Ian J Mohr, Ph.D.

A cell culture model of facial palsy resulting from reactivation of latent herpes simplex type 1
Kuhn, Maggie A; Nayak, Shruti; Camarena, Vladimir; Gardner, Jimmy; Wilson, Angus; Mohr, Ian; Chao, Moses V; Roehm, Pamela C
2012 Jan;33(1):87-92, Otology & neurotology
HYPOTHESIS: Reactivation of herpes simplex virus type 1 (HSV-1) in geniculate ganglion neurons (GGNs) is an etiologic mechanism of Bell's palsy (BP) and delayed facial palsy (DFP) after otologic surgery. BACKGROUND: Several clinical studies, including temporal bone studies, antibody, titers, and intraoperative studies, suggest that reactivation of HSV-1 from latently infected GGNs may lead to both BP and DFP. However, it is difficult to study these processes in humans or live animals. METHODS: Primary cultures of GGNs were latently infected with Patton strain HSV-1 expressing a green fluorescent protein-late lytic gene chimera. Four days later, these cultures were treated with trichostatin A (TSA), a known chemical reactivator of HSV-1 in other neurons. Cultures were monitored daily by fluorescent microscopy. Titers of media from lytic, latent, and latent/TSA treated GGN cultures were obtained using plaque assays on Vero cells. RNA was harvested from latently infected GGN cultures and examined for the presence of viral transcripts using reverse transcription-polymerase chain reaction. RESULTS: Latently infected GGN cultures displayed latency-associated transcripts only, whereas lytically infected and reactivated latent cultures produced other viral transcripts, as well. The GGN cultures displayed a reactivation rate of 65% after treatment with TSA. Media from latently infected cultures contained no detectable infectious HSV-1, whereas infectious virus was observed in both lytically and latently infected/TSA-treated culture media. CONCLUSION: We have shown that cultured GGNs can be latently infected with HSV-1, and HSV-1 in these latently infected neurons can be reactivated using TSA, yielding infectious virus. These results have implications for the cause of both BP and DFP
— id: 146264, year: 2012, vol: 33, page: 87, stat: Journal Article,

A herpesvirus kinase that masquerades as Akt: You don't have to look like Akt, to act like it
Chuluunbaatar, Uyanga; Mohr, Ian
2011 Jul 1;10(13):2064-2068, Cell cycle
The cellular protein synthesis machinery is tightly regulated and capable of rapid reaction to a variety of physiological inputs critical in stress-response, cell cycle control, cancer biology, and virus infection. One important strategy for stimulating protein synthesis involves the ser/thr kinase Akt, which subsequently triggers inactivation of the cap-dependent translational repressor 4E-BP1 by an mTOR-containing protein complex (mTORC1). A recent paper demonstrated that herpes simplex virus utilizes a remarkable tactic to activate mTOR in infected cells. Instead of using the cellular Akt, the virus produces a ser / thr kinase called Us3 that doesn't look like Akt, but masquerades as Akt. By making the Akt-like protein unrecognizable, this disguise allows it to bypass the strict limits normally imposed on the real cellular Akt. Importantly, preventing the virus Akt-imposter from triggering mTORC1 inhibited viral growth, suggesting a new way to block herpes simplex virus. This study also raises the possibility that other Akt-impersonators may lurk hidden in our own genomes, possibly contributing to diseases ranging from diabetes to cancer
— id: 136511, year: 2011, vol: 10, page: 2064, stat: Journal Article,

Mannose-6-phosphate regulates destruction of lipid-linked oligosaccharides
Gao, Ningguo; Shang, Jie; Huynh, Dang; Manthati, Vijaya L; Arias, Carolina; Harding, Heather P; Kaufman, Randal J; Mohr, Ian; Ron, David; Falck, John R; Lehrman, Mark A
2011 Sep;22(17):2994-3009, Molecular biology of the cell
Mannose-6-phosphate (M6P) is an essential precursor for mannosyl glycoconjugates, including lipid-linked oligosaccharides (LLO; glucose(3)mannose(9)GlcNAc(2)-P-P-dolichol) used for protein N-glycosylation. In permeabilized mammalian cells, M6P also causes specific LLO cleavage. However, the context and purpose of this paradoxical reaction are unknown. In this study, we used intact mouse embryonic fibroblasts to show that endoplasmic reticulum (ER) stress elevates M6P concentrations, leading to cleavage of the LLO pyrophosphate linkage with recovery of its lipid and lumenal glycan components. We demonstrate that this M6P originates from glycogen, with glycogenolysis activated by the kinase domain of the stress sensor IRE1-alpha. The apparent futility of M6P causing destruction of its LLO product was resolved by experiments with another stress sensor, PKR-like ER kinase (PERK), which attenuates translation. PERK's reduction of N-glycoprotein synthesis (which consumes LLOs) stabilized steady-state LLO levels despite continuous LLO destruction. However, infection with herpes simplex virus 1, an N-glycoprotein-bearing pathogen that impairs PERK signaling, not only caused LLO destruction but depleted LLO levels as well. In conclusion, the common metabolite M6P is also part of a novel mammalian stress-signaling pathway, responding to viral stress by depleting host LLOs required for N-glycosylation of virus-associated polypeptides. Apparently conserved throughout evolution, LLO destruction may be a response to a variety of environmental stresses
— id: 136999, year: 2011, vol: 22, page: 2994, stat: Journal Article,

Translational control of the abundance of cytoplasmic poly(a) binding protein in human cytomegalovirus-infected cells
Perez, Cesar; McKinney, Caleb; Chulunbaatar, Uyanga; Mohr, Ian
2011 Jan;85(1):156-164, Journal of virology
Irrespective of their effects on ongoing host protein synthesis, productive replication of the representative alphaherpesvirus herpes simplex virus type 1, the representative gammaherpesvirus Kaposi's sarcoma herpesvirus, and the representative betaherpesvirus human cytomegalovirus [HCMV] stimulates the assembly of the multisubunit, cap-binding translation factor eIF4F. However, only HCMV replication is associated with an increased abundance of eIF4F core components (eIF4E, eIF4G, eIF4A) and the eIF4F-associated factor poly(A) binding protein (PABP). Here, we demonstrate that the increase in translation factor concentration was readily detected in an asynchronous population of HCMV-infected primary human fibroblasts, abolished by prior UV inactivation of virus, and genetically dependent upon viral immediate-early genes. Strikingly, while increased mRNA steady-state levels accompanied the rise in eIF4E and eIF4G protein levels, the overall abundance of PABP mRNA, together with the half-life of the polypeptide it encodes, remained relatively unchanged by HCMV infection. Instead, HCMV-induced PABP accumulation resulted from new protein synthesis and was sensitive to the mTORC1-selective inhibitor rapamycin, which interferes with phosphorylation of the mTORC1 substrate p70 S6K and the translational repressor 4E-BP1. While virus-induced PABP accumulation did not require p70 S6K, it was inhibited by the expression of a dominant-acting 4E-BP1 variant unable to be inactivated by mTORC1. Finally, unlike the situation in alpha- or gammaherpesvirus-infected cells, where PABP is redistributed to nuclei, PABP accumulated in the cytoplasm of HCMV-infected cells. Thus, cytoplasmic PABP accumulation is translationally controlled in HCMV-infected cells via a mechanism requiring mTORC1-mediated inhibition of the cellular 4E-BP1 translational repressor
— id: 115423, year: 2011, vol: 85, page: 156, stat: Journal Article,

