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Casey Finnerty

Identification of Protein Interacting Partners Using Tandem Affinity Purification - 0 views

www.ncbi.nlm.nih.gov/...PMC3399501

norovirus

shared by Casey Finnerty on 19 Sep 14 - No Cached
  • Casey Finnerty on 19 Sep 14
     
    This paper includes a video that explains the use of Tandem Affinity Purification by the Goodfellow group.
apopp10

Norovirus Translation Requires an Interaction between the C Terminus of the Genome-link... - 75 views

www.jbc.org/...21738.long

shared by apopp10 on 12 Sep 14 - No Cached
  • Importantly, however, despite the interaction between the norovirus VPg protein and eIF4E, it appears to be dispensable for MNV translation initiation, at least in vitro
    • ameliaobert
      ameliaobert on 15 Sep 14
       
      I find it interesting that the MNV is depensable if incorporated in vitro. Im curious as to why this could be.
    • Casey Finnerty
      Casey Finnerty on 26 Sep 14
       
      They are saying the VPg-eIF4E interaction is dispensable in vitro, not the virus itself.
  • Mass spectrometry was used to identify proteins present within the samples with a minimum of 2 unique peptides and >90% identification probability
    • laceemarie
      laceemarie on 16 Sep 14
       
      I'm confused as to how exactly mass spectrometry was used in this experiment. If the proteins are already separated by molecular weight after the SDS-PAGE, what precisely are they looking for when running a mass spec? It article says, ".... a minimum of 2 unique peptides and >90% identification probability," but this doesn't mean much to me. I understand the identification part because based on the way the molecule splits after being shot with ions, you can get a basic idea of the structure of the molecule. My question with the 2 unique peptides is that each amino acid has a different molecular weight right? So if the proteins were all the same except at the 2 unique peptides, the mass spec would show a different mass to charge ratio for each protein with the varying peptides correct? Would the charge of the amino acids in the peptide have anything to do with the charge portion of the mass to charge ratio? I hope this question came out okay. 
    • abachman12
      abachman12 on 22 Sep 14
       
      I also found this confusing. I understand what your saying and it makes your question makes sense to me. I think that the charge of the amino acids in the peptide would have something to do with the charge portion of the mass to charge ration.
    • Casey Finnerty
      Casey Finnerty on 22 Sep 14
       
      I'll address this in class. In the meantime, check out http://en.wikipedia.org/wiki/Peptide_mass_fingerprinting
  • inability to culture many members of the virus family in immortalized cells.
    • slgoogin8981
      slgoogin8981 on 17 Sep 14
       
      What are the other options to culture these viruses?
    • Casey Finnerty
      Casey Finnerty on 22 Sep 14
       
      This is the subject of intense study. See ref 18.
  • ...34 more annotations...
  • The mutation F123A ablated the ability of VPg to co-purify eIF4G, eIF4A, and PABP but did not affect the ability of NTAP-MNV VPg to co-purify eIF4E (Fig. 5), suggesting that the eIF4E and eIF4G binding regions on VPg are distinct
    • ameliaobert
      ameliaobert on 17 Sep 14
       
      If F123A mutation is observed through data analysis (co-purification) that the eIF4E and eIF4G are distinct, so differ from each other. Then, why does the data suggest that the direct interacts of VPg with eIF4G determines the eIF4F binding capacity from the data obtained? As well as how do these purification datas are able to determine if the binding capacities for the virus VPg proteins are direct interactions?
    • Casey Finnerty
      Casey Finnerty on 22 Sep 14
       
      As written, your question is unclear. Please rewrite it.
  • These data would suggest that the direct interaction of VPg with eIF4G largely determines the eIF4F binding capacity of the norovirus VPg protein.
  • some of which function directly in translation initiation, whereas others may contribute to the regulation of host cell translation by virus infection.
    • ameliaobert
      ameliaobert on 17 Sep 14
       
      So here, they are saying that the initiation factors, some directly interact with the viruses translation to mRNA, whereas the other initiation factors present in teh virus contribute there translation factors specifically to when the host cells get involved with the virus translation factors? Basically specifically encoding for specific initiation translation factors based on the area of translation (i.e. from the virus or from the host cell)?
  • using the number of unique peptides identified for each protein (Table 1) as a semiquantitative indirect measure of protein abundance in the purified complex, it was also apparent that eIF4G was enriched in the complex with respect to the other proteins isolated.
    • becky214
      becky214 on 18 Sep 14
       
