Abstract

The aim of this research proposal is to elucidate the events in the infected cell concerned with the transcription and replication of the segmented influenza virus genome, and to determine the role of nucleus in these events. We intend to further characterize viral complementary RNA (cRNA), the product of transcription. Viral cRNA, the viral messenger RNA, is comprised of segments similar in size and number to the virion RNA (vRNA) segments. We will determine which cRNA segment codes for which virus-specific protein, and which cRNA segment is transcribed from which vRNA segment. Viral cRNA contains internal N6-methyladenosine (m6A) and a 5'-terminal 7-methylguan sine cap structure. Two different caps are present: one contains methylated adenosine, and the other methylated guanosine, as the penultimate nucleoside. We will determine the distribution of the two different caps, and of internal m6A, among the different cRNA segments. We will also determine whether the cRNA segments correspond to complete transcripts of the vRNA segments by sequence analysis of the 5' ends of cRNA and the 3' ends of vRNA. Specific probes, 125I-vRNA and 32P-complementary DNA, have enabled us to measure the number of cRNA and vRNA molecules in the nucleus and cytoplasm during infection. We have found that actinomycin D blocks the appearance in the cytoplasm of cRNA and not vRNA, whereas the cRNA resulting from primary transcription still appears in the nucleus. To identify the actinomycin D-sensitive nuclear step, we intend to further examine the appearance of cRNA during infection and to compare the characteristics (e.g., size, presence of poly A and 5' caps) of the cRNA synthesized in the presence and absence of actinomycin D. With these probes, we will also identify the defect(s) in RNA transcription and replication exhibited by temperature-sensitive viral mutants. We intend to develop an assay, employing hybridization and affinity chromatography, which will allow the detection of newly-synthesized cRNA and vRNA. With this assay, we will determine the intracellular site(s) of synthesis of cRNA and vRNA.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI011772-12
Application #
3125025
Study Section
Experimental Virology Study Section (EVR)
Project Start
1977-07-01
Project End
1987-06-30
Budget Start
1985-07-01
Budget End
1986-06-30
Support Year
12
Fiscal Year
1985
Total Cost
Indirect Cost

  Institution

Name
Sloan-Kettering Institute for Cancer Research
Department
Type
DUNS #
064931884
City
New York
State
NY
Country
United States
Zip Code
10065

  Publications

Smith, Bartram L; Chen, Guifang; Wilke, Claus O et al. (2018) Avian Influenza Virus PB1 Gene in H3N2 Viruses Evolved in Humans To Reduce Interferon Inhibition by Skewing Codon Usage toward Interferon-Altered tRNA Pools. MBio 9:
Kuo, Rei-Lin; Li, Li-Hsin; Lin, Sue-Jane et al. (2016) Role of N Terminus-Truncated NS1 Proteins of Influenza A Virus in Inhibiting IRF3 Activation. J Virol 90:4696-4705
Zhao, Chen; Sridharan, Haripriya; Chen, Ran et al. (2016) Influenza B virus non-structural protein 1 counteracts ISG15 antiviral activity by sequestering ISGylated viral proteins. Nat Commun 7:12754
Ma, Li-Chung; Guan, Rongjin; Hamilton, Keith et al. (2016) A Second RNA-Binding Site in the NS1 Protein of Influenza B Virus. Structure 24:1562-72
Liu, Chien-Hung; Zhou, Ligang; Chen, Guifang et al. (2015) Battle between influenza A virus and a newly identified antiviral activity of the PARP-containing ZAPL protein. Proc Natl Acad Sci U S A 112:14048-53
Krug, Robert M (2015) Functions of the influenza A virus NS1 protein in antiviral defense. Curr Opin Virol 12:1-6
Aramini, James M; Hamilton, Keith; Ma, Li-Chung et al. (2014) (19)F NMR reveals multiple conformations at the dimer interface of the nonstructural protein 1 effector domain from influenza A virus. Structure 22:515-525
Chen, Guifang; Liu, Chien-Hung; Zhou, Ligang et al. (2014) Cellular DDX21 RNA helicase inhibits influenza A virus replication but is counteracted by the viral NS1 protein. Cell Host Microbe 15:484-93
Krug, Robert M (2014) Viral proteins that bind double-stranded RNA: countermeasures against host antiviral responses. J Interferon Cytokine Res 34:464-8
Krug, Robert M (2014) Influenza: An RNA-synthesizing machine. Nature 516:338-9

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