Accepted Manuscript
MDA5 Plays a Critical Role in Interferon Response during Hepatitis C Virus
Infection
Xuezhi Cao, Qiang Ding, Jie Lu, Wanyin Tao, Bing Huang, Yanan Zhao, Junqi
Niu, Yong-Jun Liu, Jin Zhong
PII:S0168-8278(14)00850-2
DOI:https://www.doczj.com/doc/146622711.html,/10.1016/j.jhep.2014.11.007
Reference:JHEPAT 5426
To appear in:Journal of Hepatology
Received Date:7 July 2014
Revised Date: 5 November 2014
Accepted Date:7 November 2014
Please cite this article as: Cao, X., Ding, Q., Lu, J., Tao, W., Huang, B., Zhao, Y., Niu, J., Liu, Y-J., Zhong, J., MDA5 Plays a Critical Role in Interferon Response during Hepatitis C Virus Infection, Journal of Hepatology (2014), doi: https://www.doczj.com/doc/146622711.html,/10.1016/j.jhep.2014.11.007
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MDA5 Plays a Critical Role in Interferon Response during Hepatitis C Virus Infection 2
Short title: MDA5 senses HCV infection to induce IFN response
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Xuezhi Cao a,1, Qiang Ding a,1, Jie Lu a, Wanyin Tao a, Bing Huang a, Yanan Zhao a, Junqi Niu a,b, 5
Yong-Jun Liu a,c, and Jin Zhong a,2
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a Key Laboratory of Molecular Virology and Immunology, Institut Pasteur of Shanghai,
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Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 200031, China;
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b Department of Hepatology, First Hospital of Jilin University, Changchun, Jilin, 130021,
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China,
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c Baylor Institute for Immunology Research, 3434 Live Oak, Dallas, TX 75204, USA.
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1X.C. and Q.D. contributed equally to this work.
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2Corresponding author: Institut Pasteur of Shanghai, Chinese Academy of Sciences, 320
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Y ueyang Road, Shanghai, 200031, China. Phone: (86) 21-54923143. Fax: (86) 21-54923142.
E-mail: jzhong@https://www.doczj.com/doc/146622711.html,.
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Word counts: 5972 words
Number of figures and t ables: 7 Figures
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List of abbreviations:
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HCV: Hepatitis C Virus
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RIG-I: Retinoic Acid Inducible Gene
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MDA5: Melanoma Differentiation-Associated Protein
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IFN: Interferon
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UTR: Untranslated Region
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MAVS: Mitochondrial Antiviral Signaling Protein
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PAMP: Pathogen-associated Molecular Patterns
HCVcc: HCV cell culture
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Conflict of interest: We d eclare that we have no conflict of interest.
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Financial support: This work was supported by grants from National Natural Science 14
Foundation of China (81330039), Chinese National Science and Technology Major Project 15
(2012ZX10002007-003), the CAS/SAFEA International Partnership Program for Creative 16
Research Teams, Shanghai Pasteur Health Research Foundation (SPHRF2013002).
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Author contributions: X.C., Q.D., J.Z. contributed to study design, X.C., Q.D., J.L., W.T.,
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B.H., Y.Z. contributed to data acquisition and analysis, Q.D., X.
C., J.Z. contributed to
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manuscript writing, and J.Z., J.N., Y.L. contributed to study supervision and obtaining funding.
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ABSTRACT (243 Words)
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Background and aims: Hepatitis C virus (HCV) is a human pathogen that can evade host
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immunity to cause persistent infection, leading to liver cirrhosis and hepatocellular carcinoma.
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The transfected 3’UTR of HCV genomic RNA can be recognized by host protein RIG-I to
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activate interferon production in hepatocytes. However, it is difficult to demonstrate the
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RIG-I mediated sensing of HCV genomic RNA in the context of HCV infection because
HCV encoded-NS3-4A protease can inactivate MA VS, a critical adaptor protein in interferon 7
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signaling. Our aim was to identify the viral sensor that triggers interferon response in
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hepatocytes during HCV infection.
Methods: We generated a hepatic cell line that stably express mutant MA VS resistant to the
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NS3-4A cleavage. This cell line allowed us to investigate interferon signaling pathway in the
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context of HCV infection. By using the knockdown and knockout technology together with
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biochemical approaches, we were able to identify the actual viral sensor in hepatocytes
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during HCV infection.
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Results:We showed that HCV infection induced robust interferon response in the cells
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expressing MAVS resistant to the NS3-4A cleavage. Unexpectedly, the interaction between
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HCV’s 3’UTR and RIG-I seemed to play a minor role in this activation, while another
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helicase MDA5 played a more important role in sensing HCV infection to trigger interferon
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response.
Conclusions:Our data demonstrate that MDA5 recognizes HCV to initiate host innate
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immune response during HCV infection. This study provides insight into how host senses
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HCV to initiate innate immunity during HCV infection. 2
Keywords: HCV, MDA-5, Interferon, Innate Immunity 3
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INTRODUCTION
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The reaction of host to invading microbes is an innate immune response initiated by host
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pattern recognition receptors (PRRs) that recognize components specific to microorganisms
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called pathogen-associated molecular patterns (PAMPs) [1]. There are three major classes of
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PRRs: Toll-like receptors (TLRs), RIG-I–like helicases (RLHs) and NOD-like receptors
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(NLRs). RLHs comprise of RIG-I, MDA5, and LGP2, which all contain DExD/H helicase
domain. RIG-I and MDA5 both contain two CARD domains at the N terminal [2] . The
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binding of viral RNA to the C termini of RIG-I and MDA5 presumably induces a
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conformational change that exposes the N-terminal CARD domains, which interact with the
CARD domain of the mitochondrial adaptor protein MA VS (also known as IPS1, VISA, or 10
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CARDIF) [3, 4]. MA VS then activates the cytosolic kinases IKK and TBK1 [5], which
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activate the transcription factors NF-κB and IRF3, respectively. NF-κB and IRF3 translocate
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into the nucleus collectively eliciting innate antiviral immune responses, including production
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of type I and III interferons [6, 7]. RIG-I and MDA5 sense different types of ligands and
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distinct subsets of RNA viruses. Cytoplasmic 5’triphosphorylated ssRNA and short poly(I:C)
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are recognized by RIG-I [8, 9]. Long cytoplasmic dsRNA, such as long poly(I:C), is
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recognized by MDA5 [8]. This distinct ligand preference has been shown to confer specific
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recognition of individual viruses: RIG-I is required for interferon production in response to
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several paramyxoviruses, influenza virus, and Japanese encephalitis virus [10], whereas
MDA5 is crucial for the host defense against picornaviruses [11]. RIG-I and MDA5 are
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individually dispensable for signaling in response to reovirus or dengue virus (DEN)
infection. RIG-I and MDA5 cooperate to trigger an innate immune response to West Nile
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virus (WNV) [11].
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Hepatitis C virus (HCV) infects approximately 170 million people worldwide, and 80%
of the infected individuals develop persistent infection. HCV contains a 9.6-kb positive-sense 4
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RNA genome encoding a 3,000-aa polyprotein which is cleaved into at least ten structural
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and nonstructural (NS) proteins by cellular and viral proteases. The 3’-untranslated region
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(3’UTR) of HCV genomic RNA, essential for viral genome replication, contains a 100-nt
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polyU/UC tract flanked by a variable region at the 5’end and a conserved region at the 3’ end.
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Previous studies showed that in vitro transcribed HCV 3’UTR RNA can be recognized by
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RIG-I to trigger innate immune response after it is delivered into Huh-7 hepatic cells by
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transfection [12, 13]. Biochemical analyses revealed the direct physical interaction between
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RIG-I and HCV 3’UTR RNA, and the polyU/UC tract plays the essential role [14]. However,
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the physiological relevance of these results remains to be addressed as the RIG-I’s
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recognition of HCV RNA to initiate the interferon response was difficult to demonstrate in
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the context of HCV infection, in spite of availability of the infectious cell culture model
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(HCVcc) [15-17]. HCVcc infection in Huh-7 cells fails to trigger the production of detectable
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amounts of type I interferons [3, 18], because HCV-encoded NS3-4A serine protease can
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cleave MA VS/VISA at cysteine of position 508, right before its mitochondrial targeting
domain. This cleavage releases MAVS from the mitochondria and thus blocks the
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downstream interferon activation [3, 6, 19]. Therefore, it remains elusive whether HCV does
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possess the ability to trigger the interferon response during its replication life cycle, and
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whether this activation is mediated by RIG-I.
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To address these questions, we generated an Huh-7 hepatoma cell line that expresses a 4
MAVS mutant resistant to the NS3-4A cleavage. HCV infection induces robust interferon production in this cell line. However, we found that this interferon activation is mainly
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dependent upon MDA5. Our work provides important insight into how host cells sense HCV 7
to initiate the interferon signaling during HCV infection.