      I was confused by what they are suggesting with this data. Is it true that elF4G is a binding partner, or is it just suggested?
    • Casey Finnerty
      Casey Finnerty on 22 Sep 14
       
      Suggested. What evidence do you think would be conclusive?
    • Casey Finnerty
      Casey Finnerty on 22 Sep 14
       
      BTW, is that a golden eagle in your picture?
  • we attempted
  • to identify
  • components of the complex that interacted with VPg directly and contribute to viral translation
    • slgoogin8981
      slgoogin8981 on 18 Sep 14
       
      Is this the same as saying, they were looking for promoters?
    • Casey Finnerty
      Casey Finnerty on 22 Sep 14
       
      No. Promoters are involved with transcription. In this paper they are looking for eIF's (of translation) that interact with VPg.
  • Independent observations indicate that eIF4G phosphorylation is stimulated during norovirus replication in cell culture and that this phosphorylated form of eIF4G is associated with MNV VPg in infected cells (29). The impact and functional relevance of initiation factor phosphorylation on the norovirus life cycle is currently under study.
    • becky214
      becky214 on 19 Sep 14
       
      Maybe it is possible that this phosphorylation helps initiate and enhance translation.
    • alexridesducati
      alexridesducati on 19 Sep 14
       
      It seems in order for this virus to replicate effectively, it has to bind several different ways, at least, just to initiate protein synthesis. Is there any way to synthesize a molecule that can target any of these proteins for degradation or at least something that can inhibit the binding site (similar to a neutralizing antibody :) . It seems that if you nip this right at the bud, that its possible to shut this virus down for the most part, except for the fact that in vitro, elF4E can be dispensed for translation initiation. Makes me wonder what the key differences are between an in vitro and in vivo environment that allows this to happen.
  • high affinity interaction
    • smolizon11
      smolizon11 on 19 Sep 14
       
      Im confused to as what high affinity means?
    • Casey Finnerty
      Casey Finnerty on 22 Sep 14
       
      High affinity would mean a tighter interaction between two molecules. The high affinity nature of the VPg-eIF4G interaction is suggested by the fact that eIF4G is carried through the TAP procedure, and that it persists despite washing with 1M salt.
  • Noroviruses, members of the Caliciviridae family of small positive-strand RNA viruses, are a major cause of acute gastroenteritis
    • smolizon11
      smolizon11 on 19 Sep 14
       
      Its really interesting to be able to put a virus with being the cause of certain diseases. Even when talking about the disease in a "non-medical" setting its almost as if you have "higher hand" at the information about it. Makes for fun and interesting conversation with other people besides virology students.
  • As these proteins were not identified in both experimental systems and the focus of this study was to determine the role of canonical initiation factors in VPg-dependent translation, these additional host cell proteins factors were not examined in more detail.
    • Sean Hogan
      Sean Hogan on 19 Sep 14
       
          Is it possible that any of these are binding far upstream or downstream from the IF complex yet still affect affinity of complex compoenets such as with the eIF4E interaction with VPg protein in vitro?
    • Casey Finnerty
      Casey Finnerty on 22 Sep 14
       
      Yes.
  • recombinant MNV was performed by infecting baby hamster kidney cells with fowlpox virus expressing T7 RNA polymerase followed by transfection of MNV cDNA expression constructs as described previously
    • slgoogin8981
      slgoogin8981 on 19 Sep 14
       
      Was the fowlpox used just for the purpose of its polymerase? What is cDNA?
    • Casey Finnerty
      Casey Finnerty on 22 Sep 14
       
      cDNA stands for complementary DNA. Technically, that would mean the DNA strand that is complementary in sequence to the RNA. In practice, after the first cDNA strand is synthesized (typically using an RNA template and reverse transcriptase), the second strand is synthesized with a DNA polymerase as well. This double-stranded cDNA can then be cloned in a plasmid, which is then introduced into cells via transfection as done by the Goodfellow group. The fowlpox encoding RNA polymerase from T7 phage (this RNA pol is not error prone and highly specific for the T7 transcription start site) is used for two things: the T7 RNA polymerase drives transcription of the MNV RNA from the plasmid containing the cDNA; AND it also promotes capping of the MNV genome/transcript, which promotes translation of MNV proteins. See http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3471295/, Fig. 4.
    • slgoogin8981
      slgoogin8981 on 26 Sep 14
       
