Reforestation of Cunninghamia lanceolata changes the relative abundances of important prokaryotic families in soil.

Over the past decades, many forests have been converted to monoculture plantations, which might affect the soil microbial communities that are responsible for governing the soil biogeochemical processes. Understanding how reforestation efforts alter soil prokaryotic microbial communities will therefore inform forest management. In this study, the prokaryotic communities were comparatively investigated in a secondary Chinese fir forest (original) and a reforested Chinese fir plantation (reforested from a secondary Chinese fir forest) in Southern China. The results showed that reforestation changed the structure of the prokaryotic community: the relative abundances of important prokaryotic families in soil. This might be caused by the altered soil pH and organic matter content after reforestation. Soil profile layer depth was an important factor as the upper layers had a higher diversity of prokaryotes than the lower ones (p < 0.05). The composition of the prokaryotic community presented a seasonality characteristic. In addition, the results showed that the dominant phylum was Acidobacteria (58.86%) with Koribacteraceae (15.38%) as the dominant family in the secondary Chinese fir forest and the reforested plantation. Furthermore, soil organic matter, total N, hydrolyzable N, and NH4+-N were positively correlated with prokaryotic diversity (p < 0.05). Also, organic matter and NO3--N were positively correlated to prokaryotic abundance (p < 0.05). This study demonstrated that re-forest transformation altered soil properties, which lead to the changes in microbial composition. The changes in microbial community might in turn influence biogeochemical processes and the environmental variables. The study could contribute to forest management and policy-making.

Human Schlafen 11 exploits codon preference discrimination to attenuate viral protein synthesis of prototype foamy virus (PFV).

Recently, the Schlafen (SLFN) proteins have been identified as a novel interferon-stimulated family with antiviral properties. In this study, we reported that SLFN11 inhibited prototype foamy virus (PFV) replication. Over-expression of human SLFN11 reduced viral production, while knockdown of SLFN11 enhanced viral infectivity. In addition, SLFN11 from cattle and African green monkey also suppressed PFV production. Both the ATPase activity and helicase activity of SLFN11 were required for its inhibitory function. Dephosphorylation activated the antiviral activity of SLFN11. More importantly, SLFN11 inhibited the expression of viral protein, which was rescued by viral gene codon optimization. Together, our results demonstrated that SLFN11 impaired PFV viral protein synthesis by exploiting the distinct codon usage between the virus and the host. These findings further broaden our understanding of the antiviral properties of the SLFN family and the molecular mechanism of PFV latent infection.

[Identification of prototype foamy virus Bel1 nuclear localization signal and its corresponding importins].

Bel1, a transactivator of prototype foamy virus (PFV), plays pivotal roles in the replication of PFV. Previous studies have shown that Bel1 bears a nuclear localization signal (NLS), but its amino acid sequence remains unclear and the corresponding importins have not been identified. In this report, we inserted various fragments of Bel1 into an EGFP-GST fusion protein and investigated their subcellular localization by fluorescence microscopy. We found that the 215PRQKRPR221 fragment could direct nuclear localization, which accords with the consensus sequence K(K/R)X(K/R) of monopartite NLS. Point mutation experiments revealed that K218, R219, and R221 are essential for the nuclear localization of Bel1. The results of the GST-pulldown showed that the Bel1 fragment with residues 215-223, which bears the NLS, interacts with KPNA1, KPNA6, and KPNA7. This result suggests that KPNA1, KPNA6, and KPNA7 maybe involved in Bel1 nuclear translocation.

First report on isolation of methyl gallate with antioxidant, anti-HIV-1 and HIV-1 enzyme inhibitory activities from a mushroom (Pholiota adiposa).