Materials and methods
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Preparation of HCV 3’UTR RNA, RNA isolation, RNA transfection, 3
Coimmunoprecipitation, Western blot, and HCVcc stock preparation.
The protocols were as previously described [6, 20].
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Knockdown assays.
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The RIG-I or MDA-5 siRNAs, the mix of 3-5 siRNA targeting different sites were
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purchased from Santa Cruz. Control siRNA was a nontargeting pool (D-001810-10-20)
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from Dharmacon (Lafayette, CO, USA). siRNAs were transfected into cells following the
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manufacture’s instruction.
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Generation of RIG-I or MDA5 knockout Huh-7-MA VSR cells by lenti-CRISPR-Cas9. 11
HEK293T cells were seeded into 6-well plates one day prior to transfection at a density 12
of 7×105 cells per well. The cells were co-transfected with 1.3-μg VSV-G expressing plasmid, 13
2.5-μg pCMV-dR8.91plasmid expressing gag, pol and rev genes, and 2.5-μg 14
lenti-Cas9-sgRNAs [21] targeting EGFP, RIG-I or MDA5 using Lipofectamine 2000 15
(Invitrogen). Viral supernatants were harvested at 48 h post-transfection, filtered and used to 16
infect Huh-7-MA VSR cells. After 48h post-infection, 0.75 μg/ml puromycin was added into 17
medium for long time culture. The knockout of RIG-I and MDA5 was verified by sequencing 18
and Western blot.
Luciferase reporter assay.
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Huh-7-MASVR cells were seeded into 48-well plates overnight prior to infection with
JFH1 at the indicated moi. At 24 h post-infection, the cells were transfected with 20 ng/well 1
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of the IFN-β-Luc construct [22] and 20 ng/well of the CMV promoter driven-Renilla
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luciferase vector (pRL-CMV; Promega) used for normalizing transfection efficiency. One day
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after the plasmid transfection, the cells were transfected with 400 ng/well HCV 3’UTR or
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L-poly(I:C) and harvested at 18 h post-transfection. Cell lysates were assayed for luciferase
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activities using the Dual-Luciferase Reporter Assay System (Promega) following the
manufacturer’s instructions.
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Primary human hepatocytes culture, knockdown and infection.
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Primary human hepatocytes (purchased from Research Institute for Liver Diseases,
Shanghai, China) were plated in complete DMEM at a density of 1×105 cells/cm2 on 48-well 10
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plates coated with type 1 collagen. The medium was changed to serum-free differentiation
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(SFD) medium [23] at 12 h after plating. To generate lentiviruses expressing shRNA
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targeting RIG-I (5-‘CCA GAA TTA TCC CAA CCG ATA-3’) or MDA5 (5-‘CCA ACA AAG
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AAG CAG TGT A TA-3’), empty pLKO.1 plasmid or plasmids bearing the RIG-I or MDA5
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shRNA were transfected into HEK293T cells respectively, together with packaging plasmid
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psPAX2 and envelope plasmid pMD2.G. The produced lentiviruses were collected at day 2
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post-transfection, passed through a 0.45-μm filter, and then inoculated to primary human
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hepatocytes. Two days later the lentivirus-transduced cells were infected with HCVcc at an
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moi of 2. The cells were collected at 48 h post-infection for RT-qPCR analysis of interferon
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activation.
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RESULTS
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Generation of an Huh-7 cell line expressing MA VS mutant resistant to HCV NS3-4A
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protease cleavage.
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HCV NS3-4A protease can cleave the MAVS protein at Cys-508 to subvert the innate
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immune response [3, 19]. As the result, HCV infection does not trigger detectable amounts
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of interferon production in the recently developed HCV in vitro cell culture model [15, 16,
22]. To identify the host sensor that recognizes HCV infection, we used the lentiviral vector 7
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to generate Huh-7 cell lines that stably express the FLAG-tagged wild-type MA VS or
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mutant MA VS in which the cysteine at position 508 was changed to arginine (C508R). The
C508R mutation has been shown previously to block the NS3-4A proteolytic cleavage while
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maintain the function of MA VS in the interferon signaling [3]. It was shown that the
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exogenously introduced wild-type or C508R mutant MA VS were mildly expressed in the
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stable cell lines, designated Huh-7-MA VS and Huh-7-MAVSR respectively, and did not
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significantly increase the basal levels of IFN-β mRNA (Supplemental Figure 1).
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Next, we compared the abilities of these cell lines to produce IFN-βfollowing HCV
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3’UTR RNA or long poly(I:C) (L-poly(I:C)) stimulation, which are recognized by RIG-I or
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MDA5, respectively [8, 12, 13]. As shown in Figure 1A, the IFN-β levels in the two cells
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stimulated by HCV 3’UTR RNA or L-poly(I:C) were comparable, suggesting that the
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C508R mutation in MA VS does not alter RIG-I- or MDA5-mediated interferon signaling.
Next we tested how these two cells respond to the stimuli in the presence of the HCV
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NS3-4A. The NS3-4A expression plasmids were transduced into Huh-7-MA VS and
Huh-7-MA VSR cells prior to the stimulation with HCV 3’UTR RNA and L-poly(I:C). 1
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Immunoblotting assays were performed to analyze the NS3-4A expression and the MA VS
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cleavage, and IFN-β mRNA was determined by RT-qPCR assay. As illustrated in Figure 1A
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and B, NS3-4A cleaved MA VS and dramatically inhibited 3’UTR RNA- and
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L-poly(I:C)-induced interferon production in Huh-7-MA VS cells, but failed to do so in
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Huh-7-MA VSR cells. The slight decrease of IFN-βlevels in the NS3-4A transfected
Huh-7-MA VSR cells could result from the cleavage of endogenous MAVS by NS3-4A or 7
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other previously reported NS3-4A blocking mechanism independent of the MA VS cleavage
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[24, 25]. These results suggest that, in consistent with the previous findings, blockade of the
MA VS cleavage by the HCV NS3-4A protease could largely restore the interferon signaling
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mediated by RIG-I or MDA5. Therefore, Huh-7-MAVSR cells can be used to identify the
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host sensors for HCV during HCV infection.
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HCV infection induces strong interferon production in Huh-7-MA VSR cells.
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To exam whether HCV infection can induce interferon production in Huh-7-MA VSR
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cells, we infected Huh-7-MA VS and Huh-7-MA VSR cells with HCVcc (JFH1 strain) at an
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moi of 2. The IFN-βand HCV RNA levels were determined at the indicated time by
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RT-qPCR, and the HCV NS3 protein levels and MAVS cleavage were determined by Western
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blot. We found that HCV infection can induce strong interferon expression at 72 h
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post-infection in Huh-7-MA VSR cells but not in Huh-7-MA VS cells in which MA VS was
cleaved (Figure 2A and 2C). The interferon induction was confirmed at the protein level by
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IFN-β ELISA (Supplemental Figure 2). As expected, the interferon production was
accompanied by the phosphorylation of IRF3 and nuclear relocation of p65 subunit of NF-κB, 1
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important prerequisite for the activation of IFN-βtranscription (Figure 2D and 2E). No
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significant cytopathic effect was observed in either cell up to 96 h post-infection (data not
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shown). These results suggest that HCV does possess the ability to trigger the interferon
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production during its infection life cycle which is inhibited by the HCV encoded NS3-4A
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protease, and the blunt of MA VS cleavage by the NS3-4A protease could restore the
interferon production. In addition, as expected, the HCV RNA and NS3 levels negatively 7
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correlated with the IFN-β mRNA level (Figure 2B and 2C), likely due to the antiviral effects
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of induced interferons in the Huh-7-MA VSR cells.
To determine whether the interferon induction is dependent on HCV genomic RNA
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replication, we used increasing doses of BMS-790052, a potent HCV NS5A inhibitor [26], to
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treat the Huh-7-MA VSR cells at 48 h post-infection and harvested cells at 72 h post-infection
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to analyze the IFN-βmRNA and HCV NS3 protein levels. As shown in Figure 2F,
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BMS-790052 inhibited HCV replication and reduced the interferon production in a dose
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dependent manner, suggesting the HCV replication is necessary for the interferon induction.
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In addition, we found that higher moi infection (moi=10) induced faster and higher interferon
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production (Supplemental Figure 3).
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HCV infection induces strong interferon production in RIG-I-deficient
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Huh-7.5.1-MAVSR cells.
It has been shown that the polyU/UC tract within HCV 3’UTR is recognized by RIG-I to
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initiate innate immune cascade [12, 13]. To examine the role of RIG-I in HCV
infection-induced interferon production, we transduced Flag-MA VS or Flag-MAVS(C508R) 1
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into RIG-I deficient Huh-7.5.1 cells that carry with T55I mutation in the first CARD of RIG-I
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to disrupt the interaction of RIG-I with MA VS [15, 27] to generate Huh-7.5.1-MAVS and
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Huh-7.5.1-MA VSR cells respectively.
