物生产西尼罗河病毒抗体

西尼罗河病毒(West Nile vires,WNV)通常是由鸟类携带,经蚊子传播给人类.病毒感染者主要有发烧、肌肉疼痛等类似感冒的症状,其中有小部分人会出现脑炎和脊髓炎.常见于美国的48个州以及北、南美的温带区域.大多数人在感染病毒后会出现头疼等类似感冒的症状,但老人、慢性病患者和免疫力低的人会并发脑炎甚至导致死亡.     

Inhibition of West Nile Virus by Calbindin-D28k

Evidence indicates that West Nile virus (WNV) employs Ca²⁺ influx for its replication. Moreover, calcium buffer proteins, such as calbindin D28k (CB-D28k), may play an important role mitigating cellular destruction due to disease processes, and more specifically, in some neurological diseases. We addressed the hypothesis that CB-D28k inhibits WNV replication in cell culture and infected rodents. WNV envelope immunoreactivity (ir) was not readily co-localized with CB-D28k ir in WNV-infected Vero 76 or motor neuron-like NSC34 cells that were either stably or transiently transfected with plasmids coding for CB-D28k gene. This was confirmed in cultured cells fixed on glass coverslips and by flow cytometry. Moreover, WNV infectious titers were reduced in CB-D28k-transfected cells. As in cell culture studies, WNV env ir was not co-localized with CB-D28k ir in the cortex of an infected WNV hamster, or in the hippocampus of an infected mouse. Motor neurons in the spinal cord typically do not express CB-D28k and are susceptible to WNV infection. Yet, CB-D28k was detected in the surviving motor neurons after the initial phase of WNV infection in hamsters. These data suggested that induction of CB-D28k elicit a neuroprotective response to WNV infection.     

西尼罗病毒研究进展

西尼罗病毒 (West Nile virus,WNV) 首先在1937年乌干达的西尼罗地区的Omogo镇的一位发热病人血液中分离到,从而得名[1].西尼罗病毒属于黄病毒科黄病毒属.     

Spatial spreading of West Nile Virus described by traveling waves

In this work, we propose a spatial model to analyze the West Nile Virus propagation across the USA, from east to west. West Nile Virus is an arthropod-borne flavivirus that appeared for the first time in New York City in the summer of 1999 and then spread prolifically among birds. Mammals, such as humans and horses, do not develop sufficiently high bloodstream titers to play a significant role in the transmission, which is the reason to consider the mosquito-bird cycle. The model aims to study this propagation based on a system of partial differential reaction-diffusion equations taking the mosquito and the avian populations into account. Diffusion and advection movements are allowed for both populations, being greater in the avian than in the mosquito population. The traveling wave solutions of the model are studied to determine the speed of disease dissemination. This wave speed is obtained as a function of the model's parameters, in order to assess the control strategies. The propagation of West Nile Virus from New York City to California state is established as a consequence of the diffusion and advection movements of birds. Mosquito movements do not play an important role in the disease dissemination, while bird advection becomes an important factor for lower mosquito biting rates.     

Modelling the dynamics of West Nile Virus

In this work we formulate and analyze a mathematical model for the transmission of West Nile Virus (WNV) infection between vector (mosquito) and avian population. We find the Basic Reproductive Number in terms of measurable epidemiological and demographic parameters. is the threshold condition that determines the dynamics of WNV infection: if the disease fades out, and for the disease remains endemic. Using experimental and field data we estimate for several species of birds. Numerical simulations of the temporal course of the infected bird proportion show damped oscillations approaching the endemic value.     

MKRN1 Induces Degradation of West Nile Virus Capsid Protein by Functioning as an E3 Ligase