In this study, a compound with antioxidant and anti-HIV activities designated as HEB was first isolated from the edible mushroom Pholiota adiposa by extraction with ethanol and ethyl acetate. HEB was then purified by high performance liquid chromatography (HPLC) and identified to be methyl gallate (C8H8O5, 184.1 Da) based on data from its mass spectrum (MS) and nuclear magnetic resonance (NMR) spectrum. HEB displayed strong antioxidant potency in inhibiting, at 1.36 mM concentration, erythrocyte hemolysis and scavenging DPPH radicals and superoxide anion (O2(-)) by 82.4%, 85.6% and 71.4%, respectively. Besides exhibiting a low cytotoxicity, compound HEB demonstrated significant anti-HIV activity in that it inhibited HIV-1 replication in TZM-BL cells infected by pseudovirus with an IC50 value of 11.9 µM. Further study disclosed that HEB inhibited the viral entry process and activities of key enzymes essential for the HIV-1 life cycle. HEB inhibited HIV-1 reverse transcriptase and integrase activities with an IC50 value of 80.1 µM and 228.5 µM, respectively, and at 10 mM concentration inhibited HIV-1 protease activity by 17.1% which was higher than that achieved by the positive control pepstatin A. Interestingly, this study first revealed that H2O2 stimulation not only activated cell oxidative stress responses, but also accelerated HIV-1 long terminal repeat (LTR) promotion in TZM-BL cells, which was significantly reduced by HEB from 18.2% to about 2%. It implied a direct relationship between the antioxidant and anti-HIV activities of the natural active constituent HEB. Nuclear transcription factor kappa B (NF-κB) signal pathways plays an important role in oxidative stress responses. Meanwhile, there is κB target sequence in HIV promoter LTR which is significant for virus replication and gene expression. In this study, Western Blot assay showed that HEB could inhibit the activation of NF-κB signal pathway stimulated by H2O2 in mouse spleen cells through suppressing NF-κB (p65) translocation into nucleus and NF-kappa-B inhibitor (IκB) degradation in cytoplasm. In summary, the antioxidant HEB from P. adiposa could inhibit HIV-1 replication through multiple target sites. The data suggest that natural antioxidant compounds might have a potential for treatment of AIDS.

[Effect of RelB on HIV-1 Vpr-mediated transcription activation and cell G2/M arrest].

Vpr, an auxiliary protein of HIV-1(Human immunodeficiency virus type 1), exerts important functions to promote viral replication and AIDS progression. In this study, we performed a yeast two-hybrid screening assay using human cDNA library to further investigate the molecular mechanism of various functions of Vpr RelB, a key protein in NF-kappaB signaling pathway, was identified as a Vpr interaction protein by co-immunoprecipitation. Further investigations indicated that RelB not only promoted the Vpr-mediated activation of NF-kappaB reporter gene, but also enhanced the transactivation of HIV LTR. Moreover, the results showed that RelB promoted Vpr-induced cell cycle G2/M arrest. Collectively, these results indicated that RelB might interact with Vpr and regulate its transcriptional activation and cell cycle arrest.

Effects of adenosine extract from Pholiota adiposa (Fr.) quel on mRNA expressions of superoxide dismutase and immunomodulatory cytokines.

Pholiota adiposa is a kind of edible mushroom which has long been known for its health care applications. To reveal the exact mechanism of its protective functions in humans, in this study we isolated and identified the active compound PB3 of P. adiposa for the first time by a combination of chromatography techniques, including NKA macroporous resin and Sephadex G-15. PB3, with molecular mass of 267.2 Da and molecular formula of C10H13N5O4 discovered by mass spectrum (MS) was identified to be adenosine. Mice were injected intraperitoneally with purified fraction PB3. Seven days after injection, we found a 1.5-fold increase of IL10 at the mRNA level, while a down regulated expression of IL-2, IL-6 and IFN-γ to 49.0%, 56.9% and 73.4%, respectively, was detected in spleen by real-time quantitative PCR. What's more, SOD expression level was significantly increased by 1.6-fold compared to control. Fraction PB3 displayed anti-inflammatory potency and heightened SOD activity on the transcriptional level, which could be considered of further pharmaceutical or medication value.

Bovine herpesvirus 1 protein bICP0 represses the transcription of bISG15 in fetal bovine lung cells.

The ubiquitin-like modifier bISG15 is an antiviral protein found in fetal bovine lung (FBL) cells. Bovine Herpesvirus 1(BHV-1), which is a viral pathogen of cattle, can infect FBL cells and induce cytopathic effects. Real-time PCR assays showed that BHV-1's infection could repress the basal or inducible transcription of bISG15 in FBL cells. It demonstrates that this repression effect depends on BHV-1 viral infection and new protein synthesis. Our previous work showed that bIRF-3 was the key factor in the stimulation of bISG15 in FBL cells, so the effect of BHV-1 viral protein on bIRF-3 activating the promoter of bISG15 was confirmed. The luciferase assay showed the BHV-1 viral protein bICP0 inhibited the activation of bISG15 promoter stimulated by bIRF-3. Taken together, our work suggested that BHV-1 had some molecular mechanism to resist the cellular bISG15's antiviral functions.

