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Virus-Cell Interactions

Thiouracil Cross-Linking Mass Spectrometry: a Cell-Based Method To Identify Host Factors Involved in Viral Amplification

Erik M. Lenarcic, Dori M. Landry, Todd M. Greco, Ileana M. Cristea, Sunnie R. Thompson
Erik M. Lenarcic
aDepartment of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama, USA
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Dori M. Landry
aDepartment of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama, USA
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Todd M. Greco
bDepartment of Molecular Biology, Princeton University, Princeton, New Jersey, USA
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Ileana M. Cristea
bDepartment of Molecular Biology, Princeton University, Princeton, New Jersey, USA
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Sunnie R. Thompson
aDepartment of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama, USA
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DOI: 10.1128/JVI.00950-13
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  • Fig 1
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    Fig 1

    The TUX-MS method. (A) 4-Thiouracil can be exclusively incorporated into poliovirus RNA in vivo. HeLaUPRT cells were incubated with actinomycin D (Act D), poliovirus, or 4TU (30 min after addition of Act D or PV) as indicated. Total RNA was isolated and cross-linked to a thio-reactive biotin (lanes 1 to 5), separated on a denaturing formaldehyde-agarose gel, transferred to a membrane, and probed with streptavidin-HRP to detect thio-containing RNAs (top). Total RNA was visualized by ethidium bromide staining of the gel (bottom) prior to transfer to the membrane. (B) Diagram of the TUX-MS method. HeLaUPRT cells are mock infected or PV infected in the presence of 4TU and Act D. 4sU is exclusively incorporated into PV viral RNA. UV cross-linking of bound proteins to the thio-containing viral RNA (represented as dark gray balls or lines) is shown. Proteins that are bound to the non-thio-containing mRNA are not cross-linked (represented as light gray balls or lines). Poly(A) RNA is isolated using oligo(dT)25 magnetic beads under denaturing conditions. The RNA is degraded with RNase A releasing the proteins from the complex. The proteins are trypsinized, and the peptides are identified by LC-MS/MS. (C) Prior to RNase A digestion, mock-infected (lanes M) and PV-infected samples are normalized to one another based on levels of cellular transcripts. RNA from mock- and PV-infected cells was isolated either by TRIzol or by the TUX-MS method, and either equal micrograms (total mRNA) or equal volumes [poly(A) selected] of RNA were subjected to β-actin Northern analysis. Band intensities were quantified on a PhosphorImager, and relative mRNA levels are indicated. Similar results were obtained with α-tubulin (data not shown). (D) Cells were infected with PV in the presence or absence of 4TU. At 8 hpi, virus was harvested and the titer was determined by plaque assay.

  • Fig 2
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    Fig 2

    PV proteins identified by TUX-MS. (A) The PV genome encodes a polyprotein that is cleaved by viral proteases into functional precursors and mature proteins. The PV polyprotein is shaded to represent the location of the identified peptides (gray) that were detected by TUX-MS in samples from PV-infected cells. The cleavage of the precursor and mature PV proteins is shown below. The PV proteins that are shaded gray were experimentally identified in panel B. (B) Detection of PV proteins that cross-link to the viral RNA in cells. Cells were mock infected (lanes 1 to 4) or PV infected (lanes 5 to 8) in the presence of Act D and 4TU. Proteins were pulse-labeled with [35S]methionine for 2 h prior to cross-linking at 5 hpi, and cells were lysed. Mock-infected (lane 4) and PV-infected (lane 5) lysates were purified using oligo(dT)25 magnetic beads, RNase A treated, separated by SDS-PAGE, and visualized by autoradiography (lanes 4 and 5). Increasing amounts of whole-cell [35S]methionine-labeled lysates were obtained from mock-infected (lanes 1 to 3) and PV-infected (lanes 6 to 8) cells. PV proteins detected in the infected whole-cell lysate are indicated (right); asterisks indicate the PV proteins that cross-linked to the viral RNA (lane 5).