West Nile virus capsid protein (WNVCp) displays pathogenic toxicity via the apoptotic pathway. However, a cellular mechanism protective against this toxic effect has not been observed so far. Here, we identified Makorin ring finger protein 1 (MKRN1) as a novel E3 ubiquitin ligase for WNVCp. The cytotoxic effects of WNVCp as well as its expression levels were inhibited in U2OS cells that stably expressed MKRN1. Immunoprecipitation analyses revealed an interaction between MKRN1 and WNVCp. Domain analysis indicated that the C terminus of MKRN1 and the N terminus of WNVCp were required for the interaction. MKRN1 could induce WNVCp ubiquitination and degradation in a proteasome-dependent manner. Interestingly, the WNVCp mutant with amino acids 1 to 105 deleted WNVCp was degraded by MKRN1, whereas the mutant with amino acids 1 to 90 deleted was not. When three lysine sites at positions 101, 103, and 104 of WNVCp were replaced with alanine, MKRN1-mediated ubiquitination and degradation of the mutant were significantly inhibited, suggesting that these sites are required for the ubiquitination. Finally, U2OS cell lines stably expressing MKRN1 were resistant to cytotoxic effects of WNV. In contrast, cells depleted of MKRN1 were more susceptible to WNVCp cytotoxicity. Confirming this, overexpression of MKRN1 significantly reduced, but depletion of MKRN1 increased, WNV proliferation in 293T cells. Taken together, our results suggest that MKRN1 can protect cells from WNV by inducing WNVCp degradation.West Nile virus (WNV) is an arthropod-borne virus that is a member of the Flaviviridae family, which includes St. Louis encephalitis virus, Kunjin virus, yellow fever virus, dengue virus, and Murray Valley encephalitis virus (2). Since its first identification in the West Nile province of Uganda in 1937, WNV has spread quickly through Asia, Europe, and the United States and has caused a serious global health problem (34). The clinical manifestations of WNV usually entail neurological diseases such as meningitis and encephalitis. This might be caused by WNV genome replication after inoculation and its subsequent spread to lymph nodes and blood, followed by its entrance into the central nervous system through Toll-like receptor and tumor necrosis factor receptor (40).WNV has the genome of a single positive-sense RNA containing one open reading frame. The encoded polypeptide is processed further by viral and cellular proteases into several nonstructural and structural proteins (2). Nonstructural (NS) proteins include NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5. NS1 is involved in synthesis of viral RNA, and NS3 mediates the cleavage of nonstructural proteins (22, 25, 30, 48). NS5 functions as an RNA polymerase and methyltransferase, which are required for viral replication (14, 17, 18). NS2A, NS2B, NS4A, and NS4B promote the organization of viral replication factors and membrane permeabilization (3, 5, 6, 13, 37). The capsid, envelope (E), and premembrane (prM) proteins are the structural proteins, which are involved in virus assembly (43). E protein is a virion surface protein that regulates binding and fusion to the cell membrane (1, 11, 32). The prM protein is a precursor of the M protein, which is translocated to the endoplasmic reticulum (ER) by capsid (2, 21). Viral assembly occurs mainly in the ER membrane following release of viral particles (23).The capsid of WNV (WNVCp) localizes and is involved in nucleocapsid assembly on the ER membrane (15). However, extra roles of the flavivirus capsid in the nucleus has been reported. For example, capsid proteins of Japanese encephalitis virus (JEV) and hepatitis C virus (HCV), which are also members of the Flaviviridae family, participate in pathogenesis by localizing to the nucleus (33). Nucleolar and nuclear WNVCp is involved in pathogenesis via induction of the apoptotic process in cells through interaction with Hdm2, which results in the activation of the potent tumor suppressor p53 (47). It also induces apoptotic death of neuron cells via mitochondrial dysfunction and activation of caspase pathways when introduced into the brains of mice (46).The Makorin ring finger protein 1 (MKRN1) gene was first reported as the source gene of introns for the intronless imprinted MKRN gene family (10). The protein is an ancient protein conserved from invertebrates to vertebrates, and it contains several zinc finger motifs, including C3H, C3HC4, and unique Cys-His motifs (10). Furthermore, this gene is constitutively expressed in most human tissues, including neurons (10). The role of MKRN1 as an E3 ligase was first identified by its ability to degrade hTERT (16). Interestingly, MKRN1 functions as a coregulator of androgen and retinoic acid receptor (27), suggesting possible diverse roles of MKRN1 in human cells.In this study, we report on an ubiquitin (Ub) E3-ligase for WNVCp. MKRN1 was able to ubiquitinate and degrade WNVCp in a proteasome-dependent manner. Furthermore, degradation of WNVCp resulted in a reduction of WNV-induced cell death. Cells stably overexpressing MKRN1 were resistant to WNV-induced cell death. In contrast, ablation of MKRN1 by small interfering RNA (siRNA) renders cells more susceptible to the cytotoxicity of WNVCp. Furthermore, WNV proliferation was suppressed in 293T cells overexpressing MKRN1 but increased in MKRN1-depleted 293T cells. Based on these data, we suggest that MKRN1 might play a role in protection of cells against WNV infection.     