Cultured vestibular ganglion neurons demonstrate latent HSV1 reactivation
Roehm, Pamela C; Camarena, Vladimir; Nayak, Shruti; Gardner, James B; Wilson, Angus; Mohr, Ian; Chao, Moses V
2011 Oct;121(10):2268-2275, Laryngoscope
OBJECTIVES/HYPOTHESIS: Vestibular neuritis is a common cause of both acute and chronic vestibular dysfunction. Multiple pathologies have been hypothesized to be the causative agent of vestibular neuritis; however, whether herpes simplex type I (HSV1) reactivation occurs within the vestibular ganglion has not been demonstrated previously by experimental evidence. We developed an in vitro system to study HSV1 infection of vestibular ganglion neurons (VGNs) using a cell culture model system. STUDY DESIGN: basic science study. RESULTS: Lytic infection of cultured rat VGNs was observed following low viral multiplicity of infection (MOI). Inclusion of acyclovir suppressed lytic replication and allowed latency to be established. Upon removal of acyclovir, latent infection was confirmed with reverse-transcription polymerase chain reaction and by RNA fluorescent in situ hybridization for the latency-associated transcript (LAT). A total of 29% cells in latently infected cultures were LAT positive. The lytic ICP27 transcript was not detected by reverse-transcription polymerase chain reaction (RT-PCR). Reactivation of HSV1 occurred at a high frequency in latently infected cultures following treatment with trichostatin A (TSA), a histone deactylase inhibitor. CONCLUSIONS: VGNs can be both lytically and latently infected with HSV1. Furthermore, latently infected VGNs can be induced to reactivate using TSA. This demonstrates that reactivation of latent HSV1 infection in the vestibular ganglion can occur in a cell culture model, and suggests that reactivation of HSV1 infection a plausible etiologic mechanism of vestibular neuritis
— id: 137886, year: 2011, vol: 121, page: 2268, stat: Journal Article,

Viral subversion of the host protein synthesis machinery
Walsh, Derek; Mohr, Ian
2011 ;9(12):860-875, Nature reviews. Microbiology
Viruses are fully reliant on the translation machinery of their host cells to produce the polypeptides that are essential for viral replication. Consequently, viruses recruit host ribosomes to translate viral mRNAs, typically using virally encoded functions to seize control of cellular translation factors and the host signalling pathways that regulate their activity. This not only ensures that viral proteins will be produced, but also stifles innate host defences that are aimed at inhibiting the capacity of infected cells for protein synthesis. Remarkably, nearly every step of the translation process can be targeted by virally encoded functions. This Review discusses the diverse strategies that viruses use to subvert host protein synthesis functions and regulate mRNA translation in infected cells
— id: 141701, year: 2011, vol: 9, page: 860, stat: Journal Article,

Nature and duration of growth factor signaling through receptor tyrosine kinases regulates HSV-1 latency in neurons
Camarena, Vladimir; Kobayashi, Mariko; Kim, Ju Youn; Roehm, Pamela; Perez, Rosalia; Gardner, James; Wilson, Angus C; Mohr, Ian; Chao, Moses V
2010 Oct 21;8(4):320-330, Cell Host & Microbe
Herpes simplex virus-1 (HSV-1) establishes life-long latency in peripheral neurons where productive replication is suppressed. While periodic reactivation results in virus production, the molecular basis of neuronal latency remains incompletely understood. Using a primary neuronal culture system of HSV-1 latency and reactivation, we show that continuous signaling through the phosphatidylinositol 3-kinase (PI3-K) pathway triggered by nerve growth factor (NGF)-binding to the TrkA receptor tyrosine kinase (RTK) is instrumental in maintaining latent HSV-1. The PI3-K p110alpha catalytic subunit, but not the beta or delta isoforms, is specifically required to activate 3-phosphoinositide-dependent protein kinase-1 (PDK1) and sustain latency. Disrupting this pathway leads to virus reactivation. EGF and GDNF, two other growth factors capable of activating PI3-K and PDK1 but that differ from NGF in their ability to persistently activate Akt, do not fully support HSV-1 latency. Thus, the nature of RTK signaling is a critical host parameter that regulates the HSV-1 latent-lytic switch
— id: 113951, year: 2010, vol: 8, page: 320, stat: Journal Article,

Constitutive mTORC1 activation by a herpesvirus Akt surrogate stimulates mRNA translation and viral replication
Chuluunbaatar, Uyanga; Roller, Richard; Feldman, Morris E; Brown, Stuart; Shokat, Kevan M; Mohr, Ian
2010 Dec 1;24(23):2627-2639, Genes & development
All viruses require cellular ribosomes to translate their mRNAs. Viruses producing methyl-7 (m(7)) GTP-capped mRNAs, like Herpes Simplex Virus-1 (HSV-1), stimulate cap-dependent translation by activating mTORC1 to inhibit the translational repressor 4E-binding protein 1 (4E-BP1). Here, we establish that the HSV-1 kinase Us3 masquerades as Akt to activate mTORC1. Remarkably, Us3 displays no sequence homology with the cellular kinase Akt, yet directly phosphorylates tuberous sclerosis complex 2 (TSC2) on the same sites as Akt. TSC2 depletion rescued Us3-deficient virus replication, establishing that Us3 enhances replication by phosphorylating TSC2 to constitutively activate mTORC1, effectively bypassing S6K-mediated feedback inhibition. Moreover, Us3 stimulated Akt substrate phosphorylation in infected cells, including FOXO1 and GSK3. Thus, HSV-1 encodes an Akt surrogate with overlapping substrate specificity to activate mTORC1, stimulating translation and virus replication. This establishes Us3 as a unique viral kinase with promising drug development potential
— id: 114863, year: 2010, vol: 24, page: 2627, stat: Journal Article,

