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Coronaviruses are a large family of important pathogens that cause human and animal diseases. At the end of 2019, a pneumonia epidemic caused by a novel coronavirus brought attention to coronaviruses. Exploring the interaction between the virus and its receptor will be helpful in developing preventive vaccines and therapeutic drugs. The coronavirus spike protein (S) plays an important role in both binding to receptors on host cells and fusion of the viral membrane with the host cell membrane. This review introduces the structure and function of the S protein and its receptor, focusing on the binding mode and binding region of both.

Residues 221–239 of rubella virus E1 glycoprotein contain antibody neutralization domains, and the solvent-exposed charged amino acids at the binding interface may be crucial for binding ability. However, the role of charged amino acid residues on the E1 epitope in peptide–antibody binding is unknown. To investigate the role of single amino acid substitutions on the important neutralizing epitope, biolayer interferometry and serological tests were performed. There are three charged residues in the neutralizing epitope: D229, R237, and H238. Substitution of D229 for amino acid A had no influence on the binding activity of the antibody to the peptide. However, substitutions of R237 or H238 for charged amino acid H or R were found to abolish the binding activity. Furthermore, substitution of an uncharged amino acid Q236 for a charged amino acid D was found to reduce the binding activity significantly. Thus, R237 and H238 are key amino acids in the rubella virus E1 neutralization epitope.

Enterovirus 71 is a neuroinvasive virus that is associated with severe neurological complications. We had earlier suggested that the replication capacity of a severe strain was higher than that of a mild strain. The recombinant 3CRV and 3CDRV virus strains were successfully rescued in our previous study. In the present study, we found no difference in virulence between 3CRV and severe strains. However, the capacity of replication and to cause cell injury of 3CDRV strain decreased in vitro, especially at 39.5 °C. Replacement of 3CD region in the severe strain led to milder symptoms, less body weight loss, and lower viral load in ICR mice. Histopathological findings indicated less severe injury in mice infected with 3CDRV strain. This study suggests that the 3CD region contributes to the attenuation of the severe strain, including its replication capacity and temperature sensitivity.

To express the rubella virus E1-374 glycoprotein in Pichia pastoris and study the immunogenecity of the recombinant protein.The cDNA of protein E1-374 was cloned into the expression vector pGAPZalphaA and transformed into Pichia pastoris GS115 cells by electrotransfection. The expressed protein was confirmed by indirect immunofluorescence and demonstrated immunoreactivity by Western Blot. Rubella virus IgG antibody was assayed with ELISA after mice were inmmunized by E1-374 glycoprotein.SDS-PAGE analysis and Western Blot analysis of E1-374 protein revealed this protein to be 46.89 x 10(3). Antiserum (1:100) and E1-374 (5.5 microg/ml) was chosen for ELISA optimization. The intra-assay coefficient of variation for the ELISA was 0.36%-12.45%.Protein E1-374 was highly expressed in Pichia pastoris cells, and it was a good choice to prepare rubella virus recombinant protein vaccines.

To determine the functions of N-carbohydrate chains in human parainfluenza virus type 3 hemagglutinin-neuraminidase(HN) protein, a PCR-based site-directed mutagenesis method was used to obtain N-glycan mutants. Protein electrophoresis rate, cell surface expression,receptor binding activity, neuraminidase activity and cell fusion promotion activity were determined. The HN proteins of single mutants (G1, G2, and G4) and multiple mutants (G12, G14, G24 and G124) migrated faster than the wild-type (wt) HN protein on polyacrylamide gels, while G3-mutated protein and wt HN protein migrated at the same position. There was no statistic difference in cell surface expression and neuraminidase activity between wt and each mutant HN protein (P0.05), but receptor binding activity and cell fusion promotion activity of each mutant protein was reduced to significant extent (P0.05). G1, G2 and G4 mutants exhibited re duced receptor binding activity, which was 83.94%, 76.45% and 55.32% of the wt level, respectively. G1, G2 and G4-mutated proteins also showed reductions in fusion promotion activity, which was 80.84%, 77.83% and 64.16%, respectively. Multiple mutants with G12-, G14-, G24- and G124- substitutions could further reduce receptor binding activities, 33.07%, 20.67%, 19.96% and 15.11% of the wt HN level, respectively. G12, G14, G24 and G124 mutants exhibited levels of fusion promotion activity that were only 46.360, 12.04%, 13.43% and 4.05% of the wt amount, respectively. As N-glycans of hPIV3 HN protein play an important role in receptor binding activity and cell fusion promotion activity of HN protein. We propose that the loss of N-glycans change the conformation or orientation of globular domain that is responsible for receptor binding and lower receptor binding activity and cell fusion promotion activi ty.