A new indicator cell line established to monitor bovine foamy virus infection.

In order to improve the accuracy for quantitating the bovine foamy virus (BFV) in vitro, we developed a baby hamster kidney cell (BHK)-21-derived indicator cell line containing a plasmid that encodes the firefly luciferase driven by the BFV long terminal repeat promoter (LTR, from -7 to 1012). The BFV titer could be determined by detecting the luciferase expression since the viral trans-activator BTas protein activates the promoter activity of the LTR. One clone, designated BFVL, was selected from ten neomycin-resistant clones. BFVL showed a specific and inducible dose- and time-dependent luciferase activity in response to BFV infection. Although the changes in luciferase activity of BFVL peaked at 84 h post infection, it was possible to differentiate infected and uninfected cells at 48 h post infection. A linear relationship was established between the multiplicity of infection (MOI) of BFV and the activated ratio of luciferase expression in BFVL. Moreover, the sensitivity of the BFVL-based assay for detecting infectious BFV was 10,000 times higher than the conventional CPE-based assay at 48 h post infection. These findings suggest that the BFVL-based assay is rapid, easy, sensitive, quantitative and specific for detection of BFV infection.

Isolation of a polysaccharide with antiproliferative, hypoglycemic, antioxidant and HIV-1 reverse transcriptase inhibitory activities from the fruiting bodies of the abalone mushroom Pleurotus abalonus.

OBJECTIVE: The intent of this study was to purify and characterize a polysaccharide named LA from the fruiting bodies of the edible mushroom Pleurotus abalones. METHODS: The 120-kDa polysaccharide was obtained by extraction with boiling water, ethanol precipitation, ion exchange chromatography on Diethylaminoethyl-cellulose and gel filtration on Sephadex G-200. KEY FINDINGS: The LA was composed of glucose, rhamnose, glucuronic acid, xylose, galactose and arabinose in the molar ratio of 26.3:2.7:1:1.4:1.8:1.2. The FITR and 1H-NMR spectrum of LA disclosed that it was a saccharide with an α-configuration. Its 13C-NMR spectrum revealed that its main chain was [→6)-α-D-Gly(1→]n. The LA exhibited antioxidant activities, especially in scavenging 1,1- diphenyl-2-picryl-hydrazyl radicals and hydroxyl radicals. It manifested antiproliferative activity towards breast cancer MCF7 cells with an IC50 of 3.7 µm, and also exerted some antiproliferative activity against HepG2 cells. It manifested a hypoglycemic action on diabetic mice. It inhibited HIV-1 reverse transcriptase with an IC50 of 8.7 × 10(-2) µm. CONCLUSIONS: The polysaccharide from the abalone mushroom.

Preparation of BFV Gag antiserum and preliminary study on cellular distribution of BFV.

Viruses (e.g. Human immunodeficiency virus, Human simplex virus and Prototype foamy virus) are obligate intracellular parasites and therefore depend on the cellular machinery for cellular trafficking. Bovine foamy virus (BFV) is a member of the Spumaretrovirinae subfamily of Retroviruses, however, details of its cellular trafficking remain unknown. In this study, we cloned the BFV gag gene into prokaryotic expression vector pET28a and purified the denaturalized Gag protein. The protein was used to immunize BALB/c mouse to produce antiserum, which could specifically recognize the BFV Gag protein in BFV-infected cells through western blot assay. Additionally, these results demonstrated that both the optimal and suboptimal cleavage of Gag protein occur in BFV-infected cells. Subsequently, the Gag antiserum was used to investigate subcellular localization of BFV. In immunofluorescence microscopy assays, colocalization microtubules (MTs) and assembling viral particles were clearly observed, which implied that BFV may transport along cellular MTs in host cells. Furthermore, MTs-depolymerizing assay indicated MTs were required for the efficient replication of BFV. In conclusion, our study suggests that BFV has evolved the mechanism to hijack the cellular cytoskeleton for its replication.

A quantitative assay for measuring of bovine immunodeficiency virus using a luciferase-based indicator cell line.