Activation of host translational control pathways by a viral developmental switch
Arias, Carolina; Walsh, Derek; Harbell, Jack; Wilson, Angus C; Mohr, Ian
2009 Mar;5(3):e1000334-e1000334, PLoS pathogens
In response to numerous signals, latent herpesvirus genomes abruptly switch their developmental program, aborting stable host-cell colonization in favor of productive viral replication that ultimately destroys the cell. To achieve a rapid gene expression transition, newly minted capped, polyadenylated viral mRNAs must engage and reprogram the cellular translational apparatus. While transcriptional responses of viral genomes undergoing lytic reactivation have been amply documented, roles for cellular translational control pathways in enabling the latent-lytic switch have not been described. Using PEL-derived B-cells naturally infected with KSHV as a model, we define efficient reactivation conditions and demonstrate that reactivation substantially changes the protein synthesis profile. New polypeptide synthesis correlates with 4E-BP1 translational repressor inactivation, nuclear PABP accumulation, eIF4F assembly, and phosphorylation of the cap-binding protein eIF4E by Mnk1. Significantly, inhibiting Mnk1 reduces accumulation of the critical viral transactivator RTA through a post-transcriptional mechanism, limiting downstream lytic protein production, and impairs reactivation efficiency. Thus, herpesvirus reactivation from latency activates the host cap-dependent translation machinery, illustrating the importance of translational regulation in implementing new developmental instructions that drastically alter cell fate
— id: 98991, year: 2009, vol: 5, page: e1000334, stat: Journal Article,

Protein synthesis and translational control during viral infection
Mohr IJ; Pe'ery T; Mathews MB
Translational control in biology and medicine Cold Spring Harbor NY : Cold Spring Harbor Laboratory Press, 2007,
— id: 5267, year: 2008, vol: , page: 545, stat: Chapter,

Eukaryotic translation initiation factor 4F architectural alterations accompany translation initiation factor redistribution in poxvirus-infected cells
Walsh, Derek; Arias, Carolina; Perez, Cesar; Halladin, David; Escandon, Martin; Ueda, Takeshi; Watanabe-Fukunaga, Rie; Fukunaga, Rikiro; Mohr, Ian
2008 Apr;28(8):2648-2658, Molecular & cellular biology
Despite their self-sufficient ability to generate capped mRNAs from cytosolic DNA genomes, poxviruses must commandeer the critical eukaryotic translation initiation factor 4F (eIF4F) to recruit ribosomes. While eIF4F integrates signals to control translation, precisely how poxviruses manipulate the multisubunit eIF4F, composed of the cap-binding eIF4E and the RNA helicase eIF4A assembled onto an eIF4G platform, remains obscure. Here, we establish that the poxvirus infection of normal, primary human cells destroys the translational repressor eIF4E binding protein (4E-BP) and promotes eIF4E assembly into an active eIF4F complex bound to the cellular polyadenylate-binding protein (PABP). Stimulation of the eIF4G-associated kinase Mnk1 promotes eIF4E phosphorylation and enhances viral replication and protein synthesis. Remarkably, these eIF4F architectural alterations are accompanied by the concentration of eIF4E and eIF4G within cytosolic viral replication compartments surrounded by PABP. This demonstrates that poxvirus infection redistributes, assembles, and modifies core and associated components of eIF4F and concentrates them within discrete subcellular compartments. Furthermore, it suggests that the subcellular distribution of eIF4F components may potentiate the complex assembly
— id: 78356, year: 2008, vol: 28, page: 2648, stat: Journal Article,

Attenuation of herpes simplex virus neurovirulence with picornavirus cis-acting genetic elements
Campbell, Stephanie A; Mulvey, Matthew; Mohr, Ian; Gromeier, Matthias
2007 Jan;81(2):791-799, Journal of virology
Viral pathogenesis depends on a suitable milieu in target host cells permitting viral gene expression, propagation, and spread. In many instances, viral genomes can be manipulated to select for propagation in certain tissues or cell types. This has been achieved for the neurotropic poliovirus (PV) by exchange of the internal ribosomal entry site (IRES), which is responsible for translation of the uncapped plus-strand RNA genome. The IRES of human rhinovirus type 2 (HRV2) confers neuron-specific replication deficits to PV but has no effect on viral propagation in malignant glioma cells. We report here that placing the critical gamma(1)34.5 virulence genes of herpes simplex virus type 1 (HSV) under translation control of the HRV2 IRES results in neuroattenuation in mice. In contrast, IRES insertion permits HSV propagation in malignant glioma cell lines that do not support replication of HSV recombinants carrying gamma(1)34.5 deletions. Our observations indicate that the conditions for alternative translation initiation at the HRV2 IRES in malignant glioma cells differ from those in normal central nervous system (CNS) cells. Picornavirus regulatory sequences mediating cell type-specific gene expression in the CNS can be utilized to target cancerous cells at the level of translation regulation outside their natural context
— id: 78854, year: 2007, vol: 81, page: 791, stat: Journal Article,

Herpes simplex virus as a therapy for cancer
Mohr I
Gene therapy for cancer Totowa NJ : Humana Press, 2007,
— id: 5268, year: 2007, vol: , page: 87, stat: Chapter,

Maintenance of endoplasmic reticulum (ER) homeostasis in herpes simplex virus type 1-infected cells through the association of a viral glycoprotein with PERK, a cellular ER stress sensor
Mulvey, Matthew; Arias, Carolina; Mohr, Ian
2007 Apr;81(7):3377-3390, Journal of virology
In the efforts of viruses to dominate and control critical cellular pathways, viruses generate considerable intracellular stress within their hosts. In particular, the capacity of resident endoplasmic reticulum (ER) chaperones to properly process the acute increase in client protein load is significantly challenged. Such alterations typically induce the unfolded protein response, one component of which acts through IRE1 to restore ER homeostasis by expanding the folding capabilities, whereas the other arm activates the eIF-2alpha (alpha subunit of eukaryotic initiation factor 2) kinase PERK to transiently arrest production of new polypeptide clientele. Viruses, such as herpes simplex virus type 1 (HSV-1), however, go to great lengths to prevent the inhibition of translation resulting from eIF-2alpha phosphorylation. Here, we establish that PERK, but not IRE1, resists activation by acute ER stress in HSV-1-infected cells. This requires the ER luminal domain of PERK, which associates with the viral glycoprotein gB. Strikingly, gB regulates viral protein accumulation in a PERK-dependent manner. This is the first description of a virus-encoded PERK-specific effector and defines a new strategy by which viruses are able to maintain ER homeostasis
— id: 71416, year: 2007, vol: 81, page: 3377, stat: Journal Article,