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We firstly stimulated na?ve Huh-7.5.1, Huh-7.5.1-V ector, Huh-7.5.1-MA VS,
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Huh-7.5.1-MA VSR and Huh-7 cells with HCV 3’UTR RNA or L-poly(I:C). As shown in
Figure 3A, L-poly(I:C), recognized by MDA5, can induce significant IFN-β expression in the 7
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all tested cells, whereas 3’UTR RNA, recognized by RIG-I, can only induce significant
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IFN-βproduction in Huh-7 cells, but not in the RIG-I-deficient Huh-7.5.1 derived cells,
consistent with the previous reports that RIG-I plays an important role in the HCV 3’UTR 10
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RNA-induced interferon responses [12, 13].
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Next, we tested whether HCV infection induces the interferon response in these cells.
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The cells were infected with JFH1 at an moi of 2, and collected at 72 h post-infection to
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determine IFN-β mRNA level (Figure 3B, top) and the cleavage of MA VS by HCV NS3-4A
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(Figure 3B, bottom). To our surprise, HCV infection induced strong interferon production in
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Huh-7.5.1-MA VSR cells (Figure 3B, top), suggesting that the requirement for RIG-I is
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different between HCV infection- and HCV 3’UTR RNA transfection-induced interferon
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signaling.
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It has been demonstrated that the HCV 3’UTR binds RIG-I to activate the interferon
signaling pathway. In a previous study, we isolated a mutant 3’UTR (denoted MUT) which
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has a much shorter polyU/UC tract (Supplemental Figure 4) and induces no significant
interferon production compared to the wild-type 3’UTR after transfected into Huh-7 cells 1
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(Figure 3C). To test how HCVcc containing MUT-3’UTR induces interferon production, we
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made the recombinant HCVcc (JC1) with the wild-type or the MUT 3’UTR. Then we
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infected Huh-7-MA VS and Huh-7-MA VSR cells with these viruses for 3 days, and
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determined the IFN-βand HCV RNA levels. The sequencing analysis confirmed that the
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MUT 3’UTR was retained in the mutant virus after the 3-day infection (data not shown). As
shown in Figure 3D, MUT and wild-type viruses induced similar levels of interferon 7
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production, further confirming that HCV infection- and HCV 3’UTR RNA
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transfection-induced interferon signaling have a different requirement for RIG-I.
MDA5 is required for HCV infection-induced interferon production.
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Next we determined the role of MDA5, another RNA helicase in RIG-I-like receptor
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family [28], in HCV infection-induced interferon signaling. We used the MDA5-specific
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siRNA to knock down the MDA5 expression in Huh-7-MA VSR cells after HCVcc infection.
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The cells were harvested at 72 h post-infection to determine the MDA5 and IFN-β mRNA
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levels. As the controls to test the function of MDA5, HCV 3’UTR RNA (RIG-I ligand) and
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L-poly(I:C) (MDA5 ligand) were transfected into the MDA5-knockdown Huh-7-MAVR cells.
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As shown in Figure 4A, siRNA targeting MDA5 efficiently decreased the MDA5 mRNA
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abundance. L-poly(I:C) and HCV infection-induced IFN-βproduction was dramatically
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impaired, while the HCV 3’UTR RNA-induced IFN-βproduction was not in the
MDA5-knockdown Huh-7-MA VR cells, suggesting that MDA5 is required for HCV
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infection-induced interferon production.
Parainfluenza virus 5 (PIV5) encodes V protein that blocks induction of 1
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MDA5-mediated type I interferon but does not antagonize RIG-I [29-31]. To determine
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whether V protein can block HCV infection induced interferon production, we transfected
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plasmids expressing PIV5-encoded V protein into the JFH1-infected Huh-7-MA VR cells
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together with the IFN-βpromoter-driven luciferase reporter plasmid. The cells were
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harvested at 72 h post-infection for luciferase assay. As the controls to test the function of V
protein, HCV 3’UTR RNA and L-poly(I:C) were included in parallel. As shown in Figure 4C, 7
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V protein efficiently inhibited L-poly(I:C) or HCV infection induced IFN-β production, but
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had no effect on HCV 3’UTR RNA induced IFN-β production. Taken together, our results
suggested that MDA5 plays an important role in HCV infection-induced interferon response.
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It is known that Sendai virus (SeV) is recognized by RIG-I to induce innate immune
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response [32, 33]. Therefore, we included SeV in our assay to test the differential roles of
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RIG-I and MDA5 in the recognition of virus infection to initiate innate immune response. As
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shown in Figure 5A and B, siRNA targeting RIG-I or MDA5 can specifically and efficiently
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inhibit RIG-I and MDA5 expression. Importantly, knockdown of RIG-I dramatically reduced
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SeV-induced interferon signaling while had no effect on the HCV infection-induced
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interferon production. In contrary, knockdown of MDA5 significantly decreased HCV
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infection-induced interferon signaling while had no effect on the SeV-induced interferon
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production. Our results demonstrated that, unlike SeV that is sensed by RIG-I, HCV is
mainly recognized by MDA5 to induce innate immune response during its infection.
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Next we examined the molecular interaction of MA VS and RNA sensors in the context
of HCV infection. Huh-7-MA VSR cells were infected with JFH1 at an moi of 5, and analyzed 1
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for the interaction between MA VS and MDA5 or RIG-I by coimmunoprecipitation assay. As
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shown in Figure 5C, MDA5 but not RIG-I specifically interacted with MAVS following HCV
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infection (Figure 5C), further confirming the role of MDA5 in the interferon response to
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HCV infection in Huh-7-MA VSR cells.
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It was reported that RIG-I may contribute to the HCV-infection induced interferon
signaling in the early time of HCV infection [34]. To systemetic evaluate the role of RIG-I 7
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and MDA5 in the innate sensing of HCV infection, we generated RIG-I and MDA5 knockout
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Huh-7-MA VSR cells using the CRISPR/Cas9 technology [21]. Two knockout cells were
generated for RIG-I and MDA5 respectively by using 2 different sgRNA targeting sequences,
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and the knockout was verified by DNA sequencing (Supplemental Figure 5) and Western blot
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(Figure 6A). As expected, L-poly(I:C) but not HCV 3’UTR RNA transfection induced the
13
interferon signaling in the RIG-I knockout Huh-7-MA VSR cells, whereas HCV 3’UTR RNA
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but not L-poly(I:C) transfection induced the interferon signaling in the MDA5 knockout
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Huh-7-MA VSR cells (Figure 6B). Next we determined the interferon production at the
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different time points after the cells were infected by HCVcc at an moi of 5. Consistently, the
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knockout of MDA5 significantly impaired the induction of IFN-β, IL-28 and IL29.
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Interestingly, the knockout of RIG-I reduced the induction fold of interferons at day 2 and 3
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post-infection, but had no apparent effect on the interferon production at day 4 post-infection
(Figure 6C-E). Again, the HCV RNA levels negatively correlated with the host interferon
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21
response (Figure 6F). These results suggest that RIG-I may play some roles in early sensing
1
HCV infection, but MDA5 plays a more critical role in interferon signaling activation during 2
HCV infection.
3
Finally we tested the role of MDA5 in sensing HCV infection in primary human 4
hepatocytes. Primary human hepatocytes were first transduced with lentiviruses expressing 5
shRNA targeting RIG-I or MDA5, and then infected with JFH1 virus. The cells were 6
analyzed at 48 h post-infection for the knockdown efficiency, HCV RNA, IFN-β and ISG15 7
(Figure 7A-D respectively) by RT-qPCR assay. The results suggested that knockdown of 8
MDA5 had a more dramatic effect on the interferon activation, indicating a more 9
predominant role in innate immune response against HCV infection.
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11
DISCUSSION
12
Previous studies have shown that HCV 3’UTR polyU/UC is recognized by RIG-I to
13
trigger innate immune response in an artificial RNA transfection model [12, 13]. In this study, 14
we established a novel HCV infectious cell culture model to study host innate immune
15
response in the context of HCV infection. We demonstrated that the authentic HCV infection 16
is capable of inducing interferon production that is mainly dependent upon MDA5 rather than 17
RIG-I. First, decrease of RIG-I expression by knockdown/knockout has much less effect on 18
the HCV infection-induced interferon production than HCV 3’UTR RNA
19
transfection-induced interferon production. Second, HCV infection induces strong interferon production in Huh-7.5.1-MAVSR, a RIG-I deficient cell line in which HCV 3’UTR RNA
20
21
fails to induce the interferon signaling. In addition, we found that HCVcc consisting of a
mutant 3’UTR (MUT) unable to induce interferon signaling upon RNA transfection indeed
1
2
triggers interferon expression comparable to its wild-type HCVcc, indicating that the 3’UTR 3
of HCV genomic RNA may not be the native PAMP that triggers interferon production during natural HCV infection.