In order to quantitate the bovine immunodeficiency virus (BIV) infection in vitro, a BIV indicator cell line (BIVL) was established by transfecting baby hamster kidney cells with reporter plasmids containing the firefly luciferase gene driven by a BIV long terminal repeat promoter. The BIV activates promoter activity of the LTR to express luciferase upon infection. BIV infection could therefore by quantified by detection of luciferase activity. Compared to standard assays used to detect BIV infection, the BIVL-based assay is 10 times more sensitive than the the CPE-based assay, and has similar sensitivity with the viral capsid protein Western blot assay. BIV indicator cell line could detect BIV infection specifically. Luciferase activity of BIV infected BIVL cells showed a time dependent manner, and 60 h post infection is the optimal time to detect BIV infection. Luciferase activity of BIVL cells correlates with the BIV capsid protein expression. Moreover, a linear relationship was found between MOI and the activated intensity of luciferase expression. In brief, the BIV indicator cell line is an easy, robust and quantitive method for monitoring BIV infection.

Establishment of an indicator cell line for monitoring bovine immunodeficiency virus infection and inhibitor susceptibility.

Indicator cell lines are useful biological tools for monitoring virus infection. In order to monitor infection with bovine immunodeficiency virus (BIV) in vitro, an indicator cell line derived from baby hamster kidney cells which contains integrated copies of an enhanced green fluorescent protein gene driven by the BIV long terminal repeat was constructed. The BIV indicator cell line, designated BIVE, can detect BIV infection more easily and effectively than the established method, which involves the observation of cell cytopathic effects. Furthermore, viral titration using an assay based on the indicator cells is 100 times more sensitive than the assay based on cytopathic effect. The finding that BIV can infect the hamster cell line expands the known host range of BIV in vitro. The BIV indicator cell line could also be used for the evaluation of the inhibitory effect of antiviral agents. The fusion inhibition effect of the heptad repeat 2 region of the BIV envelope protein could also be quantified.

Cloning and characterization of a novel intracellular protein p48.2 that negatively regulates cell cycle progression.

Neurofibromatosis type 1 (NF1) microdeletion is a large genomic deletion that embraces at least 11 continuous genes at human chromosome 17q11.2. To date, most of these genes' functions still remain undefined. In this study, we report an unknown cytokine receptor like molecule (p48.2) that is frequently deleted in patients with type-1 and type-2 NF1 microdeletions in the neurofibromin locus. The cloned gene has 1317 base pair long that encodes a 438aa intracellular protein. The gene was subsequently named p48.2 based on its predicted molecular weight. A typical fibronectin type III (FNIII) domain was identified in p48.2 between Arg(176) and Pro(261) in which a palindromic Arg-Gly-Asp (RGD) repeat plus a putative Trp-Ser-X-Trp-Ser (WSXWS) motif were found at the domain's C-terminus. p48.2 mRNAs were abundant in many tumor cell lines and normal human tissues and up-regulated in some freshly isolated lung cancer and leukemia cells. Interestingly, over-expression of p48.2 in human embryo kidney 293T cells could significantly cause G0/G1 arrest and prevented S phase entry. In contrast, repressing endogenous p48.2 gene expression by specific siRNA markedly reduced G0/G1 population. Importantly, over-expression of p48.2 could significantly up-regulate rather than down-regulate cyclin D1 and cyclin D3 expressions. We further showed that the induction of cyclin D1 expression was directly due to the activation of signal transducers and activators of transcription 3 (STAT3), but was independent of RAS/mitogen-activated protein kinase (RAS/MAPK) signaling pathway. Thus, p48.2 may represent a novel type of intracellular protein functioning as a negative regulator at the G0/G1 phase.

Construction and characterization of a new simian/human immunodeficiency viruses clone carrying an env gene derived from a CRF07_BC strain.