Inhibition of cellular 2'-5' oligoadenylate synthetase by the herpes simplex virus type 1 Us11 protein
Sanchez, Ricardo; Mohr, Ian
2007 Apr;81(7):3455-3464, Journal of virology
Among the many host genes induced by virus infection and interferon, the eIF2alpha protein kinase PKR and the 2'-5' oligoadenylate synthetase (OAS) are both activated by double-stranded RNA (dsRNA) produced in virus-infected cells. Furthermore, each is a critical component that independently acts to inhibit virus replication and thereby contributes to the establishment of an antiviral state. As part of their tactics to foil host defense mechanisms, some viruses prevent the induction of interferon-responsive genes at the level of transcription. Other viruses, such as herpes simplex virus type 1 (HSV-1), can additionally replicate in interferon-treated cells and must also evade the actions of host defense proteins such as PKR and OAS that have been previously synthesized and merely await detection of an activating signal. Whereas HSV-1 gene products gamma(1)34.5 and Us11 are required for viral replication in interferon-treated cells and both act in a temporally coordinated manner during infection to counteract PKR, HSV-1 functions that target OAS have not been described. Here, we demonstrate that HSV-1 infection inhibits 2'-5' oligoadenylate synthesis in interferon-stimulated primary human cells. The OAS-inhibiting activity is generated late in the virus' productive life cycle and requires the Us11 gene product. Moreover, we establish that the Us11 protein is sufficient to block OAS activation in extracts from uninfected, interferon-treated cells. Inhibition of OAS specifically requires the Us11 dsRNA-binding domain, suggesting a mechanism that, in part, relies on sequestering available dsRNA produced during infection. Thus, in addition to PKR and its protein activator, PACT, the HSV-1 Us11 gene product is able to counteract the activity of OAS, a third cellular protein critical for host defense
— id: 71417, year: 2007, vol: 81, page: 3455, stat: Journal Article,

Phosphorylation and dephosphorylation events that regulate viral mRNA translation
Mohr, Ian
2006 Jul;119(1):89-99, Virus research
As they are completely dependent upon the protein synthesis machinery resident in the cells of their host to translate their mRNAs, it is imperative that viruses are able to effectively manipulate the elaborate cellular regulatory network that controls translation. Indeed, this exquisite dependence on host functions has made viral models attractive systems to explore translational regulatory mechanisms operative in eukaryotic cells. Central among these are an intricate array of phosphorylation and dephosphorylation events that have far reaching consequences on the activity of cellular translation factors. Not only do these modulate the activity of a given factor, but they can also determine if the translation of host proteins persists in infected cells, the efficiency with which viral mRNAs are translated and the outcome of a systemic host anti-viral response. In this review, we discuss how various viruses manipulate the phosphorylation state of key cellular translation factors, illustrating the critical nature these interactions play in virus replication, pathogenesis and innate host defense
— id: 65793, year: 2006, vol: 119, page: 89, stat: Journal Article,

Resistance of mRNA translation to acute endoplasmic reticulum stress-inducing agents in herpes simplex virus type 1-infected cells requires multiple virus-encoded functions
Mulvey, Matthew; Arias, Carolina; Mohr, Ian
2006 Aug;80(15):7354-7363, Journal of virology
Via careful control of multiple kinases that inactivate the critical translation initiation factor eIF2 by phosphorylation of its alpha subunit, the cellular translation machinery can rapidly respond to a spectrum of environmental stresses, including viral infection. Indeed, virus replication produces a battery of stresses, such as endoplasmic reticulum (ER) stress resulting from misfolded proteins accumulating within the lumen of this organelle, which could potentially result in eIF2alpha phosphorylation and inhibit translation. While cellular translation is exquisitely sensitive to ER stress-inducing agents, protein synthesis in herpes simplex virus type 1 (HSV-1)-infected cells is notably resistant. Sustained translation in HSV-1-infected cells exposed to acute ER stress does not involve the interferon-induced, double-stranded RNA-responsive eIF2alpha kinase PKR, and it does not require either the PKR inhibitor encoded by the Us11 gene or the eIF2alpha phosphatase component specified by the gamma(1)34.5 gene, the two viral functions known to regulate eIF2alpha phosphorylation. In addition, although ER stress potently induced the GADD34 cellular eIF2alpha phosphatase subunit in uninfected cells, it did not accumulate to detectable levels in HSV-1-infected cells under identical exposure conditions. Significantly, resistance of translation to the acute ER stress observed in infected cells requires HSV-1 gene expression. Whereas blocking entry into the true late phase of the viral developmental program does not abrogate ER stress-resistant translation, the presence of viral immediate-early proteins is sufficient to establish a state permissive of continued polypeptide synthesis in the presence of ER stress-inducing agents. Thus, one or more previously uncharacterized viral functions exist to counteract the accumulation of phosphorylated eIF2alpha in response to ER stress in HSV-1-infected cells
— id: 67538, year: 2006, vol: 80, page: 7354, stat: Journal Article,

Assembly of an active translation initiation factor complex by a viral protein
Walsh, Derek; Mohr, Ian
2006 Feb 15;20(4):461-472, Genes & development
Recruitment of the 40S ribosome to the 5' end of a eukaryotic mRNA requires assembly of translation initiation factors eIF4E, the cap-binding protein, together with eIF4A and eIF4G into a complex termed eIF4F. While the translational repressor 4E-BP1 regulates binding of eIF4E to eIF4G, the forces required to construct an eIF4F complex remain unidentified. Here, we establish that the herpes simplex virus-1 (HSV-1) ICP6 polypeptide associates with eIF4G to promote eIF4F complex assembly. Strikingly, release of eIF4E from the 4E-BP1 repressor is insufficient to drive complex formation, suggesting that ICP6 is an eIF4F-assembly chaperone. This is the first example of a translation initiation factor-associated protein that promotes active complex assembly and defines a new, controllable step in the initiation of translation. Homology of the N-terminal, eIF4G-binding segment of ICP6 with cellular chaperones suggest that factors capable of interacting with eIF4G and promoting eIF4F complex assembly may play important roles in a variety of processes where translation complexes need to be remodeled or assembled on populations of newly synthesized or derepressed mRNAs, including development, differentiation, and the response to a broad spectrum of environmental cues
— id: 64200, year: 2006, vol: 20, page: 461, stat: Journal Article,

To replicate or not to replicate: achieving selective oncolytic virus replication in cancer cells through translational control
Mohr, Ian
2005 Nov 21;24(52):7697-7709, Oncogene
To ensure that their mRNAs are translated and that the viral proteins necessary for assembling the next generation of infectious progeny are produced, viruses must effectively seize control of the translational machinery within their host cells. In many cases, the ability to productively engage host translational components can determine if a given cell type can support viral replication, illustrating the critical importance of this task in the viral life cycle. Failure to interface properly with the host translational apparatus can compromise the productive growth cycle, resulting in an abortive infection and radically restricting viral replication. Not only have viruses become facile at commandeering this machinery, they are also particularly adept at manipulating cellular translation control pathways for their own ends. In this review, the mechanisms by which numerous viruses manipulate host translational control circuits are discussed. Furthermore, particular attention is devoted to understanding how interfering with the ability of a virus to properly regulate translation in its host can be exploited to generate oncolytic strains that selectively replicate in cancer cells
— id: 78855, year: 2005, vol: 24, page: 7697, stat: Journal Article,