4
5
Huh-7.5 (derived from Huh-7 cells) and Huh-7.5.1 cells (derived from Huh-7.5 cells)
6
have been reported to support better HCV replication than their parental Huh-7 cells [17, 35]. 7
Because both Huh-7.5 and Huh-7.5.1 cells have a point mutation in RIG-I to impair the
8
interaction of RIG-I with MA VS [27, 36], the better permissiveness for HCV replication in
9
these two cells has been accredited to its defective RIG-I-mediated interferon signaling.
10
However, M. Binder et al. reported that neither blocking interferon activation in Huh-7 cells 11
by expression of a dominant-negative RIG-I nor reconstituting RIG-I signaling in Huh-7.5 by 12
expression of wild-type RIG-I had any impact on HCV replication [37]. These findings
13
suggest the increased permissiveness of Huh-7.5 and Huh-7.5.1 cells for HCV replication is 14
not due to the presence of a deficient RIG-I signaling pathway. It is worth of noting that the 15
interferon signaling in Huh-7.5.1 cells can be induced by L-poly(I:C), a known inducer of
16
MDA5-mediated interferon signaling[38], while other studies have shown that the interferon 17
signaling could not be induced by poly(I:C) in parental Huh-7.5 cells [37]. This discrepancy 18
could be due to the different sources of poly(I:C) and possible differences between Huh-7.5.1 and Huh-7.5 cells.
19
20
Interestingly, we found that MDA5, known to recognize dsRNA, may recognize HCV
PAMP to induce innate immune response. The exact nature of the HCV PAMP that MDA5 1
2
recognizes still needs to be further characterized. Paramyxoviruses encode V protein that 3
specifically interacts with MDA5 to block interferon induction [29-31]. We found that V 4
protein inhibits L-poly(I:C)- and HCV infection-induced IFN-β production, but has no effect 5
on HCV 3’UTR RNA-induced IFN-β production in Huh-7-MA VR cells, suggesting MDA5 is 6
required for HCV infection-induced innate immune response. In 2011, Charles Rice et al.
found that ectopic expression of paramyxovirus V proteins significantly promote HCV 7
8
infection of primary human fetal liver cells [39], which highlights the potential role of MDA5 9
in HCV induced innate immunity.
It was recently demonstrated that PKR may be also involved in sensing HCV infection
10
11
to trigger the innate immune response [34]. We determined whether PKR plays any role in 12
the interferon production in the JFH1 infected Huh-7-MA VSR cells. Indeed, knockdown of 13
PKR significantly reduces HCV infection-induced interferon production in Huh-7-MA VSR 14
cells (Supplemental Figure 6A and 6B). However, interestingly, unlike MDA5, knockdown of 15
PKR also reduces the HCV 3’UTR-induced interferon production (Supplemental Figure 6C), 16
suggesting a more general role of PKR in the interferon activation. The exact role of PKR in 17
the interferon activation in the context of HCV infection and its relationship with MDA5 will 18
need to be further studied.
19
Interestingly, we noticed that the HCV infection-induced interferon production occurred at day 3 post-infection, a significant delay compared to the kinetics of HCV genomic RNA
20
21
levels (Fig 2A-2B), suggesting that HCV RNA replication does not immediately activate the
play a part (in) 重要程度：★☆☆☆☆难易程度：★★☆☆☆ Have you realized the part computers have ___________ in the daily life? A. made B. given C. caused D. played 【参考答案】D 【拓展延伸】 1. play a part (in) 在……中扮演一个角色；参与；在……中起作用 2. play the role of 扮演……的角色 play an important role / part in...在……中起重要作用 play the leading role / part主演；起带头作用 3. take part in 参加 for the most part多半；在很大程度上 for one’s part就某人而言，对某人来说 1. Colors play an important ___________ in the way you look. A. part B. form C. effect D. pride 2. Mr. Huang will ___________ in the movement. A. play a leading part B. take parts C. play leading part D. take a part 3. __________ part that women ___________ in society is great. A. The; plays B. A; takes C. A; plays
CPU的技术参数的意思1 CPU（Central Processing Unit) 也就是我们常说的中央处理器,就一般的用户来说,它不是装机配件中最昂贵的,但它是电脑当中最核心的配件,一台电脑的性能如何跟CPU的性能有着最直接的关系.而且CPU的选择也同时关系到主板和内存的搭配问题!! 为了让大家更清晰地了解CPU,我们先来了解CPU的一些基本的概念. CPU重要参数介绍： 1）前端总线:英文名称叫Front Side Bus,一般简写为FSB.前端总线是CPU跟外界沟通的唯一通道,处理器必须通过它才能获得数据,也只能通过它来将运算结果传送出其他对应设备.前端总线的速度越快,CPU的数据传输就越迅速.前端总线的速度主要是用频率来衡量,前端总线的频率有两个概念:一就是总线的物理工作频率（即我们所说的外频）,二就是有效工作频率（即我们所说的FSB频率）.由于INTEL跟AMD采用了不同的技术,所以他们之间FSB频率跟外频的关系式也就不同了.现时的Inter是:FSB频率=外频X4;而AMD的就是:FSB频率=外频X2.举个例子：P4 2.8C的FSB频率是800MHZ,由那公式可以知道该型号的外频是200MHZ了;又如BARTON核心的Athlon XP2500+ ,它的外频是166MHZ,根据公式,我们知道它的FSB频率就是333MHZ了.目前的前端总线频率,这一点Intel还是有优势的.