To study the genetic diversity of HIV-1 nef genes from a patient with AIDS dementia complex(ADC) , so as to research the amino acid variability and the pathogenesis of ADC.The nef gene was amplified with PCR from genomic DNA which was extracted from spleen and different brain tissues(basal ganglia, frontal gray matter, meninges, temporal lobe)of a patient who died of ADC. PCR products were cloned into the pMD19-T vector, after transformation and selection by ampicillin and blue/white spotting. Five of positive clones were sequenced and confirmed with BLAST. HIV-1 nef sequences were processed with BioEdit and MEGA4 to do Neighbor-Joining tree, p-Distances, and values of ds/dn.The samples were all identified as HIV-1 B and genetic variation exists in HIV-1 nef gene isolated from different tissues compared with HXB2. In addition,part of the changes were different between periphery and brain.Variations exist in the HIV-1 nef gene extracted from the ADC patient and the variations from peripheral and central nerve tissues were different,these variations may change the function of Nef,and it needs more research.

To explore the role of HIV-1 tat gene variations in AIDS dementia complex (ADC) pathogenesis.HIV-1 tat genes derived from peripheral spleen and central basal ganglia of an AIDS patient with ADC and an AIDS patient without ADC were cloned for sequence analysis. HIV-1 tat gene sequence alignment was performed by using CLUSTAL W and the phylogentic analysis was conducted by using Neighbor-joining with MEGA4 software. All tat genes were used to construct recombinant retroviral expressing vector MSCV-IRES-GFP/tat. The MSCV-IRES-GFP/tat was cotransfected into 293T cells with pCMV-VSV-G and pUMVC vectors to assemble the recombinant retrovirus. After infection of gliomas U87 cells with equal amount of the recombinant retrovirus, TNF-α, and IL-1β concentrations in the supernatant of U87 cells were determined with ELISA.HIV-1 tat genes derived from peripheral spleen and central basal ganglia of the AIDS patient with ADC and the other one without ADC exhibited genetic variations. Tat variations and amino acid mutation sites existed mainly at Tat protein core functional area (38-47aa). All Tat proteins could induce U87 cells to produce TNF-α and IL-1β, but the level of IL-1β production was different among Tat proteins derived from the ADC patient's spleen, basal ganglia, and the non-ADC patient's spleen. The level of Tat proteins derived from the ADC patient's spleen, basal ganglia, and the non-ADC patient's spleen were obviously higher than that from the non-ADC patient's basal ganglia.Tat protein core functional area (38-47aa) may serve as the key area of enhancing the secretion of IL-1β. This may be related with the neurotoxicity of HIV-1 Tat.

To clone and express the HIV-1B gp120 genes isolated at different organizations from a patient died of AIDS dementia complex (ADC) in eukaryotic cells.Using the genomic DNA isolated from peripheral lymphnodes, choroid plexus and occipital white matter from a patient died of ADC as the template, HIV-1B gp120 gene was amplified with PCR. After sequenced, HIV-1B gp120 was inserted into pcDNA3.1 (+) and recombinant expressing vector gp120/pcDNA3.1 (+) was constructed succeffuly confirming with sequencing. Then expressing vector was transfected into eukaryotic cells U87 using liposome transfection and expression of HIV-1B gp120 gene was assayed with indirect immunofluorescence.HIV-1B gp120 genes isolated from peripheral lymphnodes, choroid plexus and occipital white matter of the ADC patient were successfully cloned and recombinant expressing vector gp120/pcDNA3; 1 (+) could express envelope glycoprotein HIV-1B gp120 in U87 cells.All the HIV-1B gp120 gene isolated at the different organizations of the same ADC patient could express in U87 cells, which may supply a valuable basis for studying the neurotoxicity and neurotoxic mechanism of HIV-1 gp120 protein.