Regulation of the translation initiation factor eIF4F by multiple mechanisms in human cytomegalovirus-infected cells
Walsh, Derek; Perez, Cesar; Notary, Joanna; Mohr, Ian
2005 Jul;79(13):8057-8064, Journal of virology
As a viral opportunistic pathogen associated with serious disease among the immunocompromised and congenital defects in newborns, human cytomegalovirus (HCMV) must engage the translational machinery within its host cell to synthesize the viral proteins required for its productive growth. However, unlike many viruses, HCMV does not suppress the translation of host polypeptides. Here, we examine how HCMV regulates the cellular cap recognition complex eIF4F, a critical component of the cellular translation initiation apparatus that recruits the 40S ribosome to the 5' end of the mRNA. This study establishes that the cap binding protein eIF4E, together with the translational repressor 4E-BP1, are both phosphorylated early in the productive viral growth cycle and that the activity of the cellular eIF4E kinase, mnk, is critical for efficient viral replication. Furthermore, HCMV replication also induces an increase in the overall abundance of eIF4F components and promotes assembly of eIF4F complexes. Notably, increasing the abundance of select eIF4F core components and associated factors alters the ratio of active eIF4F complexes in relation to the 4E-BP1 translational repressor, illustrating a new strategy through which members of the herpesvirus family enhance eIF4F activity during their replicative cycle
— id: 78856, year: 2005, vol: 79, page: 8057, stat: Journal Article,

Neutralizing innate host defenses to control viral translation in HSV-1 infected cells
Mohr, Ian
2004 Jan-Apr;23(1-2):199-220, International reviews of immunology
Lytic replication of many viruses activates an innate host response designed to prevent the completion of the viral lifecycle, thus impeding the spread of the infection. One branch of the host's complex reaction functions to incapacitate the cellular translational machinery on which the synthesis of viral polypeptides completely depends. This is achieved through the activation of specific protein kinases that phosphorylate eIF2 on its alpha subunit and inactivate this critical translation initiation factor. However, as continued synthesis of viral proteins is required to assemble the viral progeny necessary to transmit the infection to neighboring cells, viruses have developed a variety of strategies to counter this cellular response. Genetic and biochemical studies with herpes simplex virus type 1 (HSV-1) have revealed that the virus produces at least two discrete products at different times during its replicative program that act to prevent the accumulation of phosphorylated eIF2alpha. The gamma(1)34.5 gene product is expressed first, encoding a regulatory subunit that binds the cellular protein phosphatase 1alpha and regenerates pools of active eIF2 by removing the inhibitory phosphate from the alpha subunit. The second function, encoded by the product of the Us11 gene, specifies a double-stranded RNA-binding protein that prevents activation of PKR, a cellular eIF2alpha kinase. Together, both proteins cooperate to overcome the antiviral response of the host and properly regulate translation in HSV-1-infected cells
— id: 42631, year: 2004, vol: 23, page: 199, stat: Journal Article,

Full resistance of herpes simplex virus type 1-infected primary human cells to alpha interferon requires both the Us11 and gamma(1)34.5 gene products
Mulvey, Matthew; Camarena, Vladimir; Mohr, Ian
2004 Oct;78(18):10193-10196, Journal of virology
The gamma(1)34.5 gene product is important for the resistance of herpes simplex virus type 1 (HSV-1) to interferon. However, since the inhibition of protein synthesis observed in cells infected with a gamma(1)34.5 mutant virus results from the combined loss of the gamma(1)34.5 gene product and the failure to translate the late Us11 mRNA, we sought to characterize the relative interferon sensitivity of mutants unable to produce either the Us11 or the gamma(1)34.5 polypeptide. We now demonstrate that primary human cells infected with a Us11 mutant virus are hypersensitive to alpha interferon, arresting translation upon entry into the late phase of the viral life cycle. Furthermore, immediate-early expression of Us11 by a gamma(1)34.5 deletion mutant is sufficient to render translation resistant to alpha interferon. Finally, we establish that the Us11 gene product is required for wild-type levels of replication in alpha interferon-treated cells and, along with the gamma(1)34.5 gene, is an HSV-1-encoded interferon resistance determinant
— id: 46131, year: 2004, vol: 78, page: 10193, stat: Journal Article,

Phosphorylation of eIF4E by Mnk-1 enhances HSV-1 translation and replication in quiescent cells
Walsh, Derek; Mohr, Ian
2004 Apr 15;18(6):660-672, Genes & development
Although the activity of the translation initiation factor eIF4F is regulated in part by translational repressors (4E-BPs) that prevent incorporation of eIF4E, the cap-binding protein, into the initiation complex, the contribution of eIF4E phosphorylation to translational control remains controversial. Here, we demonstrate that the herpes simplex virus-1 (HSV-1) ICP0 gene product, a multifunctional transactivator of viral gene expression with ubiquitin E3 ligase activity that is important for vegetative replication and reactivation of latent infections, is required to stimulate phosphorylation of eIF4E as well as 4E-BP1, and promote assembly of eIF4F complexes in infected cells. Furthermore, 4E-BP1 is degraded by the proteasome in an ICP0-dependent manner, establishing that the proteasome can control 4E-BP1 steady-state levels. Preventing eIF4E phosphorylation by inhibiting the eIF4E kinase mnk-1 dramatically reduced viral replication and the translation of viral polypeptides in quiescent cells, providing the first evidence that phosphorylation of eIF4E by mnk-1 is critical for viral protein synthesis and replication. Thus, in marked contrast to many viruses that inactivate eIF4F, HSV-1 stimulates eIF4F complex assembly in quiescent, differentiated cells; moreover, this is important for viral replication, and may be crucial for HSV-1 to initiate its productive growth cycle in resting cells, such as latently infected neurons
— id: 46105, year: 2004, vol: 18, page: 660, stat: Journal Article,

