Mutational analysis of the influenza virus cRNA promoter and identification of nucleotides critical for replication

Replication of the influenza A virus virion RNA (vRNA) requires the synthesis of full-length cRNA, which in turn is used as a template for the synthesis of more vRNA. A "corkscrew" secondary-structure model of the cRNA promoter has been proposed recently. However the data in support of that model were indirect, since they were derived from measurement, by use of a chloramphenicol acetyltransferase (CAT) reporter in 293T cells, of mRNA levels from a modified cRNA promoter rather than the authentic cRNA promoter found in influenza A viruses. Here we measured steady-state cRNA and vRNA levels from a CAT reporter in 293T cells, directly measuring the replication of the authentic influenza A virus wild-type cRNA promoter. We found that (i) base pairing between the 5' and 3' ends and (ii) base pairing in the stems of both the 5' and 3' hairpin loops of the cRNA promoter were required for in vivo replication. Moreover, nucleotides in the tetraloop at positions 4, 5, and 7 and nucleotides forming the 2-9 base pair of the 3' hairpin loop were crucial for promoter activity in vivo. However, the 3' hairpin loop was not required for polymerase binding in vitro. Overall, our results suggest that the corkscrew secondary-structure model is required for authentic cRNA promoter activity in vivo, although the precise role of the 3' hairpin loop remains unknown.     

Cyclin/CDK regulates the nucleocytoplasmic localization of the human papillomavirus E1 DNA helicase

Cyclin-dependent kinases (CDKs) play key roles in eukaryotic DNA replication and cell cycle progression. Phosphorylation of components of the preinitiation complex activates replication and prevents reinitiation. One mechanism is mediated by nuclear export of critical proteins. Human papillomavirus (HPV) DNA replication requires cellular machinery in addition to the viral replicative DNA helicase E1 and origin recognition protein E2. E1 phosphorylation by cyclin/CDK is critical for efficient viral DNA replication. We now show that E1 is phosphorylated by CDKs in vivo and that phosphorylation regulates its nucleocytoplasmic localization. We identified a conserved regulatory region for localization which contains a dominant leucine-rich nuclear export sequence (NES), the previously defined cyclin binding motif, three serine residues that are CDK substrates, and a putative bipartite nuclear localization sequence. We show that E1 is exported from the nucleus by a CRM1-dependent mechanism unless the NES is inactivated by CDK phosphorylation. Replication activities of E1 phosphorylation site mutations are reduced and correlate inversely with their increased cytoplasmic localization. Nuclear localization and replication activities of most of these mutations are enhanced or restored by mutations in the NES. Collectively, our data demonstrate that CDK phosphorylation controls E1 nuclear localization to support viral DNA amplification. Thus, HPV adopts and adapts the cellular regulatory mechanism to complete its reproductive program.     

Lytic replication-defective Kaposi's sarcoma-associated herpesvirus: potential role in infection and malignant transformation

Defective viruses often have pivotal roles in virus-induced diseases. Although Kaposi's sarcoma-associated herpesvirus (KSHV) is etiologically associated with Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL), defective KSHV has not been reported. Using differential genetic screening methods, we show that defective KSHV is present in KS tumors and PEL cell lines. To investigate the role of defective viruses in KSHV-induced pathogenesis, we isolated and characterized a lytic replication-defective KSHV, KV-1, containing an 82-kb genomic deletion of solely lytic genes. Cells harboring KV-1 escaped G(0)/G(1) apoptosis induced by spontaneous lytic replication occurred in cells infected with regular KSHV but maintained efficient latent replication. Consequently, KV-1-infected cells had phenotypes of enhanced cell proliferation and transformation potentials. Importantly, KV-1 was packaged as infectious virions by using regular KSHV as helpers, and KV-1-like variants were detected in cultures of two of five KSHV cell lines and 1 of 18 KS tumors. These results point to a potential role for defective viruses in the regulation of KSHV infection and malignant transformation.     

Small molecules blocking the entry of severe acute respiratory syndrome coronavirus into host cells

Severe acute respiratory syndrome coronavirus (SARS-CoV) is the pathogen of SARS, which caused a global panic in 2003. We describe here the screening of Chinese herbal medicine-based, novel small molecules that bind avidly with the surface spike protein of SARS-CoV and thus can interfere with the entry of the virus to its host cells. We achieved this by using a two-step screening method consisting of frontal affinity chromatography-mass spectrometry coupled with a viral infection assay based on a human immunodeficiency virus (HIV)-luc/SARS pseudotyped virus. Two small molecules, tetra-O-galloyl-beta-D-glucose (TGG) and luteolin, were identified, whose anti-SARS-CoV activities were confirmed by using a wild-type SARS-CoV infection system. TGG exhibits prominent anti-SARS-CoV activity with a 50% effective concentration of 4.5 microM and a selective index of 240.0. The two-step screening method described here yielded several small molecules that can be used for developing new classes of anti-SARS-CoV drugs and is potentially useful for the high-throughput screening of drugs inhibiting the entry of HIV, hepatitis C virus, and other insidious viruses into their host cells.     