      Thank you for the link! I am very unskilled with the lab results and procedures done in lab. I appreciate the guidance and opportunity to learn. I have only had three biology labs: 241, 242 and Cell.
    • Casey Finnerty
      Casey Finnerty on 01 Oct 14
       
      You're welcome! And thanks for the question!
  • human noroviruses have yet to be cultivated in the laboratory (18).
  • Our understanding of norovirus biology has been greatly enhanced by the discovery of murine norovirus (MNV) in 2003 (19),
  • Previous reports have also highlighted a potential association of the norovirus VPg protein with components of the eIF3 complex (28) and eIF4E, PABP, and eIF4G as well as the ribosomal protein S6 (29), although the functional relevance of these interactions has yet to be demonstrated. Here we describe the proteomic characterization of the murine norovirus translation initiation factor complex, demonstrating that VPg associates directly with the core components of the eIF4F complex and PABP. We further demonstrate that the interaction between eIF4G and VPg is essential for norovirus translation. Furthermore, we demonstrate that eIF4G is required for efficient virus replication in cell culture.
  • although these cells are not permissive to MNV infection due to the lack of a suitable receptor, robust MNV translation and replication occurs upon transfection of viral RNA into the cytoplasm (22).
    • slgoogin8981
      slgoogin8981 on 19 Sep 14
       
      It is very cool that this virus can still use human translation equipment even though it is a marine from of the virus. How did they enter the virus genome into the cytoplasm?
  • It is worth noting, however, that in addition to disrupting electrostatic interactions, increasing sodium chloride concentrations stabilize hydrophobic interactions.
  • It is important to note that in both these approaches low levels of eIF4E remained (Fig. 8C) and 4E-BP1 expression in the MNV permissive cells did not completely block the eIF4E-4G interaction. Therefore, further studies on the role of eIF4E during the norovirus life cycle are clearly warranted, but it is worth noting that in addition to a direct role in cap binding for translation initiation, eIF4E plays numerous roles in the regulation of gene expression.
    • jpolanco10
      jpolanco10 on 19 Sep 14
       
      Why did this not block the eIF4E-4G interaction? Is there a really high affinity or was it able to recruit other proteins to aide it? It seems that the low level of eIF4E was still able to do its job, mainly regulation of the genes
  • Murine macrophage RAW264.7 and microglial BV2 cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% (v/v) fetal calf serum (FCS), penicillin (100 units/ml), streptomycin (100 μg/ml), and 10 mm HEPES buffer. Baby hamster kidney cells (BHK-21) expressing T7 RNA polymerase (BSRT7 cells) were cultured in similar media lacking HEPES but containing 1 mg/ml G418. Similarly, HEK 293T cells were maintained in media lacking HEPES. HEK 293T cells stably expressing pMEP4-NTAP or pMEP4-NTAP MNV VPg plasmids were supplemented with 50 μg/ml hygromycin B and nonessential amino acids. The HEK293 TREX cells stably expressing pcDNA4/TO-NTAP derivatives of MNV VPg were supplemented with 5 μg/ml Blasticidin and 200 μg/ml Zeocin. All cell lines were maintained at 37 °C and 10% CO2.
    • nkasdagly
      nkasdagly on 19 Sep 14
       
      Are they setting up the primary and secondary antibodies here or am i totally off? Just wondering because i see they used fetal calf serum and baby hampster kidney cells... 
  • Noroviruses, members of the Caliciviridae family of small positive-strand RNA viruses, are a major cause of acute gastroenteritis in man (8) but have also been identified in a number of other species including dogs (9, 10), cats (11), sheep (12), and cattle (13
    • Sean Hogan
      Sean Hogan on 19 Sep 14
       
