In human cells infected with herpes simplex virus 1 the double-stranded RNA-dependent protein kinase (PKR) is activated but phosphorylation of the ␣ subunit of eukaryotic translation initiation factor 2 (eIF-2) and total shutoff of protein synthesis is observed only in cells infected with ␥ 1 z34.5 ؊ mutants. The carboxyl-terminal 64 aa of ␥ 1 34.5 protein are homologous to the corresponding domain of MyD116, the murine growth arrest and DNA damage gene 34 (GADD34) protein and the two domains are functionally interchangeable in infected cells. This report shows that (i) the carboxyl terminus of MyD116 interacts with protein phosphatase 1␣ in yeast, and both MyD116 and ␥ 1 34.5 interact with protein phosphatase 1␣ in vitro; (ii) protein synthesis in infected cells is strongly inhibited by okadaic acid, a phosphatase 1 inhibitor; and (iii) the ␣ subunit in purified eIF-2 phosphorylated in vitro is specifically dephosphorylated by S10 fractions of wild-type infected cells at a rate 3000 times that of mock-infected cells, whereas the eIF-2␣-P phosphatase activity of ␥ 1 34.5 ؊ virus infected cells is lower than that of mock-infected cells. The eIF-2␣-P phosphatase activities are sensitive to inhibitor 2. In contrast to eIF-2␣-P phosphatase activity, extracts of mock-infected cells exhibit a 2-fold higher phosphatase activity on [ 32 P]phosphorylase than extracts of infected cells. These results indicate that in infected cells, ␥ 1 34.5 interacts with and redirects phosphatase to dephosphorylate eIF-2␣ to enable continued protein synthesis despite the presence of activated PKR. The GADD34 protein may have a similar function in eukaryotic cells. The proposed mechanism for maintenance of protein synthesis in the face of double-stranded RNA accumulation is different from that described for viruses examined to date.

The protein encoded by the y,34.

The carboxyl-terminal domain of the ␥ 1 34.5 protein of the herpes simplex virus 1 binds to protein phosphatase 1␣ (PP1) and is required to prevent the shut-off of protein synthesis resulting from phosphorylation of the ␣ subunit of eIF-2 by the double-stranded RNA-activated protein kinase. The corresponding domain of the conserved GADD34 protein homologous to ␥ 1 34.5 functionally substitutes for ␥ 1 34.5. This report shows that ␥ 1 34.5 and PP1 form a complex in the infected cells, that fractions containing this complex specifically dephosphorylate eIF-2␣, and that both ␥ 1 34.5 and GADD34 proteins contain the amino acid sequence motif common to subunits of PP1 that is required for binding to the PP1 catalytic subunit. An oligopeptide containing this motif competes with ␥ 1 34.5 for binding to PP1. Substitution of Val 193 and Phe 195 in the PP1-binding motif abolished activity. These results suggest that the carboxyl-terminal domain of ␥ 1 34.5 protein has the structural and functional attributes of a subunit of PP1 specific for eIF-2␣, that it has evolved to preclude shut-off of protein synthesis, and that GADD34 may have a similar function.The ␥ 1 34.5 gene of herpes simplex virus 1 (HSV-1) 1 encodes two functions. The first enables the virus to replicate in vivo and particularly to multiply and spread in the central nervous system of experimental animal systems (1,2). This function appears to map throughout the coding domain of the gene (3, 4). The second blocks the shut-off of protein synthesis resulting from phosphorylation of the ␣ subunit of the translation initiation factor eIF-2 by the double-stranded RNA-activated protein kinase (PKR). This function maps in the 3Ј-terminal domain of the 263-codon gene (5, 6). Earlier studies have shown that in HSV-1-infected cells PKR is activated but that in cells infected with wild-type virus or virus carrying in-frame deletions of the 5Ј-terminal coding domain of the gene eIF-2␣ was not phosphorylated (7). In subsequent studies (8) we have shown that the ␥ 1 34.5 protein interacts with the protein phosphatase 1␣ (PP1). Indeed, infected cells contain a phosphatase activity that specifically dephosphorylates eIF-2␣ at a rate 3000-fold greater than that measured in uninfected cells. This phosphatase activity is inhibited by inhibitors of PP1. The hypothesis that emerged from these studies is that transcription of complementary sequences of the HSV-1 DNA results in the formation of double-stranded RNA, that PKR is activated in cells infected with both wild-type and mutant viruses, and that a domain of the ␥ 1 34.5 protein binds PP1 and redirects its activity to dephosphorylate eIF-2␣. This report centers on one aspect of this hypothesis: we show that in infected cells ␥ 1 34.5 protein is a component of a multi-protein, cytoplasmic complex containing PP1 and that the interacting domain of the ␥ 1 34.5 protein is near the carboxyl terminus of the protein and has an amino acid motif shared with accessory proteins or subunits interacting with the catalytic subunit of PP1...