2）二级缓存:也就是L2 Cache,我们平时简称L2.主要功能是作为后备数据和指令的存储.L2容量的大小对处理器的性能影响很大.因为L2需要占用大量的晶体管,是CPU晶体管总数中占得最多的一个部分,高容量的L2成本相当高!!所以INTEL和AMD都是以L2容量的差异来作为高端和低端产品的分界标准! 3）制造工艺:我们经常说的0.18微米、0.13微米制程,就是指制造工艺.制造工艺直接关系到CPU的电气性能.而0.18微米、0.13微米这个尺度就是指的是CPU核心中线路的宽度.线宽越小,CPU的功耗和发热量就越低,并可以工作在更高的频率上了.所以0.18微米的CPU 能够达到的最高频率比0.13微米CPU能够达到的最高频率低,同时发热量更大都是这个道理. 4）流水线:流水线也是一个比较重要的概念.CPU的流水线指的就是处理器内核中运算器的设计.这好比我们现实生活中工厂的生产流水线.处理器的流水线的结构就是把一个复杂的运算分解成很多个简单的基本运算,然后由专门设计好的单元完成运算.CPU流水线长度越长,运算工作就越简单,CPU的工作频率就越高,不过CPU的效能就越差,所以说流水线长度并不是越长越好的.由于CPU的流水线长度很大程度上决定了CPU所能达到的最高频率,所以现在INTEL为了提高CPU的频率,而设计了超长的流水线设计.Willamette和Northwood核心的流水线长度是20工位,而如今上市不久的Prescott 核心的P4则达到了让人咋舌的30(如果算上前端处理，那就是31)工位.而现在AMD的Clawhammer K8,流水线长度仅为11工位,当然
3.1中介语理论产生的历史背景 60年代是对比分析的兴盛时期。70年代初开始衰落,反映了一种历史的必然,因为这种理论方法无论在理论上还是实践上都面临着严重的危机。 因此,语言学家们为语言教师勾画了这样一幅图景:首先,语言学家们通过两种语言系统(L1和L2)的对比,为语言教师提供一个详细的菜单。这个菜单包括两种语言的相同点与不同点。然后,语言教师便依据这些不同点来预测学习者的难点,并据此来编写教学大纲和教材。但是后来的教学研究和实践证明,语言学家的许诺仅仅是一幅理想的图画而已。70年代初,对比分析遭到激烈的批评。如果第二语言学习者产生的错误完全可以通过两种语言的对比来预测。由此推论,语言的差异等于学习的难点，学习的难点必然导致语言表达的错误。问题是,语言差异是语言学上的概念,学习的难点则是心理学上的概念。学习的难点无法直接从两种语言差异的程度来推测。教学实践也证明,依据对比分析确认的难点事实上并不完全导致错误的产生。对比分析的理论方法存在的致命弱点,如果归结为一句话,那就是,人们试图用简单的语言学的方法去解决复杂的心理学的问题。语言习得涉及到学习的主体和客体的方方面面,对比分析却仅仅局限于语言系统的对比,忽略了学习者这一主体以及作为学习客体的学习过程。由于对比分析在理论与实践上的危机,人们呼吁一种新的理论的诞生,并要求这种新的理论把目光投向学习的主体和客体。早期的中介语理论正是在这种历史背景下产生的。 3.2中介语的概念 于根元、鲁健骥等是在中国应用语言学领域，最早进行了介绍、评述和研究中介语的意义、特点和研究方法。我们来看他们是怎么界定中介语的。于根元认为，所谓中介语就是介于习得语和目的语之间的独立的语言系统，他是第二语言习得者创造的语言系统。鲁健骥认为，中介语指的是由于学习外语的人在学习过程中对于目的语的规律所做的不正确的归纳与推论而产生的一个语言系统,这个语言系统既不同于学习者的母语,又区别于他所学的目的语。 3.3中介语出现的根源 我们着重重复一下鲁健骥对中介语的定义：中介语指的是由于学习外语的人在学习过程中对于目的语的规律所做的不正确的归纳与推论而产生的一个语言
CPU的主要性能参数 主频 通常所说的某某CPU是多少兆赫的，而这个多少兆赫就是“CPU的主频”。主频也叫时钟频率，单位是GHZ，用来表示CPU的运算速度。CPU的主频＝外频×倍频系数。 有人以为认为CPU的主频指的是CPU运行的速度，实际上这个认识是很片面的。CPU的主频表示在CPU内数字脉冲信号震荡的速度，与CPU实际的运算能力是没有直接关系的。当然，主频和实际的运算速度是有关的，但目前还没有一个确定的公式能够定量两者的数值关系，因为CPU的运算速度还要看CPU的流水线的各方面的性能指标（缓存、指令集，CPU的位数等等）。由于主频并不直接代表运算速度，所以在一定情况下，很可能会出现主频较高的CPU实际运算速度较低的现象。因此主频仅仅是CPU性能表现的一个方面，而不代表CPU的整体性能。 外频 外频是CPU与主板上其它设备进行数据传输的物理工作频率，也就是系统总线的工作频率。它代表着CPU与主板和内存等配件之间的数据传输速度。单位也是MHz。CPU标准外频主要有66MHz、100MHz、133MHz、166MHz、200MHz几种。 外频也是内存与主板之间的同步运行的速度，在这种方式下，可以理解为CPU的外频直接与内存相连通，实现两者间的同步运行状态。 倍频 倍频系数是指CPU主频与外频之间的相对比例关系。在相同的外频下，倍频越高CPU的频率也越高。但实际上，在相同外频的前提下，高倍频的CPU本身意义并不大。这是因为CPU与系统之间数据传输速度是有限的，一味追求高倍频而得到高主频的CPU就会出现明显的“瓶颈”效应——CPU从系统中得到数据的极限速度不能够满足CPU运算的速度。 理论上倍频是从1.5一直到无限的，但需要注意的是，倍频是以以0.5为一个间隔单位。 倍频一般是不能改的，现在的CPU基本都对倍频进行了锁定。 CPU的其它参数
中介语简介中介语理论是二语习得中的一个重要理论，它产生于20世纪70年代初并于80年代初被介绍到我国，对我国的外语教学产生了巨大的推动作用，人们也逐步认识到中介语在外语教学中的积极作用。Selinker在其论文Language Transfer （1969）中首次使用了“interlanguage”一词，并于1972发表了题为Interlanguage的研究论文。Selinker认为，中介语是第二语言学习者独立的语言系统，在结构上处于母语和目的语的中间状态（1972）。 中介语在英语学习中的干扰作用 一、中介语定义及特点 中介语（Interlanguage, 简称IL）一词是英国语言学家Selinker 1969 年首次提出. 现在又被译为过渡语、中间语、中继语、语际语等。它是指学习者在某一段时间内所创建的内在语言体系或学习者在整个学习过程中所构建的相互关联的语言体系。学习者在学习和使用第二语言时，不断接受和理解新的语音、语法知识，在此基础上逐渐形成自己的语言结构。并不断对其进行系统的预测调整，通过归纳和推论产生中介语。中介语的语言系统在结构上处于母语（Native Language）和目的语（Target Language）之间，具有独立性，并兼有两者的特点。综合国内外近来的研究,中介语有如下一些特征: 1．独立性 中介语是一个独立的语言系统，它既不同于母语, 又区别于外语, 也不能单纯的把它地看作外语学习过程中由于受母语的干扰而形成的混合体。中介语有其独特的语言规则，这些规则常常被学习者用来解释外语中固有而不规则的语言现象。 2．阶段性 中介语在逐渐进化的过程中，具有一定的阶段性。它是一个开放的体系，不是固定的一成不变的。这个体系在不断被新知识渗透的同时，修正原有知识，逐渐接近目的语。 3．动态性 在外语学习过程中，学习者的中介语在不断的发展变化。虽然它充满了错误，但由于新的语言规则有及强的扩展能力，它们处于不断的组合和变化中，因此中介语随着学习者的努力和交际需要而不断变化，由简而繁，由低而高，逐渐离开母语而接近目的语。如果我们设在母语与目的语之间的中介语为一个连续体，那么，在某一特定阶段，学习者的中介语可以用连续体上的某一点。中介语越接近目的语，说明学习者的外语水平越高。 4．系统性 中介语在每个阶段都表现出较强的系统性和内部一致性。它也是一个由内部要素构成的系统，就是说它有语音的、词汇的、语法的规则系统，而且自成体系。学习者对中介语的使
play a part in的用法 Do you know this pretty girl Right! She is Audrey Hepburn who played many classic roles in a great many famous films. For example, < Roman Holiday> is the one of the films which earned Hepburn her first Academy Award for Best Actress. Audrey Hepburn played the part/role of Princess Ann in this film. play the part/role of…扮演……角色
Audrey Hepburn played a leading part in
Audrey Hepburn also played leading roles in < Funny Face>and < My Fair Lady〉.( My Fair Lady 窈窕淑女影片讲述下层卖花女被语言学教授改造成优雅贵妇的故事，从头至尾洋溢着幽默和雅趣 .Funny Face 甜姐儿) Play a role in…在……中扮演角色，在……中起作用 play a part/role in doing sth. 在做某事方面起作用,参与做某事 We can?all?play?a?role/part?in?reducing?our?dependence?on?plastic, if we started to?take some small?steps?in?our?everyday?lives?to?be
