The R1 subunit of herpes simplex virus ribonucleotide reductase protects cells against apoptosis at, or upstream of, caspase-8 activation

Abstract

The R1 subunit of herpes simplex virus (HSV) ribonucleotide reductase, which in addition to its Cterminal reductase domain possesses a unique N-terminal domain of about 400 amino acids, is thought to have an additional, as yet unknown, function. Here, we report that the full-length HSV-2 R1 has an anti-apoptotic function able to protect cells against death triggered by expression of R1(∆2-357), an HSV-2 R1 subunit with its first 357 amino acids deleted. We further substantiate the R1 anti-apoptotic activity by showing that its accumulation at low level could completely block apoptosis induced by TNF-receptor family triggering. Activation of caspase-8 induced either by TNF or by Fas ligand expression was prevented by the R1 protein. As HSV R1 did not inhibit cell death mediated by several agents acting via the mitochondrial pathway (Bax overexpression, etoposide, staurosporine and menadione), it is proposed that it functions to interrupt specifically death receptor-mediated signalling at, or upstream of, caspase-8 activation. The N-terminal domain on its own did not exhibit anti-apoptotic activity, suggesting that both domains of R1 or part(s) of them are necessary for this new function. Evidence for the importance of HSV R1 in protecting HSV-infected cells against cytokine-induced apoptosis was obtained with the HSV-1 R1 deletion mutants ICP6∆ and hrR3. These results show that, in addition to its ribonucleotide reductase function, which is essential for virus reactivation, HSV R1 could contribute to virus propagation by preventing apoptosis induced by the immune system.

The ribonucleotide reductase R1 subunits of herpes simplex virus types 1 and 2 protect cells against TNFα- and FasL-induced apoptosis by interacting with caspase-8

“…However, as 50% of cells infected with R1 deletion mutants were resistant to TNFa, we also postulated that other viral protein(s) contribute(s) to protection. Since deletion of the vhs gene reduced antiapoptotic potential by 30%, we hypothesized that vhs protein, by decreasing the amount of DRs at the cell surface, could play a significant role in resistance [7]. Our hypothesis was later substantiated by a report showing that, owing to the very short half-life of TNFR1 protein, vhs causes a rapid reduction of TNFR1 early after HSV-1 infection [8].…”

“…The conditions for culture of HeLa, A549-tTA and A549-tTA-HSV-R1-GFP cells have been described [7,28]. When cultured in the absence of anhydrotetracyclin (Tet), A549-tTA-HSV-R1-GFP cells express HSV-2 R1 fused to green fluorescent protein (GFP) [28].…”

“…Our group has shown that the individually expressed HSV-2 R1 blocks DR-induced apoptosis but does not impair apoptosis triggered via the mitochondrial pathway [7]. The RR domain of HSV-2 R1, but not the NH 2 domain, is essential for protection against TNFa-induced apoptosis [28].…”

“…The importance for HSVs to counteract DR activation is furthermore evidenced by the diverse strategies used by these viruses to block DR signaling pathways. At least two viral proteins, the ribonucleotide reductase (RR) R1 subunit of HSV-2 (HSV-2 R1) and the virion host shutoff protein (vhs), have been hypothesized to interfere with these pathways [7,8].…”

“…The RR domain of HSV-2 R1, but not the NH 2 domain, is essential for protection against TNFa-induced apoptosis [28]. HSV-2 R1, which does not act as an enzymatic inhibitor of active caspase-8, interrupts DR-mediated signaling at, or upstream of, caspase-8 activation [7]. As two HSV-1 UL39 deletion mutants, ICP6D and hrR3, exhibited a 50% reduction in protection from TNFa, we had proposed that HSV-1 R1 could be important for protecting HSV-infected cells against this DR ligand.…”