  • Fig 3
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    Fig 3

    Functional analysis of known and putative host factors reveals shared cellular functions in nucleocytoplasmic shuttling, spliceosome assembly, and pre-mRNA editing. (A) Venn diagrams were constructed based on the subcellular localization(s) of known (left) and TUX-MS (right) host factors classified by gene ontology annotation. (B) STRING functional association network representing 54/81 host proteins (see Tables 1 and 2). Nodes are labeled with the proteins' primary gene names. Bold node outlines indicate proteins previously identified as PV host factors (Table 1). Node colors represent a range of average spectral count fold enrichment (1.0- to ≥5-fold) in PV-infected versus mock-infected samples (n = 2). Red nodes correspond to host factors detected only in PV-infected samples. Node shape corresponds to its cellular localization: circles, nucleus; diamonds, cytoplasm; squares, both. Functional associations were retained with a combined score of >0.5. Associations represented by multiple lines of evidence were collapsed to a single edge. (C) Proteins identified as unique or enriched in PV-infected samples (n = 82) were uploaded to ProteinCenter (version 3.7). Functional overrepresentation was determined versus the Swiss-Prot reference gene set (35,613 entries). The most statistically significant terms for molecular function (GO MF), biological processes (GO BP), and Pfam domains are indicated as percentages of annotated genes. RRM_1, RNA recognition motif, RNP-1. FDR-corrected P values were 1.8e−60, 5.7e−39, and 2.7e−47, respectively. (D) Proteins from panel C were analyzed by ClueGO functional clustering. The network highlights functional clusters of ribonucleoprotein, spliceosomal, and CRD-mediated mRNA stability complexes.

  • Fig 4
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    Fig 4

    Depletion of host proteins found to interact with PV RNA has an impact on PV amplification. HeLa cells were transfected with either control or specific siRNAs as indicated. (A) Quantitative Western analysis of PCBP2 and hnRNP U knockdown. (Top) A β-actin Western blot is shown as a loading control. (Middle) qRT-PCR of mRNA levels following knockdowns normalized to both β-actin and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) mRNA levels. (Bottom) Cell viability was measured using an MTT assay 48 h following knockdown by the indicated siRNAs; the amount of mRNA remaining following knockdown as determined by qRT-PCR is indicated. (B) Knockdown cells were infected at an MOI of 0.1 with PV (white bars) or adenovirus 5 (dark gray bars), and the virus titer after a single round of amplification (6 or 30 hpi, respectively) was determined by plaque assay. (C) Cells were mock infected (lanes 1 to 4) or PV infected (lanes 5 to 8) in the presence of Act D and 4TU. Cross-linking with long-wavelength UV light was performed at 5 hpi prior to cell lysis. RNA-protein complexes were isolated using oligo(dT25) magnetic beads, and then RNA was degraded with RNase A and the proteins were separated by SDS-PAGE (lanes 4 and 5) along with total protein whole-cell lysates from mock-infected (lanes 1 to 3) and poliovirus-infected (lanes 6 to 8) cells. hnRNP U was detected by Western analysis. (D) PV RNA immunoaffinity purifies with NONO. PV-infected HeLaUPRT cells were harvested at 5 hpi and subject to immunoprecipitation with NONO, c-myc, or no antibody (No-Ab). RNA was isolated from input, supernatant (Sup), and immunoprecipitation (IP) and detected by reverse transcription and PCR. Shown is a representative result (n = 3).