BACKGROUND: The CRF07_BC recombinant strain has been one of the most predominantly circulated HIV-1 strains in China, it is therefore necessary and urgent to develop a relevant animal model to evaluate candidate vaccines targeting HIV-1 CRF07_BC. A highly replication-competent simian/human immunodeficiency viruses (SHIV) construct containing the Chinese CRF07_BC HIV-1 env gene with the ability to infect Chinese rhesus monkeys would serve as an important tool in the development of HIV vaccines. The aim of this study was to examine whether SHIV XJDC6431 with the env fragment from a Chinese HIV-1 isolate virus could infect the human and monkey peripheral blood mononuclear cell (PBMC), establish infection in Chinese rhesus macaque. METHODS: A SHIV strain was constructed by replacing the rev/env genes of SHIV KB9 with the corresponding fragment derived from the HIV-1 CRF07_BC strain. The infectious activity of the SHIV clones was determined in vitro in PBMCs from both non-human primate animals and humans. Finally, one Chinese rhesus macaques (Macaca mulatta) was infected with one SHIV via intravenous infusion. RESULTS: One SHIV clone designated as SHIV XJDC6431, was generated that could infect macaque and human PBMC. The virus produced from this clone also efficiently infected the CCR5-expressing GHOST cell lines, indicating that it uses CCR5 as its coreceptor. Finally, the virus was intravenously inoculated into one Chinese rhesus macaque. Eventually, the animal became infected as shown by the occurrence of viremia within 3 of infection. The viral load reached 105 copies of viral RNA per ml of plasma during the acute phase of infection and lasted for 10 weeks post infection. CONCLUSIONS: We conclude that SHIV XJDC6431 is an R5-tropic chimeric virus, which can establish infection not only in vitro but also in vivo in the Chinese rhesus macaque. Although the animal inoculated with SHIV XJDC6431 became infected without developing a pathologic phenotype, the virus efficiently replicated with a persistent level of viral load in the plasma. This suggested that the SHIV could be used as a tool to test candidate AIDS vaccines targeting the Chinese HIV-1 CRF_07BC recombinant strain.

Detection and analysis of bovine foamy virus infection by an indicator cell line.

AIM: To determine the infectivity and replication strategy of bovine foamy virus (BFV) in different cultured cells using the BFV indicator cell line (BICL) system. METHODS: BFV infection was induced by the co-culture method or the transient transfection of the infectious BFV plasmid [pCMV (cytomegalovirus) - BFV] clone. The infectivity of BFV was monitored by the percentage of green fluorescent protein-positive cells in the BICL. The effect of reverse transcriptase inhibitor zidovudine (AZT) on BFV replication was also evaluated in the BICL. RESULTS: The titer of BFV in fetal bovine lung cells was 4-5-folds more than that in either 293T or HeLa (Cells from Henrietta lacks) cells using the co-culture method, and in the meantime was significantly higher than that produced by the infectious clone pCMV-BFV in the same cells. AZT had only a minor effect on viral titers when added to cells prior to the virus infection. In contrast, viral titers reduced sharply to the level of the negative control when the virus was produced from cells in the presence of AZT. CONCLUSIONS: BICL can be used for the titration of the BFV viral infection in non-cytopathic condition. In addition, we provide important evidence to show that reverse transcription is essential for BFV replication at a late step of viral infection.

Construction and characterization of chimeric BHIV (BIV/HIV-1) viruses carrying the bovine immunodeficiency virus gag gene.

AIM: To explore the possibility of the replacement of the gag gene between human immunodeficiency virus and bovine immunodeficiency virus, to achieve chimeric virions, and thereby gain a new kind of AIDS vaccine based on BHIV chimeric viruses. METHODS: A series of chimeric BHIV proviral DNAs differing in the replacement regions in gag gene were constructed, and then were transfected into 293T cells. The expression of chimeric viral genes was detected at the RNA and protein level. The supernatant of 293T cell was ultra centrifuged to detect the probable chimeric virion. Once the chimeric virion was detected, its biological activities were also assayed by infecting HIV-sensitive MT4 cells. RESULTS: Four chimeric BHIV proviral DNAs were constructed. Genes in chimeric viruses expressed correctly in transfected 293T cells. All four constructs assembled chimeric virions with different degrees of efficiency. These virions had complete structures common to retroviruses and packaged genomic RNAs, but the cleavages of the precursor Gag proteins were abnormal to some extent. Three of these virions tested could attach and enter into MT4 cells, and one of them could complete the course of reverse transcription. Yet none of them could replicate in MT4 cells. CONCLUSION: The replacement of partial gag gene of HIV with BIV gag gene is feasible. Genes in chimeric BHIVs are accurately expressed, and virions are assembled. These chimeric BHIVs (proviral DNA together with virus particles) have the potential to become a new kind of HIV/AIDS vaccine.