Mosaic RNA Phage VLPs Carrying Domain III of the West Nile Virus E Protein

The virus-neutralising domain III (DIII) of the West Nile virus glycoprotein E was exposed on the surface of RNA phage AP205 virus-like particles (VLPs) in mosaic form. For this purpose, a 111 amino acid sequence of DIII was added via amber or opal termination codons to the C-terminus of the AP205 coat protein, and mosaic AP205-DIII VLPs were generated by cultivation in amber- or opal-suppressing Escherichia coli strains. After extensive purification to 95 % homogeneity, mosaic AP205-DIII VLPs retained up to 11–16 % monomers carrying DIII domains. The DIII domains appeared on the VLP surface because they were fully accessible to anti-DIII antibodies. Immunisation of BALB/c mice with AP205-DIII VLPs resulted in the induction of specific anti-DIII antibodies, of which the level was comparable to that of the anti-AP205 antibodies generated against the VLP carrier. The AP205-DIII-induced anti-DIII response was represented by a significant fraction of IgG2 isotype antibodies, in contrast to parallel immunisation with the DIII oligopeptide, which failed to induce IgG2 isotype antibodies. Formulation of AP-205-DIII VLPs in alum adjuvant stimulated the level of the anti-DIII response, but did not alter the fraction of IgG2 isotype antibodies. Mosaic AP205-DIII VLPs could be regarded as a promising prototype of a putative West Nile vaccine.     

Comparison of the Neuropathology Induced by Two West Nile Virus Strains

Some strains of West Nile virus (WNV) are neuroinvasive and may induce fatal encephalitis/meningitis in a variety of animal species including humans. Whether, however, there is a strain-specific signature in the brain is as yet unknown. Here we investigated the neuropathogenesis induced by two phylogenetically distant WNV strains of lineage 1, WNV_IS98 and WNV_KUN35 911. While four-week old C57Bl/6J mice were susceptible to both strains and succumbed rapidly after intraperitoneal inoculation, differences were observed in virulence and clinical disease. WNV_KUN35 911, the less virulent strain as judged by determination of LD₅₀, induced typical signs of encephalitis. Such signs were not observed in WNV_IS98-infected mice, although they died more rapidly. Histological examination of brain sections also revealed differences, as the level of apoptosis and inflammation was higher in WNV_KUN35 911- than WNV_IS98-infected mice. Moreover, staining for cleaved caspase 3 showed that the two WNV strains induced apoptotic death through different molecular mechanisms in one particular brain area. Finally, the two strains showed similar tropism in cortex, striatum, brainstem, and cerebellum but a different one in hippocampus. In summary, our data show that, upon peripheral administration, WNV_IS98 and WNV_KUN35 911 strains induce partially distinct lesions and tissue tropism in the brain. They suggest that the virulence of a WNV strain is not necessarily correlated with the severity of apoptotic and inflammatory lesions in the brain.     

The Roles of Endoplasmic Reticulum Overload Response Induced by HCV and NS4B Protein in Human Hepatocyte Viability and Virus Replication

Hepatitis C virus (HCV) replication is associated with endoplasmic reticulum (ER) and its infection triggers ER stress. In response to ER stress, ER overload response (EOR) can be activated, which involves the release of Ca²⁺ from ER, production of reactive oxygen species (ROS) and activation of nuclear factor κB (NF-κB). We have previously reported that HCV NS4B expression activates NF-κB via EOR-Ca²⁺-ROS pathway. Here, we showed that NS4B expression and HCV infection activated cancer-related NF-κB signaling pathway and induced the expression of cancer-related NF-κB target genes via EOR-Ca²⁺-ROS pathway. Moreover, we found that HCV-activated EOR-Ca²⁺-ROS pathway had profound effects on host cell viability and HCV replication. HCV infection induced human hepatocyte death by EOR-Ca²⁺-ROS pathway, whereas activation of EOR-Ca²⁺-ROS-NF-κB pathway increased the cell viability. Meanwhile, EOR-Ca²⁺-ROS-NF-κB pathway inhibited acute HCV replication, which could alleviate the detrimental effect of HCV on cell viability and enhance chronic HCV infection. Together, our findings provide new insights into the functions of EOR-Ca²⁺-ROS-NF-κB pathway in natural HCV replication and pathogenesis.