Association of the herpes simplex virus type 1 Us11 gene product with the cellular kinesin light-chain-related protein PAT1 results in the redistribution of both polypeptides
Benboudjema, Louisa; Mulvey, Matthew; Gao, Yuehua; Pimplikar, Sanjay W; Mohr, Ian
2003 Sep;77(17):9192-9203, Journal of virology
The herpes simplex virus type 1 (HSV-1) Us11 gene encodes a multifunctional double-stranded RNA (dsRNA)-binding protein that is expressed late in infection and packaged into the tegument layer of the virus particle. As a tegument component, Us11 associates with nascent capsids after its synthesis late in the infectious cycle and is delivered into newly infected cells at times prior to the expression of viral genes. Us11 is also an abundant late protein that regulates translation through its association with host components and contains overlapping nucleolar retention and nuclear export signals, allowing its accumulation in both nucleoli and the cytosol. Thus, at various times during the viral life cycle and in different intracellular compartments, Us11 has the potential to execute discrete tasks. The analysis of these functions, however, is complicated by the fact that Us11 is not essential for viral replication in cultured cells. To discover new host targets for the Us11 protein, we searched for cellular proteins that interact with Us11 and have identified PAT1 as a Us11-binding protein according to multiple, independent experimental criteria. PAT1 binds microtubules, participates in amyloid precursor protein trafficking, and has homology to the kinesin light chain (KLC) in its carboxyl terminus. The carboxyl-terminal dsRNA-binding domain of Us11, which also contains the nucleolar retention and nuclear export signals, binds PAT1, whereas 149 residues derived from the KLC homology region of PAT1 are important for binding to Us11. Both PAT1 and Us11 colocalize within a perinuclear area in transiently transfected and HSV-1-infected cells. The 149 amino acids derived from the KLC homology region are required for colocalization of the two polypeptides. Furthermore, although PAT1 normally accumulates in the nuclear compartment, Us11 expression results in the exclusion of PAT1 from the nucleus and its accumulation in the perinuclear space. Similarly, Us11 does not accumulate in the nucleoli of infected cells that overexpress PAT1. These results establish that Us11 and PAT1 can associate, resulting in an altered subcellular distribution of both polypeptides. The association between PAT1, a cellular trafficking protein with homology to KLC, and Us11, along with a recent report demonstrating an interaction between Us11 and the ubiquitous kinesin heavy chain (R. J. Diefenbach et al., J. Virol. 76:3282-3291, 2002), suggests that these associations may be important for the intracellular movement of viral components
— id: 39108, year: 2003, vol: 77, page: 9192, stat: Journal Article,

Genetic metamorphosis of herpes simplex virus-1 into a biological therapeutic for human cancer
Mohr, Ian
2003 Feb;3(1):113-125, Expert opinion on biological therapy
Over the past decade, rapid progress has been made in engineering safe, replicating herpes simplex virus-1 (HSV-1) mutants for use as biological oncolytic agents in the treatment of human cancer. While initial efforts demonstrated the potential of HSV-1 mutants as antitumour agents, they relied on viruses that were not sufficiently attenuated. Following its identification as the major viral neurovirulence determinant, mutations in the gamma34.5 gene were subsequently incorporated into oncolytic strains. Despite the fact that gamma34.5 mutant derivatives can be safely administered to mice, non-human primates and humans, their efficacy is limited because, like many weakened viral strains, they replicate poorly in a number of cell types, including cancer cells. Strategies to improve the oncolytic properties of gamma34.5 mutant derivatives through further genetic manipulation are reviewed. In addition, traditional treatment modalities that incorporate viral inoculation, along with efforts to elicit an antitumour immune response following treatment with gamma34.5 derivatives, are discussed
— id: 39236, year: 2003, vol: 3, page: 113, stat: Journal Article,

Regulation of eIF2alpha phosphorylation by different functions that act during discrete phases in the herpes simplex virus type 1 life cycle
Mulvey, Matthew; Poppers, Jeremy; Sternberg, David; Mohr, Ian
2003 Oct;77(20):10917-10928, Journal of virology
Multiple herpes simplex virus type 1 functions control translation by regulating phosphorylation of the initiation factor eIF2 on its alpha subunit. Both of the two known regulators, the gamma(1)34.5 and Us11 gene products, are produced late in the viral life cycle, although the gamma(1)34.5 gene is expressed prior to the gamma(2) Us11 gene, as gamma(2) genes require viral DNA replication for their expression while gamma(1) genes do not. The gamma(1)34.5 protein, through a GADD34-related domain, binds a cellular phosphatase (PP1alpha), maintaining pools of active, unphosphorylated eIF2. Infection of a variety of cultured cells with a gamma(1)34.5 mutant virus results in the accumulation of phosphorylated eIF2alpha and the inhibition of translation prior to the completion of the viral lytic program. Ectopic, immediate-early Us11 expression prevents eIF2alpha phosphorylation and the inhibition of translation observed in cells infected with a gamma(1)34.5 mutant by inhibiting activation of the cellular kinase PKR and the subsequent phosphorylation of eIF2alpha; however, a requirement for the Us11 protein, produced in its natural context as a gamma(2) polypeptide, remains to be demonstrated. To determine if Us11 regulates late translation, we generated two Us11 null viruses. In cells infected with a Us11 mutant, elevated levels of activated PKR and phosphorylated eIF2alpha were detected, viral translation rates were reduced 6- to 7-fold, and viral replication was reduced 13-fold compared to replication in cells infected with either wild-type virus or a virus in which the Us11 mutation was repaired. This establishes that the Us11 protein is critical for proper late translation rates. Moreover, it demonstrates that the shutoff of protein synthesis observed in cells infected with a gamma(1)34.5 mutant virus, previously ascribed solely to the gamma(1)34.5 mutation, actually results from the combined loss of gamma(1)34.5 and Us11 functions, as the gamma(2) Us11 mRNA is not translated in cells infected with a gamma(1)34.5 mutant
— id: 39054, year: 2003, vol: 77, page: 10917, stat: Journal Article,

Identification of a lytic-cycle Epstein-Barr virus gene product that can regulate PKR activation
Poppers, Jeremy; Mulvey, Matthew; Perez, Cesar; Khoo, David; Mohr, Ian
2003 Jan;77(1):228-236, Journal of virology
The Epstein-Barr virus (EBV) SM protein is a posttranscriptional regulator of viral gene expression. Like many transactivators encoded by herpesviruses, SM transports predominantly unspliced viral mRNA cargo from the nucleus to the cytosol, where it is subsequently translated. This activity likely involves a region of the protein that has homology to the herpes simplex virus type 1 (HSV-1) ICP27 gene product, the first member of this class of regulators to be discovered. However, SM also contains a repetitive segment rich in arginine and proline residues that is dispensable for its effects on RNA transport and splicing. This portion of SM, comprised of RXP triplet repeats, shows homology to the carboxyl-terminal domain of Us11, a double-stranded RNA (dsRNA) binding protein encoded by HSV-1 that inhibits activation of the cellular PKR kinase. To evaluate the intrinsic ability of SM to regulate PKR, we expressed and purified several SM protein derivatives and examined their activity in a variety of biochemical assays. The full-length SM protein bound dsRNA, associated physically with PKR, and prevented PKR activation. Removal of the 37-residue RXP domain significantly compromised all of these activities. Furthermore, the SM RXP domain was itself sufficient to inhibit PKR activation and interact with the kinase. Relative to its Us11 counterpart, the SM RXP segment bound dsRNA with reduced affinity and responded differently to single-stranded competitor polynucleotides. Thus, SM represents the first EBV gene product expressed during the lytic cycle that can prevent PKR activation. In addition, the RXP repeat segment appears to be a conserved herpesvirus motif capable of associating with dsRNA and modulating activation of the PKR kinase, a molecule important for the control of translation and the cellular antiviral response
— id: 39350, year: 2003, vol: 77, page: 228, stat: Journal Article,