  • Fig 5
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    Fig 5

    Knockdown of NONO or CNBP affects PV replication or translation but not mengovirus amplification. (A). HeLaUPRT cells were transfected with scrambled control, NONO, or PCBP2 siRNAs. Forty-eight hours posttransfection of the siRNA, the cells were infected with PV at an MOI of 0.1. Virus was harvested at 4 hpi, and the titer was determined by plaque assay on HeLaUPRT cells. (B) HeLaUPRT cells were transfected with scrambled control, NONO, CNBP, or PCBP2 siRNAs, and 48 h posttransfection, the cells were transfected with a dicistronic reporter plasmid containing the PV IRES in the intercistronic region and a control plasmid expressing a cap-dependent β-galactosidase. Twenty-four hours later, luciferase and β-galactosidase activities were determined. The amount of mRNA remaining following knockdown was determined by qRT-PCR (CNBP, 9.9%; NONO, 30%; and PCBP2, 23%). IRES-driven translation was normalized to β-galactosidase activity and expressed as a percentage of the control siRNA. (C and D) HeLaUPRT cells were transfected with scrambled control, NONO, or PCBP2 siRNA. Forty-eight hours posttransfection, the cells were infected with PV at an MOI of 0.1. qRT- PCR was used to determine the number of plus-strand (C) and minus-strand (D) RNAs at 4 hpi. Copy numbers were determined using an in vitro-transcribed template of a known amount (plus strand, control, 7.7 × 109, NONO, 5.4 × 108, and PCBP2, 1.4 × 109 copies/ng RNA; minus strand, control, 3.0 × 104, NONO, 1.3 × 104, and PCBP2, 2 × 104 copies/ng RNA). (E) Knockdown cells were infected at an MOI of 0.1 with mengovirus (MV), and the virus titer after a single round of amplification (5.5 hpi) was determined by plaque assay (left). mRNA levels after qRT-PCR of mRNA following knockdown were normalized to β-actin (right). The same results were obtained by normalization to GAPDH mRNA levels. qRT-PCR and titer results are percentages relative to the control siRNA (represented by the horizontal dotted lines). Errors bars are standard errors for n ≥ 3. The P value for viral amplification compared to the control siRNA is indicated. **, P < 0.005.

Tables

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  • Additional Files
  • Table 1

    The gene product and primer designations, accession numbers, and primer sequences used in this study

    Gene product or primer designationAccession no.Primer sequence (5′→3′)
    ForwardReverse
    ACTBNM_001101.3GCACTCTTCCAGCCTTCCTGTCCACGTCACACTTCATG
    GAPDHNM_002046.3ACATCGCTCAGACACCATGTGTAGTTGAGGTCAATGAAGGG
    HNRNPLNM_001533.2TCAGTGAATCCCGGAACAATCGGTTCCCAGCTCATCGCAGATCTCAAA
    RSL1D1NM_015659.2AGAGAAGTGGGAGAGCGTGAAACTCAGCAATTGGGATGAAGCCACCAA
    XRCC6NM_001469.3GTCTTCTGTCCAAGTTGGTCGCTTGGCGATGAAGAAGCAGAGGAAGAA
    DDX17NM_006386.4CAACAGGATAGGGACATCAGACACTGCATTCTTTGGCTAAGG
    DDX5NM_004396.3AGCGTGACTGGGTTCTAAATGAGGAGTTAGGGTAGTCATAATTGATG
    NONONM_001145408.1AAGAAGAAATGATGCGGCGACAGCTGGGAGGTGCTATGGGCATAAACA
    CNBPNM_001127192.1GCCATCAACTGCAGCAAGACAAGTTTGCACGGGAATGCACAATTGAGG
    PVposV01148.1ATGTTCCTGTCGGTGCTGTGCACTGTCCTGCTCTGGTTGG
    PVnegV01148.1GCGGGAACACAAAGGCATTCACTCCTGACAACAACCAGACATC
    PV_pRFV01148.1TACGAATTCACTCCGGTATTGCGGTACCCTTGTACGATACATGTTGATACAATTGTCTGATTGAAATAACTG
  • Table 2

