The Stress-Inducible Immediate-Early Responsive Gene IEX-1 Is Activated in Cells Infected with Herpes Simplex Virus 1, but Several Viral Mechanisms, Including 3′ Degradation of Its RNA, Preclude Expression of the Gene

Abstract

The accumulation of cellular transcripts from cells infected with herpes simplex virus 1 (HSV-1) as measured with the aid of Affymetrix microchips has been reported elsewhere. Among these transcripts were genes that respond to stress and that could have a noxious effect on viral replication. We have selected the stress-inducible cellular gene encoding the immediate-early response protein IEX-1 to verify and determine the significance of the accumulation of these transcripts in infected cells. We report that we verified the increase in accumulation of IEX-1 transcripts after infection by Northern analyses and real-time PCR. These transcripts reach peak levels between 3 and 7 h after infection and decrease thereafter. However, IEX-1 protein was detected in cells 1 h after infection but not at later intervals. Studies designed to elucidate the failure of IEX-1 protein to be synthesized revealed the following points.

The U L 41 protein of herpes simplex virus 1 degrades RNA by endonucleolytic cleavage in absence of other cellular or viral proteins

“…The results described above were in agreement with the pattern of degradation of IEX-1 mRNA observed in HSV-1-infected cells and specifically with the lingering of truncated 5Ј domains, generated by vhs-dependent endonucleolytic cleavage within the AREs (19,21). The objective of the experiments described in this section was to test whether a substrate, consisting of only IEX-1 coding sequence domain (CDS, substrate 3), was cleaved by the purified GST-vhs fusion protein.…”

“…The choice of this specific substrate was based on earlier studies showing that, in infected cells, IEX-1 mRNA was transcribed but that the protein was not made (21). Extensive analyses showed that IEX-1 mRNA was degraded in a unique, vhs-dependent manner (19,21).…”

“…The choice of this specific substrate was based on earlier studies showing that, in infected cells, IEX-1 mRNA was transcribed but that the protein was not made (21). Extensive analyses showed that IEX-1 mRNA was degraded in a unique, vhs-dependent manner (19,21). Specifically, the mRNA was deadenylated and subjected to 3Ј-to-5Ј degradation and the truncated 5Ј mRNA fragments tended to 5) or GST (lanes 6 -9) were incubated at 30°C with internally labeled substrate 3.…”

“…HeLa or HEp-2 cells were mock infected or infected with 10 PFU of the wild-type or mutant viruses per cell. The procedures for harvesting, solubilization, protein quantification, SDS͞PAGE, transfer to nitrocellulose membranes and reaction with antibody to specific protein were as reported (21). The anti-vhs polyclonal rabbit serum was used at a 1:1,000 dilution.…”

“…Examination of the relative rates of degradation of several cellular mRNAs upon HSV-1 infection led to the conclusion that cellular RNAs form at least three classes based on their fate after infection. There are (i) constitutively expressed mRNAs (i.e., GAPDH, ␤-actin) that were rapidly degraded (20); (ii) some inducible mRNAs containing AU-rich elements (AREs) in their 3Ј UTR (i.e., stress-inducible immediate-early responsive gene or IEX-1, I B␣, c-fos) that were deadenylated, subjected to endonucleolytic cleavage near the 3Ј end and to 3Ј-to-5Ј degradation, with the 5Ј-truncated mRNA fragments persisting in wild-type virus-infected cells for many hours (20,21), and (iii) some inducible mRNAs, exemplified by GADD45␤ and tristetraprolin, which showed no evidence of degradation and whose proteins readily accumulated in infected cells (19,22). All of these processes require vhs because none of them was detected in cells infected with the ⌬U L 41-mutant virus.…”