HIV-1 infected and immune-activated macrophages and microglia secrete neurotoxins, such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), which play major role in the neuronal death. It has been shown that different HIV-1 variants have varying abilities to elicit secretion of TNF-α by peripheral blood mononuclear cell (PBMC); however, whether the difference of gp120 gene could affect the secretion of TNF-α and IL-1β by glial cells is unknown. The aim of this study was to explore the association between gene diversity and induction of neurotoxic cytokines.In this study, we constructed retroviral vectors MSCV-IRES-GFP/gp120 using HIV-1 gp120 genes isolated from four different tissues of one patient who died of AIDS dementia complex (ADC). Recombinant retroviruses produced by cotransfection of MSCV-IRES-GFP/gp120, pCMV-VSV-G and pUMVC into 293T cells were collected and added into U87 glial cells. Concentrations of TNF-α and IL-1β secreted by transduced U87 cells were assayed with ELISA separately.The four HIV-1 gp120 were in the different branch of the neighbor-joining tree. Compared to the pMIG retrovirus (gp120-negative) or U87 cells, all the gp120-positive recombinant retroviruses induced more TNF-α (P0.01) and IL-1β (P0.01). In addition, compared with the L/MIG retrovirus, all the three brain gp120-positive recombinant retroviruses induced less TNF-α (P0.01) and IL-1β (P0.01).HIV-1 gp120 could induce U87 cells secret more TNF-α and IL-1β again. The more important is that difference of HIV-1 gp120, especially cell-tropism may account for the different ability in eliciting secretion of TNF-α and IL-1β, which might supply a novel idea helping understand the pathogenesis of ADC.

To study the diversity of HIV-1 tat gene in CNS and peripheral tissue of a patient with ADC and a patient with non-ADC, so as to research HIV evolution, the mechanism of CNS invasion and the pathogenesis of ADC.The tat gene was amplified with nested PCR from genomic DNA which was extracted from spleen and basal ganglia of one non-ADC patient with a wide range of cerebral artery atherosclerosis and one ADC patient. PCR products were cloned into the PGEM-T vector, after transformation and selection by ampicillin and blue/white spotting. Five of positive clones were sequenced. HIV-1 tat sequences were processed with BioEdit and MEGA4. With the softwares, neighbor-joining tree, p-distances, values of ds/dn, and analysis of amino acid motifs were all done, so as to research the diversity of HIV-1 tat gene in CNS and peripheral tissue.Gene mutation of HIV-1 tat exist in the two patients, the mutation process of tat isolated from ADC patient suffered more compartmentalization than tat isolated from non-ADC patient, the differences of tat genes between CNS and peripheral tissue in ADC patient were greater than the non-ADC patient. Ds/dn showed that the virus gene mutation played a major role, the body intend to remove harmful non-synonymous mutations.The compartmentation of tat gene in CNS and peripheral tissue of the two patients was different, the reason may be related to the pathway of HIV into the CNS, the relationship between HIV gene mutation in CNS and ADC still need more investigation.

To explore the relationship between the genetic diversity and biological functional site of human immunodeficiency virus HIV-1 gp120 and the pathogenesis of AIDS dementia complex (ADC), the full length sequences of gp120 gene was amplified with PCR from genomic DNA which was extracted from lymphoid and different brain department (periaortic lymphoid, temporal gray/white matter junction, periventricular tissue, choroids plexus, occipital white matter and occipital gray/white matter junction.) of a patient who died of ADC. PCR products were cloned into the pGEM-T vector and positive clones were sequenced. The analysis of neighbor-joining tree, N-glycosylation sites, values of ds/dn, and loop were then all performed. The samples were all identified as HIV-1 B and genetic variation existed in HIV-1 gp120 isolated from different tissues. Compared with standard HIV-1B gp120, biological functional sites of HIV-1 gp120 isolated from the patient changed to some extent. In addition, there were differences in some biological functional sites of HIV-1 gp120 between lymphoid and brain. Therefore, genetic diversity and alterations of some biological functional sites of HIV-1 gp120 might be associated with the pathogenesis of ADC.