    Known picornaviral host factors identified by TUX-MS

    Protein nameDesignationAccession no.aFold increase (no. of spectra)bCellular localizationcFunction(s)Reference
    CellulardVirale
    NucleolinNCLP1933850 (50)N/CRNA bindingTranslation79
    Lupus La proteinSSBP0545511 (11)NRNA bindingTranslation13
    Interleukin enhancer-binding factor 2 (NF45)ILF2Q129053.9 (16)N/CTranscriptional regulationTranslation, inhibition80
    ATP-dependent RNA helicase ADHX9Q082113.3 (56)N/CRNA helicaseReplication81
    Splicing factor, arginine/serine-rich 3 (SRP20)SFRS3P841033.3 (20)NRNA bindingTranslation82
    Interleukin enhancer-binding factor 3 (DRBP76)ILF3Q129062.1 (65)N/CTranscriptional regulationTranslation, inhibition80
    Heterogeneous nuclear ribonucleoprotein KHNRNPKP619782.1 (54)N/CRNA bindingReplication56
    Poly(rC)-binding protein 2PCBP2Q153661.9 (21)N/CRNA bindingTranslation, replication83
    Poly(rC)-binding protein 1PCBP1Q153651.7 (22)N/CRNA bindingReplication60
    Heterogeneous nuclear ribonucleoproteins C1/C2HNRNPCP079101.6 (28)NRNA bindingReplication84
    Cold shock domain-containing protein E1 (UNR)CSDE1O755341.2 (32)CRNA bindingTranslation85
    Polypyrimidine tract-binding protein 1PTBP1P265991.2 (25)NRNA bindingTranslation86
    Far upstream element-binding protein 2KHSRPQ929451.1 (28)N/CRNA bindingTranslation87
    Polyadenylate-binding protein 1PABPC1P119400.8 (55)N/CPoly(A) bindingTranslation88
    Proliferation-associated protein 2G4 (ITAF45)PA2G4Q9UQ80N/A (3)N/CRNA bindingTranslation89
    • ↵a UniProt-SwissProt accession number.

    • ↵b Average fold increase in total spectra for PV-infected sample versus mock-infected control.

    • ↵c Cellular localization is indicated as nuclear (N) or cytoplasmic (C). N/C, proteins known to shuttle between the nucleus and the cytoplasm.

    • ↵d General cellular functions of the protein.

    • ↵e Stage of the virus life cycle in which the protein is active.

  • Table 3

    The 66 TUX-MS-identified host factors that have not been previously implicated in the enteroviral life cyclea