      Work?
  • the first step in translation is the binding of the initiation factor eIF4E, a component of the eIF4F complex, to the 5′ cap structure.
  • eIF4F is a complex of three initiation factors; eIF4E is the cap-binding protein, eIF4A functions as an RNA helicase, and eIF4G acts as a scaffold to bridge the mRNA to the 40 S ribosomal subunit via its interaction with eIF3 (3).
  • 14 individual point mutations in VPg were introduced into the MNV infectious clone,
  • the effect of mutations on the ability of VPg to associate with initiation factors was assessed by the enrichment of the eIF4F complex using m7GTP-Sepharose followed by Western blotting for VPg.
  • the mutations V115A, D116A, and F123A altered the levels of infectious virus produced;
  • the mutants V115A, D116A, and F123A showed a consistently reduced interaction with eIF4F (Fig. 4).
  • The degree to which the mutation affected virus recovery appeared to correlate with the ability of VPg to be co-purified with initiation factors (Fig. 4).
  • These data would indicate that the disordered C terminus of the norovirus VPg protein contains amino acids involved in the interaction with eIF4G.
  • We recently analyzed the solution structure of the core domain of VPg using nuclear magnetic resonance (NMR) spectroscopy and found that the MNV VPg protein consists of a compact structured core formed by a pair of α-helices that is flanked by long, flexible N and C termini (35). The region we have identified as being involved in the direct interaction with eIF4G, namely the C-terminal domain, is disordered and, therefore, is likely to adopt a fixed structure only upon interaction with eIF4G.
    • rmeloche10
      rmeloche10 on 22 Sep 14
       
      Using this advanced spectroscopy to find the structured core is an awesome break in technology. It shows the C-terminal is disordered until interaction with the initiation factor, does this disorder provide any advantage within the virus?
    • Casey Finnerty
      Casey Finnerty on 22 Sep 14
  • eIF4GI
    • Casey Finnerty
      Casey Finnerty on 22 Sep 14
       
      eIF4GI and eIF4GII are related forms of eIF4G.
  • This approach was used in place of authentic infection to enable the use of the F123A VPg mutant as a specificity control as this cDNA clone does not produce infectious virus (Fig. 4).
    • Casey Finnerty
      Casey Finnerty on 22 Sep 14
       
      Throughout the paper, the authors have included helpful explanatory text like this.
  • These data indicate that the VPg binding site lies within residues 654–1131 of eIF4GI, a region known to contain both eIF4A and eIF3 binding sites (7).
    • Casey Finnerty
      Casey Finnerty on 22 Sep 14
       
      Couldn't the binding site be narrowed further to 675-1131?
  • Noroviruses, a major cause of gastroenteritis in man, have evolved a mechanism that relies on the interaction of translation initiation factors with the virus-encoded VPg protein covalently linked to the 5′ end of the viral RNA.
    • apopp10
      apopp10 on 08 Dec 14
       
      I guess this comment never got adhered to my original highlight. Here it is once more. As someone who has gastrointestinal issues including inflammation, i wonder if this virus could have played a role in the cause of my condition, Ulcerative Colitis. I think interrupting this interaction could provide an effective treatment against this virus.
  • Casey Finnerty on 12 Sep 14
     
    Journal Article for our Week 4 discussion.
sdahlseng10

Production and purification of lentiviral vectors generated in 293T suspens... - 6 views

web.ebscohost.com.wsuproxy.mnpals.net/...pdfviewer

lentivirus vector production purification vectors baculovirus 293T gene therapy

shared by sdahlseng10 on 02 Oct 12 - No Cached
Trevor F liked it
  • sdahlseng10 on 02 Oct 12
     
    Focus Article; Journal club 1:This is an article that details a novel technique for production of HIV (lentiviral) vectors for use as tools of gene therapy. The fascinating approach that was developed by the authors uses baculovirus as a vector to infect human embryonic kidney cells in culture with the genes necessary to produce a lentiviral vector with therapeutic capabilities.
Casey Finnerty

Are all virus particles infectious? - 0 views

www.virology.ws/...all-virus-particles-infectious

shared by Casey Finnerty on 06 Nov 14 - No Cached
  • A high particle-to-pfu ratio is sometimes caused by the presence of noninfectious particles with genomes that harbor lethal mutations or that have been damaged during growth or purification. Another explanation is that although all viruses in a preparation are in fact capable of initiating infection, not all of them succeed because of the complexity of the infectious cycle. Failure at any one step in the cycle prevents completion. A high particle-to-pfu ratio does not indicate that most particles are defective, but that they failed to complete the infection.
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