Emergence of a drug-dependent human immunodeficiency virus type 1 variant during therapy with the T20 fusion inhibitor

The fusion inhibitor T20 belongs to a new class of anti-human immunodeficiency virus type 1 (HIV-1) drugs designed to block entry of the virus into the host cell. However, the success of T20 has met with the inevitable emergence of drug-resistant HIV-1 variants. We describe an evolutionary pathway taken by HIV-1 to escape from the selective pressure of T20 in a treated patient. Besides the appearance of T20-resistant variants, we report for the first time the emergence of drug-dependent viruses with mutations in both the HR1 and HR2 domains of envelope glycoprotein 41. We propose a mechanistic model for the dependence of HIV-1 entry on the T20 peptide. The T20-dependent mutant is more prone to undergo the conformational switch that results in the formation of the fusogenic six-helix bundle structure in gp41. A premature switch will generate nonfunctional envelope glycoproteins (dead spikes) on the surface of the virion, and T20 prevents this abortive event by acting as a safety pin that preserves an earlier prefusion conformation.     

TGF-beta1 induces alveolar epithelial to mesenchymal transition in vitro

The aim of this study was to investigate whether transforming growth factor-beta1 (TGF-beta1) could induce alveolar epithelial to mesenchymal transition (EMT) in vitro. Alveolar epithelial cells (AECs) from SD rats were isolated by elastase cell dispersion and IgG panning. Expression of alpha-smooth muscle actin (alpha-SMA) was assayed using Western blotting and immunostaining analysis. Morphological changes, the markers of epithelial cell (E-cadherin), and stress fiber by actin reorganization were detected by an indirect immunostaining. The contents of collagen I were determined by spectrophotometry. The levels of endogenous TGF-beta1 were measured with ELISA. Incubation of AECs with TGF-beta1 (0.1 approximately 10 ng/mL) induced abundant expression of alpha-SMA protein, and alpha-SMA expression in AECs reached a plateau when TGF-beta1 was > 3 ng/mL. Furthermore, we found that TGF-beta1 (3 ng/mL) exposure of AECs induced an authentic EMT characterized by abundant expression of alpha-smooth muscle actin, transformation of myofibroblastic morphology, increased formation of stress fiber by actin reorganization, and loss of epithelial marker E-cadherin. Meanwhile, significant increase in the levels of collagen I from 32.0 +/- 6.6 mg/g in control to 98 +/- 10.8 mg/g in TGF-beta1-treated group was found over a 72 h incubation period. Moreover, following stimulated by TGF-beta1 (3 ng/mL), a marked and time-dependent increase in endogenous TGF-beta1 released from AECs was observed. At time points 72 h, TGF-beta1 release mounted to 3451 pg/ml, which was much enough to induce EMT in vitro. These results demonstrated that AECs, under stimulation of TGF-beta1, underwent a conversion process into myofibroblasts in vitro.     

超声彩色血流成像的计算机快速仿真方法

研究超声彩色血流成像的快速仿真方法,克服原先仿真方法非常耗时的缺点。方法超声彩色血流成像计算机仿真中,血流信号是对成像区间内所有点散射体的回波信号累加而得到的。通过引入新的等效散射体模型,可以大大降低散射体的密度,从而减少计算回波信号所需时间。在计算机上用Matlab编程来进行仿真实验,对以往仿真方法和基于等效散射体模型方法的性能进行比较。结果实验表明:基于等效散射体模型的仿真,在保证相同流速精度的前提下,仿真速度比传统方法提高了10倍以上。结论基于等效散射体模型的仿真方法能极大地提高超声彩色血流成像的仿真速度,可以为超声彩色血流成像的方法研究提供便利。     

Arabidopsis CONSTANS-LIKE3 is a positive regulator of red light signaling and root growth

CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1) is an E3 ubiquitin ligase that represses photomorphogenesis in the dark. Therefore, proteins interacting with COP1 could be important regulators of light-dependent development. Here, we identify CONSTANS-LIKE3 (COL3) as a novel interaction partner of COP1. A green fluorescent protein-COL3 fusion protein colocalizes with COP1 to nuclear speckles when transiently expressed in plant cells. This localization requires the B-box domains in COL3, indicating a novel function of this domain. A loss-of-function col3 mutant has longer hypocotyls in red light and in short days. Unlike constans, the col3 mutant flowers early and shows a reduced number of lateral branches in short days. The mutant also exhibits reduced formation of lateral roots. The col3 mutation partially suppresses the cop1 and deetiolated1 (det1) mutations in the dark, suggesting that COL3 acts downstream of both of these repressors. However, the col3 mutation exerts opposing effects on cop1 and det1 in terms of lateral roots and anthocyanin accumulation, suggesting that COL3 also has activities that are independent of COP1 and DET1. In conclusion, we have identified COL3 as a positive regulator of photomorphogenesis that acts downstream of COP1 but can promote lateral root development independently of COP1 and also function as a daylength-sensitive regulator of shoot branching.     

Mouse oocytes derived from fetal germ cells are competent to support the development of embryos by in vitro fertilization

Mouse oocyte development in vitro has been studied in the past several years, but no evidence showed that the fertilizable oocytes could be obtained from the fetal mouse germ cells before the formation of the primordial follicles. In this study, an efficient and simple method has been established to obtain the mature oocytes from the fetal mouse germ cells at 16.5 days post-coitum (dpc). For the initial of follicular formation, fetal mouse 16.5 dpc ovaries were transplanted to the recipient under the kidney capsule, and the ovaries were recovered after 14 days. Subsequently, the growing preantral follicles in the ovarian grafts were isolated and cultured in vitro for 12 days. Practically, the mature oocytes ovulated from the antral follicles were able to be fertilized in vitro and support the embryonic development. The results demonstrate that the fetal mouse 16.5 dpc germ cells are able to form primordial follicles with the ovarian pregranulosa cells during the period of transplantation in the ectopic site, and the oocytes within the growing follicles are able to mature in vitro, then are able to support the embryonic development.