Human immunodeficiency virus type 1 Vpr interacts with spliceosomal protein SAP145 to mediate cellul

1
Human immunodeficiency virus type 1 Vpr interacts
with spliceosomal protein SAP145 to mediate
cellular pre-mRNA splicing inhibition

• Chieko Hashizume, Madoka Kuramitsua, Xianfeng
  Zhang, Terue Kurosawa, Masakazu Kamata and Yoko
  Aida
• Microbes Infect. 2007 Apr9(4)490-7

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HIV-1 Vpr Viral Protein R

• Regulates nuclear import of the PIC
• contains an NLS, binds importin-a
• Stimulates viral gene expression
• acts as an adaptor in assembly of transcription
  machinery, binds steroid receptor coactivators
• Induces apoptosis by
• translocating mitochondrial ptn. HAX-1 into
  cytoplasm
• Arrests host cell cycle at G2-to-M transition
• inactivates Cdc2

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Viral inhibition of splicing

• HSV-1 ICP27 inhibits a kinase of splicing
  factors, also associated with SAP145
• HIV
• Tat (transactivator) inhibits splicing
• Rev can export incompletely spliced vRNA

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Pre-mRNA splicing
Sanford, J. R. et al. J Cell Sci
20041176261-6263
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• Authors previously found in a yeast 2-hybrid
  screen that Vpr interacts with SAP145 (component
  of SF3b splicing factor), and inhibits splicing.
• re-confirm previous results in an in vitro
  splicing assay
• determine which domains of Vpr are responsible
  for splicing inhibition and interaction with
  SAP145
• does Vpr inhibit splicing by interfering with
  SAP145-SAP49 interaction?

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In vitro splicing assays

• Nuclear extracts prepared from control HeLa or
  HeLa expressing Vpr
• Splicing activity percentage of spliced mRNA
  over total

Graph shows average splicing activity over at
least 3 experiments bars show standard error.
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• Lane 3 Untransfected HeLa with purified
  FLAG-Vpr.

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Identification of Vpr domain involved in splicing
inhibition

• FLAG-GFP fusions of 4 Vpr domains separately
  constructed and expressed in HeLA
• Splicing products of a-globin 2 detected by
  nested RT-PCR of NE
• Note unspliced pre-mRNA disappears in all
  treatments

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Vpr-SAP145 interaction is via Leu67

• Single aa mutations created in the aH-3 and
  Arg-rich domains
• Double mutants fail to bind SAP, do not inhibit
  splicing efficiently

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Effect on SAP145-SAP49

• SAP49 interacts directly with SAP145 and
  substrate RNA.
• binding is abolished by Vpr
• a-H3 and double mutants are impaired in blocking
  interaction
• Binding assay with purified proteins shows
  concentration-dependent inhibition of SAP49-SAP145

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Discussion

• Inhibition of splicing by Vpr and interaction
  with SAP145 confirmed in vitro
• Leucine 67 is essential for SAP145 binding
• Arginine-rich CTD essential, but the two residues
  examined in this study were not.
• Vpr interferes with SAP145-49 -gt uncouples U2
  snRNA from branch site of substrate