Evidence for Translational Regulation by the Herpes Simplex Virus Virion Host Shutoff Protein▿
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FIG. 1.Schematic representation of the bicistronic reporter constructs used in this study (not drawn to scale). The test IRES elements were inserted between the 5′ β-galactosidase (βgal) and 3′ CAT cistrons. The locations of the CMV IE promoter and two alternative polyadenylation signals are indicated.
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FIG. 2.Effects of vhs on 5′ and 3′ cistron expression from reporter constructs bearing wild-type and mutant EMCV IRES elements. HeLa cells were transfected with the indicated reporter plasmids and increasing amounts of a vhs expression vector (pCMVvhs). Cell extracts were then assayed for β-galactosidase (left panel) and CAT (right panel) enzymatic activities.
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FIG. 3.The EMCV GW3 mutant IRES retains vhs targeting activity in vitro. Uniformly labeled EMCV transcripts containing wild-type (A), GW3 (B), and GW8 (C) IRES elements at their 5′ ends were added to RRL containing pretranslated vhs, and aliquots were removed after the indicated times (minutes) and analyzed by gel electrophoresis. neg, RRL lacking pretranslated vhs.
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FIG. 4.Effects of vhs on 5′ and 3′ cistron expression from reporter constructs bearing various IRES elements. HeLa cells were transfected with the indicated reporter plasmids and increasing amounts of pCMVvhs. Cell extracts were then assayed for β-galactosidase (left panel) and CAT (right panel) enzymatic activities.
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FIG. 5.vhs does not activate the BiP IRES in Vero cells. HeLa and Vero cells were transfected with the pβgal/BiP/CAT reporter plasmid and increasing amounts of pCMVvhs. Cell extracts were then assayed for β-galactosidase (left panel) and CAT (right panel) enzymatic activities.
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FIG. 6.Several HSV-1 5′ UTR sequences serve as vhs positive response elements. HeLa cells were transfected with the reporter plasmids bearing the indicated elements inserted into the intercistronic regions, along with 0, 20, or 200 ng of pCMVvhs. Cell extracts were then assayed for β-galactosidase and CAT enzymatic activities.
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FIG. 7.vhs modulates 5′ and 3′ cistron expression without greatly altering reporter mRNA levels or structure. (A) Northern blot analysis of poly(A)+ RNA extracted from HeLa cells transfected with pβgal/BiP/CAT and 0, 20, or 200 ng of pCMVvhs, probed for CAT sequences. The electrophoretic mobilities of marker RNAs of the indicated sizes (kb) are displayed on the left. (B) β-Galactosidase and CAT activities in extracts of the transfected cells.
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FIG. 8.vhs does not alter the 5′ end of the reporter mRNA. Total RNA extracted from HeLa cells transfected with pβgal/BiP/CAT in the presence or absence of pCMVvhs was analyzed by primer extension as described in Materials and Methods, using a 32P-labeled oligonucleotide primer complementary to residues 70 to 93 of the βgal/BiP/CAT reporter RNA. Products were resolved on 8% polyacrylamide sequencing gels. HeLa cells were transfected with pUC19 (lane 1), βgal/BiP/CAT and pUC19 (lane 2), pCMVvhs and pUC19 (lane 3), or βgal/BiP/CAT, pCMVvhs, and pUC19 (lane 4). Lane M, sizes of marker pUC19 MspI cleavage fragments in nt.
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FIG. 9.vhs suppresses expression from pSV3CAT without triggering a corresponding loss of reporter mRNA in HeLa cells. (A) Northern blot analysis of poly(A)+ RNA extracted from HeLa cells transfected with pSV3CAT and 0, 20, or 200 ng of pCMVvhs, probed for CAT sequences. The electrophoretic mobilities of marker RNAs of the indicated sizes (kb) are displayed on the left. (B) CAT activity in extracts of the transfected cells.
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FIG. 10.Rhinovirus 2A protease does not activate 3 cistron expression. HeLa cells were transfected with the βgal/BiP/CAT bicistronic reporter plasmid and increasing amounts of expression vectors encoding vhs or wild-type (wt) or mutant (mut) HRV2 2A protease. Cell extracts were then analyzed for β-galactosidase (left panel) and CAT (right panel) enzymatic activities.
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FIG. 11.Translation of the 5′ cistron is not required for 3′ cistron activation. HeLa cells were transfected with the βgal/BiP/CAT bicistronic reporter plasmid (BiP) or a derivative bearing a stem-loop inserted into the 5′ UTR (SL+BiP) along with the indicated amounts of pCMVvhs. Cell extracts were then analyzed for β-galactosidase (left panel) and CAT (right panel) enzymatic activities.
- American Society for Microbiology