中介语理论研究与第二语言教学 [摘要]中介语理论是第二语言习得研究领域中的一个认知理论。分析和研究中介语产生的根源和特点有助于了解第二语言习得机制,揭示第二语言习得的发展过程和规律。对提高教学效果有重大意义。[关键词]中介语;特点;教学 第二语言习得研究在近40年间取得了令人瞩目的成就。随着研究的不断深入和发展,人们越来越重视第二语言习得的心理过程。中介语理论把第二语言学习者的语言看作是一个内在的语言行为。因此,中介语的研究对外语教学方法论有着重要的意义。 一、中介语的概念 中介语(interlanguage)，也有人译为"过渡语"或"语际语",指的是第二语言学习者特有的一种目的语系统。是指在第二语言习得过程中,学习者通过一定的学习策略,在目的语输入的基础上所形成的一种既不同于其第一语言也不同于目的语,随着学习的进展向目的语逐渐过渡的动态的语言系统. 中介语理论认为，第二语言学习者在学习过程中所掌握和使用的目的语是一种特定的语言系统，这种语言系统在语音、词汇，语法、文化和交际等方面既不同于自己的第一语言，也不同于目的语，而是一种随着学习的进展向目的语的正确形式逐渐靠拢的动态的语言系统。由于这是一种介乎第一语言和目的语之间的语言系统，所以称之为“中介语”。 与lnterlanguage (中介语)相近的概念最早由Corder在论文《学习者错误的意义》中提出,他把学习者尚未达到的目的语语
言能力的外语能力称为过渡能力( transitional competence)。美国语言学Selinker于1969年在论文《语言迁移》中首先提出中介语假说(interlanguage)的概念。1971年,W. Nemsers在《外语学习者的相似系统》中提出了“approximative system”的概念。1972年Selinker在其著名论文《中介语》中提出的中介语假说, 对“中介语”这一概念进行较详细的阐述,是试图探索第二语言习得者在习得过程中的语言系统和习得规律的假说,在第二语言习得的研究史上有重大意义。从而确立了它在第二语言习得研究中的地位。Selinker指出:“中介语是一个独立的语言系统,它产生于学习者试图掌握第二语言所做的努力。”根据塞格林的定义,中介语既可是指第二语言学习者在学习过程中某一特定阶段中认知目标语的方式和结果的特征系统,即一种特定、具体的中介语言,也可以指反映所有学习者在第二语言习得整个过程中认知发生和发展的特征性系统,即一种普遍、抽象的中介语语言体系interlanguage continuum塞格林还指出中介语本身是一个阶段到过程的双重系统和庞大体系,即母语→中介语→目标语系统中的一个必然成分和过程。在这个系统中二语学习者从母语出发经过中介语到达目标语。并指出要到目标语必须经过中介语,中介语是第二语言认知中的必经之路。 二、中介语的产生 应用语言学领域中产生了对比分析方法(20世纪中期)。它通过对人们的母语以及所要学习的第二语言的语音、语法、词法、
CPU的簡介及主要性能指標 什麽是CPU？ CPU是英語※Central Processing Unit/中央處理器§的縮寫， CPU一般由邏輯運算單元、控制單元和存儲單元組成。在邏輯運算和控制單元中包括一些寄存器，這些寄存器用於CPU在處理資料過程中資料的暫時保存。 CPU主要的性能指標有： 主頻即CPU的時鐘頻率(CPU Clock Speed)。 這是我們最關心的，我們所說的233、300等就是指它，一般說來，< 主頻越高，CPU的速度就越快，整機的就越高。 時鐘頻率: CPU的外部時鐘頻率，由電腦主板提供，以前一般是66MHz，也有主板支援75各83MHz，目前Intel公司最新的晶片組BX以使用100 MHz的時鐘頻率。另外VIA 公司的MVP3、MVP4等一些非Intel的晶片組也開始支援100MHz的外頻。精英公司的BX主板甚至可以支援133 MHz的外頻。 內部緩存（L1 Cache）： 封閉在CPU晶片內部的快取記憶體，用於暫時存儲CPU運算時的部分指令和資料，存取速度與CPU主頻一致，L1緩存的容量單位一般爲KB。L1緩存越大，CPU 工作時與存取速度較慢的L2緩存和記憶體間交換資料的次數越少，相對電腦的運算速度可以提高。 外部緩存（L2 Cache）： CPU外部的快取記憶體，PentiumPro處理器的L2和CPU運行在相同頻率下的，但成本昂貴，所以 PentiumII運行在相當於CPU頻率一半下的，容量爲512K。爲降低成本Inter公司生産了一種不帶L2的CPU命爲賽揚，性能也不錯。 MMX技術是※多媒體擴展指令集§的縮寫。 MMX是Intel公司在1996年爲增強Pentium CPU在音像、圖形和通信應用方面而採取的新技術。爲CPU增加57條MMX指令，除了指令集中增加MMX指令外，還將CPU晶片內的L1緩存由原來的 16KB增加到32KB（16K指命+16K資料），因此MMX CPU 比普通 CPU在運行含有MMX指令的程式時，處理多媒體的能力上提高了 60％左右。
中介语石化现象
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浅谈中介语石化现象 1．引言 外语学习中存在一个普遍现象,绝大多数学习者的外语学习达到了一定程度后,就不再像学习的最初阶段稳步提高,而是处于停滞不前的徘徊状态,很难达到目的语这个理想的终点。1972年Selinker把这个现象定义为中介语的石化 (fossilization),此后相关研究、论著相继问世,中介语石化也成为目前二语和外语教育界研究的热门课题之一。 2．中介语石化现象的定义 中介语的石化概念是Selinker(1972)首先提出的:“石化就是母语的词条、规则和词系统倾向保留在与目的语相关的中介语中,不管学习者的年龄有多大,也不管学习者接受的解释和指导有多少,这种倾向都不会改变。”在外语学习中,学习者的语言是处于一种动态的发展变化状态,中介语是一种介于学习者母语与目的语之间的一个逐渐积累和逐渐完善的过程,整个过程形成一个连续体(continuum) 。其理论假设是,中介语的始点是学习者的母语,然后随着目的语、知识的不断摄人,中介语逐渐向目的语靠拢。外语学习过程就是一种以目的语为标准的不断调整和重组的连续体,是学习者在学习新语言过程中所使用的过渡语言。中介语是第二语言认知中的必经之路。理论上,随着语言习得的逐步发展,中介语会渐渐接近直到达到目的语水平。然而大量实验表明,当学习者达到一定程度后,中介语的某些特征就会趋于停滞状态,很难甚至无法消除,从而形成语言石化。 后来,Selinker (1992) 对石化现象进一步阐释:“语言的石化现象是指外语学习者的中介语的一些语言项目,语法规则和系统性知识趋向与固定下来的状态,年龄的增长和学习量的变化对改变这种固定状态不起作用。”Selinker 认为所有外语学习
计算机性能指标 (1)运算速度。运算速度是衡量计算机性能的一项重要指标。通常所说的计算机运算速度（平均运算速度），是指每秒钟所能执行的指令条数，一般用“百万条指令／秒”（mips，Million Instruction Per Second）来描述。同一台计算机，执行不同的运算所需时间可能不同，因而对运算速度的描述常采用不同的方法。常用的有CPU时钟频率（主频）、每秒平均执行指令数(ips)等。微型计算机一般采用主频来描述运算速度，例如，Pentium/133的主频为133 MHz，Pentium Ⅲ/800的主频为800 MHz，Pentium 4 1.5G的主频为1.5 GHz。一般说来，主频越高，运算速度就越快。 (2)字长。计算机在同一时间内处理的一组二进制数称为一个计算机的“字”，而这组二进制数的位数就是“字长”。在其他指标相同时，字长越大计算机处理数据的速度就越快。早期的微型计算机的字长一般是8位和16位。目前586（Pentium， Pentium Pro， PentiumⅡ，PentiumⅢ，Pentium 4）大多是32位，现在的大多数人都装64位的了。 (3)内存储器的容量。内存储器，也简称主存，是CPU可以直接访问的存储器，需要执行的程序与需要处理的数据就是存放在主存中的。内存储器容量的大小反映了计算机即时存储信息的能力。随着操作系统的升级，应用软件的不断丰富及其功能的不断扩展，人们对计算机内存容量的需求也不断提高。目前，运行Windows 95或Windows 98操作系统至少需要 16 M的内存容量，Windows XP则需要128 M以上的内存容量。内存容量越大，系统功能就越强大，能处理的数据量就越庞大。 （4)外存储器的容量。外存储器容量通常是指硬盘容量（包括内置硬盘和移动硬盘）。外存储器容量越大，可存储的信息就越多，可安装的应用软件就越丰富。目前，硬盘容量一般为10 G至60 G，有的甚至已达到120 G。 (5)I/O的速度 主机I／O的速度，取决于I/O总线的设计。这对于慢速设备（例如键盘、打印机）关系不大，但对于高速设备则效果十分明显。例如对于当前的硬盘，它的外部传输率已可达20MB／S、4OMB／S以上。 （6）显存
Unit 7 Will people have robots?知识 点整理 Unit7willpeoplehaverobots?知识点整理 一、词组、短语： 、oncomputers在电脑上， 2、onpaper在纸上， 3、livetobe200yearsold活到200岁， 4、freetime空闲时间， 5、indanger 在危险中， 6、ontheearth在世界上 7、playapartinsth在某方面出力/做贡献， 8、spacestation太空站， 8、lookfor寻找， 9、computerprogrammer电脑程序师， 10、inthefuture 在将来， 11、hundredsof成百上千的， 12、thesame…as与…一样， 13、overandoveragain反复， 14、getbored 无聊，
15、wakeup醒来/唤醒， 16、looklike 看起来像， 17、falldown倒下/落下 二、重要句子（语法） 、will+动词原形 将要做 2、fewer/more+可数名词复数更少/更多… 3、less/more+不可数名词 更少/更多 4、trytodosth. 尽力做某事 5、havetodosth 不得不做某事 6、agreewithsb. 同意某人的意见 7、such+名词（词组） 如此
8、playapartindoingsth 参与做某事 9、makesbdosth 让某人做某事 10、helpsbwithsth 帮助某人做某事 11、Therewillbe+主语+其他 将会有…. 12、Thereis/are+sb.+doingsth 有…正在做… 13、Itis +形容词+forsb+todosth 做某事对某人来说… 语法： whatwillthefuturebelike? citieswillbemorepolluted.Andtherewillbefewertrees. willpeopleusemoneyin100years?