    Protein nameDesignationAccession no.bMol mass (kDa)No. of unique peptidescTotal no. of spectradFold incorporatedeGene ontologyfCellular localizationgRelative abundancehInteractionReference(s)
    VirusiIRESj
    Non-POU domain-containing octamer-binding proteinNONOQ1523354.21421INFRRMN++++HIV-1, HDVc-Myc90–92
    Nucleolar RNA helicase 2DDX21Q9NR3087.31517INFRBPN+++BDV93
    Ribosomal L1 domain-containing protein 1RSL1D1O7602155.01315INFRBPN/C+++
    Ribonucleases P/MRP protein subunit POP1POP1Q99575114.71214INFRBPN/C++
    Myb-binding protein 1AMYBBP1AQ9BQG0148.91212INFDBPN/C+++
    SAFB-like transcription modulatorSLTMQ9NWH9117.2811INFRRMN++
    KH domain-containing, RNA-binding, signal transduction-associated protein 1KHDRBS1Q0766648.21011INFRBPN++++HIV-194
    TATA-binding protein-associated factor 2NTAF15Q9280461.8410INFRRMN/C+++
    Cellular nucleic acid-binding protein (ZNF9)CNBPP6263319.579INFRBPN/C++++JCVODC95–97
    Putative rRNA methyltransferase NOP2NOP2P4608789.389INFRBPN++
    Fragile X mental retardation syndrome-related protein 1FXR1P5111469.777INFRBPN/C+++
    Fragile X mental retardation syndrome-related protein 2FXR2P5111674.247INFRBPN/C++
    X-ray repair cross-complementing protein 5XRCC5P1301082.767INFDBPN/C++++
    X-ray repair cross-complementing protein 6XRCC6P1295669.856INFDBPN/C++++PDGF2, VEGF98
    Heterogeneous nuclear ribonucleoprotein L-likeHNRPLLQ8WVV960.145INFRRMN++
    RNA-binding protein 28RBM28Q9NW1385.755INFRRMN/C++
    Bcl-2-associated transcription factor 1BCLAF1Q9NYF8106.155INFDBPN/C+++
    Histone H2B type 1-DHIST1H2BDP5887613.925INFDBPN+++++
    RNA-binding protein EWSEWSR1Q0184468.545INFRRMN/C+++HCVHCV99
    Splicing factor, arginine/serine-rich 5SFRS5Q1324331.334INFRRMN+++
    Importin subunit alpha-2/karyopherin alpha 2KPNA2P5229257.944INFRBPN/C++++
    A-kinase anchor protein 8AKAP8O4382376.144INFDBPN/C++
    Nucleolysin TIA-1 isoform p40TIA1P3148343.0310INFRRMN/C++
    RNA-binding protein FUSFUSP3563753.4122625.5RRMN/C+++
    Polyubiquitin-BUBBP0CG4725.872525N/C++++
    Splicing factor, proline- and glutamine-rich (PSF)SFPQP2324676.1152121.0RRMN++++HDVc-Myc92, 100
    Zinc finger protein 326ZNF326Q5BKZ165.7101211.5DBPN+++
    Zinc finger RNA-binding proteinZFRQ96KR1117.0101111.0RBPN/C++
    Zinc finger protein 638ZNF638Q14966220.615188.8RRMN/C+
    Heterogeneous nuclear ribonucleoprotein U-like protein 2HNRNPUL2Q1KMD385.115217.0RBPN+++
    Heterogeneous nuclear ribonucleoprotein GRBMXP3815942.318355.8RRMN+++
    Ras GTPase-activating protein-binding protein 2G3BP2Q9UN8654.1565.5RRMC+++
    rRNA 2′-O-methyltransferase fibrillarinFBLP2208733.8565.5RBPN+++
    Transcriptional activator protein Pur-alphaPURAQ0057734.9555.0DBPN/C+++
    RNA-binding protein 4RBM4Q9BWF340.3894.3RRMN/C+++
    Probable ATP-dependent RNA helicase DDX17DDX17Q9284172.423504.1RBPN+++
    Heterogeneous nuclear ribonucleoprotein RHNRNPRO4339070.922654.0RRMN/C++++
    Zinc finger CCCH-type antiviral protein 1ZC3HAV1Q7Z2W4101.411124.0RBPN/C+++RNA viruses65
    Heterogeneous nuclear ribonucleoprotein U (SAF-A)HNRNPUQ0083990.5551383.8RBPN/C++++
    Heterogeneous nuclear ribonucleoprotein LHNRNPLP1486664.1251023.5RRMN/C++++HDV, HSV, HCVHCV, Cat-167–69, 91, 101
    RNA-binding protein 39RBM39Q1449859.4443.5RRMN/C+++
    Splicing factor 3B subunit 4SF3B4Q1542744.4343.5RRMN++
    Heterogeneous nuclear ribonucleoprotein A0HNRNPA0Q1315130.89113.5RRMN++++
    Protein FAM98AFAM98AQ8NCA555.4343.5+++
    Peptidyl-prolyl cis-trans-isomerase APPIAP6293718.0343.5N/C+++++
    Probable ATP-dependent RNA helicase DDX5DDX5P1784469.128513.4RBPN+++HCV, IAV66, 102
    Heterogeneous nuclear ribonucleoprotein D0 (AUF1)HNRNPDQ1410338.417313.4RRMN/C++++
    Ubiquitin-associated protein 2-likeUBAP2LQ14157114.59143.4DBPN+++
    La-related protein 1LARP1Q6PKG0123.59103.3RBPN/C++
    Serine/arginine-rich splicing factor 10SFRS13AO7549431.3573.3RRMN/C++
    Splicing factor, arginine/serine-rich 7SFRS7Q1662927.38133.3RRMN++++HSV-1103
    RNA-binding protein 47RBM47A0AV9664.1563.0RRMN+
    Serine/arginine-rich splicing factor 2SFRS2Q0113025.5393.0RRMN+++
    RNA-binding protein RalyRALYQ9UKM932.5563.0RRMN+++
    Splicing factor, arginine/serine-rich 9SFRS9Q1324225.5692.8RRMN+++
    Nuclease-sensitive element-binding protein 1YBX1P6780935.917512.6RBPN/C++++DENc-Myc92, 104
    Splicing factor U2AF 65-kDa subunitU2AF2P2636853.5682.5RRMN++++
    Nucleolysin TIARTIAL1Q0108541.613182.5RRMN/C+
    Heterogeneous nuclear ribonucleoprotein D-likeHNRPDLO1497946.413292.4RRMN/C++++NRF105, 106
    Heterogeneous nuclear ribonucleoprotein A3HNRNPA3P5199139.625432.4RRMN++++
    Serine/arginine-rich splicing factor 1 (ASF-1, SF2-P33)SFRS1Q0795527.711192.3RRMN/C++++HDV91
    Heterogeneous nuclear ribonucleoprotein U-like protein 1HNRNPUL1Q9BUJ296.015232.3RBPN+++Adenovirus107
    Transformer-2 protein homolog betaTRA2BP6299533.7692.3RRMN+++
    Serine/arginine-rich splicing factor 6SFRS6Q1324739.68112.2RRMN+++
    Heterogeneous nuclear ribonucleoprotein QSYNCRIPO6050669.630642.2RRMN/C++++HCVHCV, BiP108–110
    Plasminogen activator inhibitor 1 RNA-binding proteinSERBP1Q8NC5145.017222.2RBPN/C++++
    • ↵a The mass spectroscopy was performed on two poliovirus-infected samples, and the data presented represent an average of results between those data sets. The data sets were highly reproducible (see Data Set S1 in the supplemental material).