The herpes simplex virus type 1 ␥ 1 34.5 gene product precludes the host-mediated protein shutoff response induced by activated protein kinase R (PKR). Earlier studies demonstrated that recombinant viruses lacking the ␥ 1 34.5 gene (⌬␥ 1 34.5) developed secondary mutations that allowed earlier U S 11 expression and enabled continued protein synthesis. Further, in vitro studies demonstrated that a recombinant expressed U S 11 protein binds PKR, blocks the phosphorylation of the ␣ subunit of eukaryotic initiation factor 2 (eIF-2␣) by activated PKR, and, if provided prior to PKR activation, precluded PKR autophosphorylation. The present study furthers the hypothesis that early U S 11 production precludes PKR-mediated host protein shutoff by demonstrating that (i) U S 11 and PKR interact in the context of viral infection, (ii) this interaction is RNA dependent and requires a 30-amino-acid domain (amino acids 91 to 121) in the carboxyl domain of the U S 11 protein, (iii) the proteins biochemically colocalize in the S100 ribosomal fraction, and (iv) there is a PKR substrate domain immediately adjacent to the binding domain. The results suggest that the U S 11 interaction with PKR at the ribosome is RNA dependent and that the U S 11 protein contains a substrate domain with homology to eIF-2␣ in close proximity to an essential binding domain.

Introduction Penile prosthesis infections remain challenging despite advancements in surgical technique, device improvements, and adoption of antibiotic prophylaxis guidelines. Aim To investigate penile prosthesis infection microbiology to consider which changes in practice could decrease infection rates, to evaluate current antibiotic prophylaxis guidelines, and to develop a proposed algorithm for penile prosthesis infections. Methods This retrospective institutional review board–exempt multi-institutional study from 25 centers reviewed intraoperative cultures obtained at explantation or Mulcahy salvage of infected three-piece inflatable penile prostheses (IPPs). Antibiotic usage was recorded at implantation, admission for infection, and explantation or salvage surgery. Cultures were obtained from purulent material in the implant space and from the biofilm on the device. Main Outcome Measures Intraoperative culture data from infected IPPs. Results Two hundred twenty-seven intraoperative cultures (2002–2016) were obtained at salvage or explantation. No culture growth occurred in 33% of cases and gram-positive and gram-negative organisms were found in 73% and 39% of positive cultures, respectively. Candida species (11.1%), anaerobes (10.5%) and methicillin-resistant Staphylococcus aureus (9.2%) constituted nearly one third of 153 positive cultures. Multi-organism infections occurred in 25% of positive cultures. Antibiotic regimens at initial implantation were generally consistent with American Urological Association (AUA) and European Association of Urology (EAU) guidelines. However, the micro-organisms identified in this study were covered by these guidelines in only 62% to 86% of cases. Antibiotic selection at admissions for infection and salvage or explantation varied widely compared with those at IPP implantation. Conclusion This study documents a high incidence of anaerobic, Candida, and methicillin-resistant S aureus infections. In addition, approximately one third of infected penile prosthesis cases had negative cultures. Micro-organisms identified in this study were not covered by the AUA and EAU antibiotic guidelines in at least 14% to 38% of cases. These findings suggest broadening antibiotic prophylaxis guidelines and creating a management algorithm for IPP infections might lower infection rates and improve salvage success.