Translation initiation and viral tricks
Schneider, Robert J; Mohr, Ian
2003 Mar;28(3):130-136, Trends in biochemical sciences
A variety of viral strategies are utilized for dominance of the host-cell protein synthetic machinery, optimization of viral mRNA translation and evasion of host-cell antiviral responses that act at the translational level. Many viruses exploit regulated steps in the initiation of cellular protein synthesis to their own advantage. They have developed some rather unconventional means for mRNA translation, which were probably adapted from specialized cellular mRNA translation systems. Regardless of the type of translational tricks exploited, viruses typically ensure efficient viral translation, often at the expense of host-cell protein synthesis
— id: 44998, year: 2003, vol: 28, page: 130, stat: Journal Article,

In vivo replication of an ICP34.5 second-site suppressor mutant following corneal infection correlates with in vitro regulation of eIF2 alpha phosphorylation
Ward, Stephen L; Scheuner, Donalyn; Poppers, Jeremy; Kaufman, Randal J; Mohr, Ian; Leib, David A
2003 Apr;77(8):4626-4634, Journal of virology
In animal models of herpes simplex virus type 1 (HSV-1) infection, ICP34.5-null viruses are avirulent and also fail to grow in a variety of cultured cells due to their inability to prevent RNA-dependent protein kinase (PKR)-mediated inhibition of protein synthesis. We show here that the inability of ICP34.5 mutants to grow in vitro is due specifically to the accumulation of phosphorylated eIF2 alpha. Mutations suppressing the in vitro phenotype of ICP34.5-null mutants have been described which map to the unique short region of the HSV-1 genome, resulting in dysregulated expression of the US11 gene. Despite the inability of the suppressor mutation to suppress the avirulent phenotype of the ICP34.5-null parental virus following intracranial inoculation, the suppressor mutation enhanced virus growth in the cornea, trigeminal ganglia, and periocular skin following corneal infection compared to that with the ICP34.5-null virus. The phosphorylation state of eIF2 alpha following in vitro infection with the suppressor virus was examined to determine if in vivo differences could be attributed to differential regulation of eIF2 alpha phosphorylation. The suppressor virus prevented accumulation of phosphorylated eIF2 alpha, while the wild-type virus substantially reduced eIF2 alpha phosphorylation levels. These data suggest that US11 functions as a PKR antagonist in vivo, although its activity may be modulated by tissue-specific differences in translation regulation
— id: 78857, year: 2003, vol: 77, page: 4626, stat: Journal Article,

Characterization of RNA determinants recognized by the arginine- and proline-rich region of Us11, a herpes simplex virus type 1-encoded double-stranded RNA binding protein that prevents PKR activation
Khoo, David; Perez, Cesar; Mohr, Ian
2002 Dec;76(23):11971-11981, Journal of virology
The herpes simplex virus Us11 gene product inhibits activation of the cellular PKR kinase and associates with a limited number of unrelated viral and cellular RNA molecules via a carboxyl-terminal 68-amino-acid segment rich in arginine and proline. To characterize the determinants underlying the recognition of an RNA target by Us11, we employed an in vitro selection technique to isolate RNA ligands that bind Us11 with high affinity from a population of molecules containing an internal randomized segment. Binding of Us11 to these RNA ligands is specific and appears to occur preferentially on conformational isoforms that possess a higher-order structure. While the addition of unlabeled poly(I. C) reduced binding of Us11 to a selected radiolabeled RNA, single-stranded homopolymers were not effective competitors. Us11 directly associates with poly(I. C), and inclusion of an unlabeled selected RNA in the reaction reduces poly(I. C) binding, while single-stranded RNA homopolymers have no effect. Finally, Us11 binds to defined, double-stranded RNA (dsRNA) molecules that exhibit greater sequence complexity. Binding to these dsRNA perfect duplexes displays a striking dependence on length, as 39-bp or shorter duplexes do not bind efficiently. Furthermore, this interaction is specific for dsRNA as opposed to dsDNA, implying that the Us11 RNA binding domain can distinguish nucleic acid duplexes containing 2' hydroxyl groups from those that do not. These results establish that Us11 is a dsRNA binding protein. The arginine- and proline-rich Us11 RNA binding domain is unrelated to known dsRNA binding elements and thus constitutes a unique recognition motif that interacts with dsRNA. The ability of Us11 to bind dsRNA may be important for inhibiting activation of the cellular PKR kinase in response to dsRNA
— id: 39570, year: 2002, vol: 76, page: 11971, stat: Journal Article,

Inhibition of PACT-mediated activation of PKR by the herpes simplex virus type 1 Us11 protein
Peters, Gregory A; Khoo, David; Mohr, Ian; Sen, Ganes C
2002 Nov;76(21):11054-11064, Journal of virology
PACT, a protein activator of PKR, can cause inhibition of cellular protein synthesis and apoptosis. Here, we report that the Us11 protein of herpes simplex virus type 1 can block PKR activation by PACT both in vitro and in vivo. Although Us11 can bind to both PKR and PACT, mutational analyses revealed that the binding of Us11 to PKR, and not to PACT, was essential for its inhibitory action. Similar analyses also revealed that the inhibitory effect was mediated by an interaction between the C-terminal half of Us11 and the N-terminal domain of PKR. The binding of Us11 to PKR did not block the binding of PKR to PACT but prevented its activation. Us11 is the first example of a viral protein that can inhibit the action of PACT on PKR
— id: 78858, year: 2002, vol: 76, page: 11054, stat: Journal Article,

A herpes simplex virus type 1 gamma34.5 second-site suppressor mutant that exhibits enhanced growth in cultured glioblastoma cells is severely attenuated in animals
Mohr I; Sternberg D; Ward S; Leib D; Mulvey M; Gluzman Y
2001 Jun;75(11):5189-5196, Journal of virology
We describe here the neurovirulence properties of a herpes simplex virus type 1 gamma34.5 second-site suppressor mutant. gamma34.5 mutants are nonneurovirulent in animals and fail to grow in a variety of cultured cells due to a block at the level of protein synthesis. Extragenic suppressors with restored capacity to replicate in cells that normally do not support the growth of the parental gamma34.5 deletion mutant have been isolated. Although the suppressor virus reacquires the ability to grow in nonpermissive cultured cells, it remains severely attenuated in mice and is indistinguishable from the mutant gamma34.5 parent virus at the doses investigated. Repairing the gamma34.5 mutation in the suppressor mutant restores neurovirulence to wild-type levels. These studies illustrate that (i) the protein synthesis and neurovirulence defects observed in gamma34.5 mutant viruses can be genetically separated by an extragenic mutation at another site in the viral chromosome; (ii) the extragenic suppressor mutation does not affect neurovirulence; and (iii) the attenuated gamma34.5 mutant, which replicates poorly in many cell types, can be modified by genetic selection to generate a nonpathogenic variant that regains the ability to grow robustly in a nonpermissive glioblastoma cell line. As this gamma34.5 second-site suppressor variant is attenuated and replicates vigorously in neoplastic cells, it may have potential as a replication-competent, viral antitumor agent
— id: 20691, year: 2001, vol: 75, page: 5189, stat: Journal Article,