    • ↵b UniProt-SwissProt accession number.

    • ↵c Average number of unique peptides identified by MS.

    • ↵d Average total number of spectra that were assigned to that protein from the MS analysis.

    • ↵e Average fold increase in total spectra for PV-infected versus mock-infected control. For proteins only detected in the PV-infected samples, fold change is indicated as “infinite” (INF).

    • ↵f Most of the identified host factors are RNA binding proteins (RBP) or have RNA recognition motifs (RRMs), which are indicated in the gene ontology column. Some are DNA binding proteins (DBP). Blank table cells indicate they are not known to bind either RNA or DNA to date.

    • ↵g Cellular localization is indicated as nuclear (N) or cytoplasmic (C). N/C, proteins known to shuttle between the nucleus and the cytoplasm.

    • ↵h Protein abundances according to the Protein Abundance Across Organisms Database (pax-db.org). Abundances are expressed relative to β-actin (5,120 ppm). +++++ represents the range of 9,999 to 1,000 ppm. Stepwise 10-fold decreases down to 0.99 to 0.1 ppm (+) are indicated.

    • ↵i Viruses whose life cycle has been shown to be impacted in some manner by the host factor. HCV, hepatitis C virus; HDV, hepatitis D virus; BDV, Borna disease virus; JCV, JC virus; HSV, herpes simplex virus; IAV, influenza A virus; DEN, dengue virus.

    • ↵j Host factors that have been implicated as an IRES-trans-acting factor (ITAF) for other IRESs are indicated. ODC, ornithine decarboxylase; PDGF2, platelet-derived growth factor 2; VEGF, vascular endothelial growth factor; NRF, nuclear respiratory factor.

Additional Files

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    • Supplemental file 1 -

      Data set 1 (Host factors identified using TUX-MS.)

      XLS, 112K

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Thiouracil Cross-Linking Mass Spectrometry: a Cell-Based Method To Identify Host Factors Involved in Viral Amplification
Erik M. Lenarcic, Dori M. Landry, Todd M. Greco, Ileana M. Cristea, Sunnie R. Thompson
Journal of Virology Jul 2013, 87 (15) 8697-8712; DOI: 10.1128/JVI.00950-13

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Thiouracil Cross-Linking Mass Spectrometry: a Cell-Based Method To Identify Host Factors Involved in Viral Amplification
Erik M. Lenarcic, Dori M. Landry, Todd M. Greco, Ileana M. Cristea, Sunnie R. Thompson
Journal of Virology Jul 2013, 87 (15) 8697-8712; DOI: 10.1128/JVI.00950-13
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Print ISSN: 0022-538X; Online ISSN: 1098-5514