浅析中介语产生的原因 【摘要】中介语是对第二语言习得者使用目的语时所产生的不完全正确的语言系统的描述，是第二语言学习过程中学习者所使用的一种独立的语言系统。本文从母语对中介语的影响，有限的目的语知识的干扰，本族或外族文化因素的干扰，学习或交际方式、态度等的影响，教师或教材对目的语语言现象的不恰当或不充分的讲解和训练等方面分析了中介语产生的原因。 【关键词】中介语产生原因 【中图分类号】H314 【文献标识码】A 【文章编号】1674－4810（2011）13－0077－02 中介语是对第二语言习得者使用目的语时所产生的不完全正确的语言系统的描述，是第二语言学习过程中学习者所使用的一种独立的语言系统。研究者们对第二语言习得者的这一语言系统的描述曾使用过诸如：“近似语言系统”（approximative systems）、“学习者固有的内在的掌握语言的课程大纲”（learner’s built－in syllabus）、“习得者独有的语言”（idiosyncratic dialects）、“中继能力”（transitional competence）等术语。然而，广为人知，影响最大的是塞林克（Selinker）的“中介语”（interlanguage）。中介语一词最早是由塞林克首先使用，1972年他在论文“Interlanguage”中对中介语作出了全面的阐述，此后该词确立了其在第二语言习得理论中的重要地位，并为语言学界和外语教学界所接受。中介语理论于80年代初被介绍到我国。 中介语是介于母语和目的语之间的过渡性语言，以母语为出发点，不断发展并逐渐向目的语靠近。中介语作为独立的语言体系有着自身的特点。不同的学者对中介语本身的特点也有不同的概括。纵观学者们的看法，中介语具有系统性、可变性、渗透性、可创造性及石化性等特点。中介语的产生是多方心理因素影响的结果，也即产生中介语的根源。本文试对中介语的成因进行阐述。 一母语的负迁移，即干扰 在第二语言或外语学习中，学习者由于不熟悉目的语的语法规则或表达习惯，会自觉或不自觉地运用母语的规则或表达方式来处理目的语中的一些信息，会依赖其原有的母语知识辅助其意思的表达。而过多的母语干扰会产生语际错误（interlingual errors），也会出现中国式英语（Chinglish）。套用母语所出现的中介语主要反映在语音、词汇和语法等语言系统的几个层面上。例如，初学英语的中国学生把thing读成sing，因为汉语语音系统中没有齿间部位的摩擦音/θ/这一发音，中国学生习惯套用汉语的/s/来处理这一发音。又如，中国学生往往容易在读英语闭音节的同时，在结尾的辅音后面不自觉地加上一个元音，如把cook读成cooker，这是因为汉字基本上都属于开音节的缘故。在词汇方面，套用母语的例子也不少，例如，有人把汉语中的“爱人”（wife）说成“lover”（在英语中指的是情夫或情妇），把“公费医疗”说成“the public medical”（正确的说法应为the free medical care）等。在语法方面，由于受母语的影响，中国学生容易犯这样的错误：I buy it yesterday.这是因为汉语的词汇没有时态之分。又如：Though the task was difficult, but I finished it on time. 这也是受汉语习惯说法影响的结果，还有把I don’t think it is good enough. 说成I think it is not good enough. 二所学有限目的语知识的干扰 学习者因为所学目的语的知识有限，把目标语中的个别语言规则当成普遍性规则来使用，创造出不具有母语特征又不是目标语的中介语形式。学习者常将一些语言规则当成普遍性的规则滥用，将目的语的语言结构系统简单化，从而创造出既不带母语特征，目的语中又没有的结构变体。例如：What did he intended to say? 这类中介语产生的原因在于学习者把表示过去时的动词加后缀－ed的规则推而广之。又如：I don’t know when is the plane going to take off. 这是由于学习者滥用英语特殊疑问句的语序造成的。
人教版八年级英语上册Unit7知识点归纳整 理 Unit7illpeoplehaverobots? 短语归纳 onputer在电脑上2.onpaper在纸上3.aeup醒 livetodo200yearsold活动200岁5.freetie空闲时间indanger处于危险之中7.ontheearth在地球上 playapartinsth.参与某事9.inthefuture在未 0spacestation太空站11.puterprograer电脑编程员 loofor寻找13.hundredsof许多；成百上千 thesae…as…与……一样15.getbored感到厌烦的 overandoveragain多次；反复地17.falldon倒塌 ill+动词原形将要做…… feer/ore+可数名词复数更少/更多…… 0.less/ore+不可数名词更少/更多…… 1.havetodosth.不得不做某事 2.agreeithsb.同意某人的意见 3.such+名词如此…… playapartindoingsth.参与做某事 Thereillbe+主语+其他将会有…… Thereis/are+sb./sth.+doingsth.有……正在做某事
aesb.dosth.helpsb.ithsth.帮助某人做某事 trytodosth.尽力做某事 It’s+ad+forsb.todosth.对某人来说，做某事……的。 语法讲解 Boosillonlybeonputers,notonpaper.书将只在电脑里，而不是在纸上。 Thereillbeorepollution.将会有更多的污染。 ).Thereillbe+n=Thereis/aregoingtobe+n将会有…ThereisgoingtobeafootballatchthisFriday. ).pollution:污染；公害pollute：污染；弄脏polluted：受污染的 Everyoneshouldplayapartinsavingtheearth.每个人应该参与挽救地球。 Todaytherearealreadyrobotsoringinfactories.现在已经有机器人在工厂里工作了。 Therebesb.doingsth.有某人正在做…Thereisabirdsinginginthetree. Theyagreeitaytaehundredsofyears.他们同意这可能花费几百年的时间。 Ittaes+时间+todosth.某人花费时间区做某事。 Ittooehalfanhourtofinishyhoeor. agreetodosth.eagreetoeetuplaterandtalthingsover
中介语是指第二语言学习者特有的一种目的语系统，这种语言系统在语音、词汇、语法、文化和交际等方面既不同于学习者自己的第一语言，也不同于目的语，而是一种随着学习的进展向目的语的正确形式逐渐靠拢的动态语言系统。（吕必松）自然界和人类社会中都存在着大量的中间状态，人类的语言也是如此。人们学习语言的过程中，以及语言接触融合的过程中，都有所谓的中间状态。现代应用语言学理论把语言中的这些中间状态称为中介现象。语言中存在着大量的“中介物”语音方面,词语方面,语法方面还有御用方面都存在着大量的中介语，在语言规范的过程中，也存在着中介状态。 中介语的特点 1.是一个独立的语言系统 中介语在其发展过程中的任何一个阶段都是学习者创造的一种介于第一语言和目的语之间的独特的语言系统。它有一套自身的规律，在语音、词汇、语法等系统方面都有表现。学习者有意识地使用这套规则去生成或理解他们从未接触过的话语。中介语具有人类其他语言所具有的特点和功能，可以用作交际的工具。 2，,是一个动态的语言系统，新知识和新规则不断注入；原有的尚未学好的规则和结构也在不断修正调整。随着学习者语言水平的提高和交际需要的增长，中介语不断发展，并呈一定的阶段性，由简单到复杂、由低级到高级、逐渐离开第一语言向目的语靠拢。 3.反复性。 在对目的语的学习过程中，随着水平的提高，中介语是在逐步地向目的语的规范运动的，但这并不是说，这种接近是直线前进的，而是有反复、有曲折的，这就是中介语的反复性。已经纠正了的偏误还可能有规律地重现。 .具有顽固性（“化石化”） 语言的某些具体形式上学到了一定程度就停滞不前了。比如在语音方面，有的学生学了很长时间，到了高年级，还是掌握不了某几个音。我们把这种现象也称作“化石化”。 中介语理论在对外汉语教学中的运用主要体现在以下方面: (一)研究不同母语的留学生的中介语情况及其特点 (二)对外汉语教师要善于发现留学生母语和汉语的相同和相异之处,运用迁移理论恰当引导 (三) 采用多种教学方法,运用适当的教学策略,使留学生的中介语不断接近目的语
CPU，全称“Central Processing Unit”，中文名为“中央处理器”，在大多数网友的印象中，CPU只是一个方形配件，正面是金属盖，背面是一些密密麻麻的针脚或触点，可以说毫无美感可言。但在这个小块头的东西上，却是汇聚了无数的人类智慧在里面，我们今天能上网、工作、玩游戏等全都离不开这个小小的东西，它可谓是小块头有大智慧。 作为普通用户、网友，我们并不需要解读CPU里的所有“大智慧”，但CPU既然是电脑中最重要的配件、并且直接决定电脑的性能，了解它里面的部分知识还是有必要的。下面笔者将给大家介绍CPU里最重要的基础知识，让大家对CPU有新的认识。 1、CPU的最重要基础：CPU架构 CPU架构： 采用Nehalem架构的Core i7/i5处理器 CPU架构，目前没有一个权威和准确的定义，简单来说就是CPU核心的设计方案。目前CPU大致可以分为X86、IA64、RISC等多种架构，而个人电脑上的CPU架构，其实都是基于X86架构设计的，称为X86下的微架构，常常被简称为CPU架构。 更新CPU架构能有效地提高CPU的执行效率，但也需要投入巨大的研发成本，因此CPU 厂商一般每2-3年才更新一次架构。近几年比较著名的X86微架构有Intel的Netburst （Pentium 4/Pentium D系列）、Core（Core 2系列）、Nehalem（Core i7/i5/i3系列），以及AMD的K8（Athlon 64系列）、K10（Phenom系列）、K10.5（Athlon II/Phenom II 系列）。
Intel以Tick-Tock钟摆模式更新CPU 自2006年发布Core 2系列后，Intel便以“Tick-Tock”钟摆模式更新CPU，简单来说就是第一年改进CPU工艺，第二年更新CPU微架构，这样交替进行。目前Intel正进行“Tick”阶段，即改进CPU的制造工艺，如最新的Westmere架构其实就是Nehalem架构的工艺改进版，下一代Sandy Bridge架构将是全新架构。AMD方面则没有一个固定的更新架构周期，从K7到K8再到K10，大概是3-4年更新一次。 制造工艺：
中介语产生的语言心理原因 刘利民,刘 爽 (四川大学外语学院四川成都610064) 摘 要:本文从描述第二语言习得过程的中介语现象及其特征入手,通过解释人类语言的普适性心理机制,探讨了中介语现象的产生原因,得出了中介语现象产生于学习者构建目的语的心理句法系统时其语言习得机制的自主创造性。关键词:第二语言习得;中介语;语言心理 中图分类号:H0 文献标识码:A 文章编号:1000-5544(2003)01-0006-05 Abstract:T his paper descr ibed the pro per ties of int er language,a phenomeno n in second language acquisitio n,ex-plor ed the univ ersal mechanism o f hum an languag e behav io r and discussed the r easo ns of the occurr ence o f the inter-languag e pheno menon.T he paper concluded that inter lang uag e resulted fro m t he autono mous cr eativ ity o f t he lan-g uage acquisitio n mechanism o f the seco nd languag e lea rners in the co nst ructio n of a new mental gr ammar system o f the targ et languag e. Key words:second lang uage acquisitio n;inter lang ua ge;psycho ling uistic 1.中介语的特性 中介语(interlanguag e)这一概念是Selinker(1972)于20世纪70年代初正式提出的,指的是第二语言学习者自己创造的一种介于母语和目的语之间的过渡性语言系统,这种语言系统是动态的,并且随着学习的进展逐渐接近目的语。80年代初,我国学术界引进了中介语的概念,其中比较完整和详细的表述为(鲁健骥,1984):“中介语指的是由于学习外语的人在学习过程中对于目的语的规律所做的不正确的归纳与推论而产生的一种语言系统。这个语言系统既不同于学习者的母语,又区别于他所学的目的语。中介语系统在语音、词汇、语法、文化等方面都有表现。但它又不是固定不变的,而是随着学习的发展,逐渐向目的语的正确形式靠拢。” 随着相关研究的进展,学术界对于中介语的特性有了比较系统的认识,L arsen-F reem an等人(1994)在其专著中较详细地讲述了三点。首先,中介语具有变异性,包括随意变体(free var iation)和系统性变异(systemat ic v ariability)两种现象。随意变体指的是中介语所呈现的相当大的共时变异性;即学习者在相同的时间、等值的语境,为达到同样的交际目的而使用所学的目的语的时候出现的交替使用标准和非标准句法 参考文献 [1]Brow n,G.&Yu le,G.Discourse A nalysis[M].Cambridge: C UP,1983. [2]Coulthar d,M.A n I ntroduction to Discourse A naly sis[M].Lon- don:Longman,1977. [3]Fries,P.On the Status of T heme in En glish[A].In J an os,S. Petodi an d Ermel S ozer(ed.)M acr o-and M icro-Connex ity of Dis-course[C].Hamburg:Dusde,1983. [4]Halliday,M. A.K.Exp lorations in the F unctions of L angu ag e [M].London:Edw ard Arnold,1973. [5]Halliday,M.A.K.&Has an,R.Cohesion in Eng lish[M].Lon- don:Longman,1976. [6]Halliday,M.A.K.&Hasan,R.L ang uage,Context and T ex t [M].Victoria:Deak in U nivers ity Press,1985a. [7]Halliday,M.A.K.Introduction to F unctional G rammar[M]. L on don:Ed ward Arnold,1985c/1994. [8]Halliday,M. A.K.L ang uag e as S ocial S emiotic:A n Social In- terpr etation of M eaning[M].London:Edw ard Arn old,1978. [9]M artin,J.Eng lish T ex t:System and S tructure[M].Ams terdam and Philadelphia:Benjamins,1992. [10]S inclair,J.M cH.&M.Coulth ard.T ow ard s an A nalysis o f Discour se[M].London:Oxford U nivers ity Press,1975. [11]Ventola, E.Discour se in S ociety:Sy stemic Functional P ersp ec- tiv es[M].Ablex,1995. [12]Widdow s on,H.G.T eaching L anguag e as Communication[M]. Ox ford:OUP,1978. [13]胡壮麟.语篇的衔接与连贯[M].上海:上海外语教育出版社, 1994. [14]苗兴伟.论衔接与连贯的关系[J].外国语,1998(4). [15]张德禄.功能文体学[M].济南:山东教育出版社,1998. [16]张德禄.话语基调的范围及其体现[J].外语教学与研究,1998 (1). [17]张德禄.语篇连贯研究纵横谈[J].外国语,1999(6). [18]张德禄.论衔接[J].外国语,2001(2). 作者简介:张德禄,教授,博士生导师,曾于80年代赴澳大利亚悉尼大学留学,师从韩礼德教授,主要研究系统功能语言学、文体学、符号学、外语教学等。 收稿日期 2002-02-18 责任编校 薛旭辉
CPU性能指标排名 高端CPU - 更新至2018年1月19日 Processor CPU Mark Price (USD) Intel Core i9-7980XE @ 2.60GHz278191982.15 Intel Xeon Gold 6154 @ 3.00GHz27789$3,543.00* Intel Xeon W-2195 @ 2.30GHz26470$2,553.00* Intel Core i9-7960X @ 2.80GHz264471634.99 Intel Xeon E5-2679 v4 @ 2.50GHz25236$2,702.00* Intel Core i9-7940X @ 3.10GHz251001386.6 Intel Core i9-7920X @ 2.90GHz234791110.99 Intel Xeon E5-2699 v4 @ 2.20GHz233623010 Intel Core i9-7900X @ 3.30GHz22570959.99 Intel Xeon E5-2696 v3 @ 2.30GHz22542$2,498.00* Intel Xeon E5-2699 v3 @ 2.30GHz22358$3,805.02* AMD Ryzen Threadripper 1950X22031899 Intel Xeon E5-2698 v4 @ 2.20GHz21789$3,226.00* Intel Xeon E5-2697 v4 @ 2.30GHz217292805 Intel Xeon E5-2673 v4 @ 2.30GHz21625NA Intel Xeon W-2150B @ 3.00GHz21581NA Intel Xeon E5-2696 v4 @ 2.20GHz215193005.95 Intel Xeon E5-2697 v3 @ 2.60GHz21502$2,661.90* Intel Xeon E5-2690 v4 @ 2.60GHz21323$2,133.99* Intel Xeon Gold 6136 @ 3.00GHz21313NA Intel Xeon E5-2698 v3 @ 2.30GHz211493424.02 Intel Xeon E5-2695 v3 @ 2.30GHz20297$2,499.99* Intel Xeon E5-2695 v4 @ 2.10GHz20258$2,682.50* Intel Core i7-6950X @ 3.00GHz199931574.96 Intel Xeon E5-2687W v4 @ 3.00GHz19979$2,211.95* Intel Xeon E5-2680 v4 @ 2.40GHz19953$1,762.90* Intel Xeon Gold 6144 @ 3.50GHz19833$2,925.00* Intel Xeon E5-2689 v4 @ 3.10GHz19521NA Intel Xeon E5-2686 v3 @ 2.00GHz19255NA Intel Xeon E5-2690 v3 @ 2.60GHz192402359.99 AMD EPYC 7551190033699.99 Intel Xeon W-2145 @ 3.70GHz18968$1,113.00* Intel Xeon E5-2680 v3 @ 2.50GHz187151909.95 Intel Core i7-7820X @ 3.60GHz18656569 Intel Xeon Gold 6130 @ 2.10GHz18587$1,969.99* Intel Core i7-7900X @ 3.30GHz18539NA AMD Ryzen Threadripper 1920X18475674.99 AMD EPYC 7401P18465NA Intel Xeon E5-1681 v3 @ 2.90GHz18238NA Intel Xeon Gold 6134 @ 3.20GHz18083$2,348.26* Intel Xeon E5-4660 v3 @ 2.10GHz18007$4,800.00* AMD EPYC 7351P17991799.99 Intel Xeon E5-2687W v3 @ 3.10GHz178432509.95 Intel Xeon E5-2676 v3 @ 2.40GHz17795NA Intel Xeon E5-2660 v4 @ 2.00GHz17786$1,498.77* Intel Core i7-6900K @ 3.20GHz176971020.89 Intel Xeon E5-2683 v3 @ 2.00GHz17407$1,984.99* Intel Xeon E5-2697 v2 @ 2.70GHz174052235.02 Intel Xeon E5-2673 v3 @ 2.40GHz16982$700.00* Intel Xeon E5-1680 v4 @ 3.40GHz16979NA Intel Xeon E5-2678 v3 @ 2.50GHz16905NA Intel Xeon E5-1680 v2 @ 3.00GHz16769NA Intel Xeon E5-2696 v2 @ 2.50GHz16681NA Intel Xeon E5-1680 v3 @ 3.20GHz16673NA