New ways of initiating translation in eukaryotes?
Schneider, R; Agol, VI; Andino, R; Bayard, F; Cavener, DR; Chappell, SA; Chen, JJ; Darlix, JLE; Dasgupta, A; Donze, O; Duncan, R; Elroy-Stein, O; Farabaugh, PJ; Filipowicz, W; Gale, M; Gehrke, L; Goldman, E; Groner, Y; Harford, JB; Hatzoglou, M; He, B; Hellen, CUT; Hentze, MW; Hershey, J; Hershey, P; Hohn, T; Holcik, M; Hunter, CP; Igarashi, K; Jackson, R; Jagus, R; Jefferson, LS; Joshi, B; Kaempfer, R; Katze, MG; Kaufman, RJ; Kiledjian, M; Kimball, SR; Kimchi, A; Kirkegaard, K; Koromilas, AE; Krug, RM; Kruys, V; Lamphear, BJ; Lemon, S; Lloyd, RE; Maquat, LE; Martinez-Salas, E; Mathews, MB; Mauro, VP; Miyamoto, S; Mohr, I; Morris, DR; Moss, EG; Nakashima, N; Palmenberg, A; Parkin, NT; Pe'ery, T; Pelletier, J; Peltz, S; Pestova, TV; Pilipenko, EV; Prats, AC; Racaniello, V; Read, GS; Rhoads, RE; Richter, JD; Rivera-Pomar, R; Rouault, T; Sachs, A; Sarnow, P; Scheper, GC; Schiff, L; Schoenberg, DR; Semler, BL; Siddiqui, A; Skern, T; Sonenberg, N; Sossin, W; Standart, N; Tahara, SM; Thomas, AAM; Toulme, JJ; Wilusz, J; Wimmer, E; Witherell, G; Wormington, M
2001 DEC ;21(23):8238-8241, Molecular & cellular biology
— id: 54812, year: 2001, vol: 21, page: 8238, stat: Journal Article,

Enhanced antitumor efficacy of a herpes simplex virus mutant isolated by genetic selection in cancer cells
Taneja S; MacGregor J; Markus S; Ha S; Mohr I
2001 Jul 17;98(15):8804-8808, Proceedings of the National Academy of Sciences of the United States of America
Replication-competent, attenuated herpes simplex virus-1 (HSV-1) derivatives that contain engineered mutations into the viral gamma34.5 virulence gene have been used as oncolytic agents. However, as attenuated mutants often grow poorly, they may not completely destroy some tumors and surviving cancer cells simply regrow. Thus, although HSV-1 gamma34.5 mutants can reduce the growth of human tumor xenografts in mice and have passed phase I safety studies, their efficacy is limited because they replicate poorly in many human tumor cells. Previously, we selected for a gamma34.5 deletion mutant variant that regained the ability to replicate efficiently in tumor cells. Although this virus contains an extragenic suppressor mutation that confers enhanced growth in tumor cells, it remains attenuated. Here, we demonstrate that the suppressor virus replicates to greater levels in prostate carcinoma cells and, importantly, is a more potent inhibitor of tumor growth in an animal model of human prostate cancer than the gamma34.5 parent virus. Thus, genetic selection in cancer cells can be used as a tool to enhance the antitumor activity of a replication-competent virus. The increased therapeutic potency of this oncolytic virus may be useful in the treatment of a wide variety of cancers
— id: 21150, year: 2001, vol: 98, page: 8804, stat: Journal Article,

Inhibition of PKR activation by the proline-rich RNA binding domain of the herpes simplex virus type 1 Us11 protein
Poppers J; Mulvey M; Khoo D; Mohr I
2000 Dec;74(23):11215-11221, Journal of virology
Upon activation by double-stranded RNA in virus-infected cells, the cellular PKR kinase phosphorylates the translation initiation factor eukaryotic initiation factor 2 (eIF2) and thereby inhibits protein synthesis. The gamma 34.5 and Us11 gene products encoded by herpes simplex virus type 1 (HSV-1) are dedicated to preventing the accumulation of phosphorylated eIF2. While the gamma 34.5 gene specifies a regulatory subunit for protein phosphatase 1 alpha, the Us11 gene encodes an RNA binding protein that also prevents PKR activation. gamma 34.5 mutants fail to grow on a variety of human cells as phosphorylated eIF2 accumulates and protein synthesis ceases prior to the completion of the viral life cycle. We demonstrate that expression of a 68-amino-acid fragment of Us11 containing a novel proline-rich basic RNA binding domain allows for sustained protein synthesis and enhanced growth of gamma 34.5 mutants. Furthermore, this fragment is sufficient to inhibit activation of the cellular PKR kinase in a cell-free system, suggesting that the intrinsic activities of this small fragment, notably RNA binding and ribosome association, may be required to prevent PKR activation
— id: 39520, year: 2000, vol: 74, page: 11215, stat: Journal Article,

A herpesvirus ribosome-associated, RNA-binding protein confers a growth advantage upon mutants deficient in a GADD34-related function
Mulvey M; Poppers J; Ladd A; Mohr I
1999 Apr;73(4):3375-3385, Journal of virology
The herpes simplex virus type 1 gamma34.5 gene product and the cellular GADD34 protein both contain similar domains that can regulate the activity of eukaryotic initiation factor 2 (eIF2), a critical translation initiation factor. Viral mutants that lack the GADD34-related function grow poorly on a variety of malignant human cells, as activation of the cellular PKR kinase leads to the accumulation of inactive, phosphorylated eIF2 at late times postinfection. Termination of translation prior to the completion of the viral reproductive cycle leads to impaired growth. Extragenic suppressors that regain the ability to synthesize proteins efficiently in the absence of the viral GADD34-related function have been isolated. These suppressor alleles are dominant in trans and affect the steady-state accumulation of several viral mRNA species. We demonstrate that deregulated expression of Us11, a virus-encoded RNA-binding, ribosome-associated protein is necessary and sufficient to confer a growth advantage upon viral mutants that lack a GADD34-related function. Ectopic expression of Us11 reduces the accumulation of the activated cellular PKR kinase and allows for sustained protein synthesis. Thus, an RNA-binding, ribosome-associated protein (Us11) and a GADD34-related protein (gamma34.5) both function in a signal pathway that regulates translation by modulating eIF2 phosphorylation
— id: 57545, year: 1999, vol: 73, page: 3375, stat: Journal Article,

Suppression of the phenotype of gamma(1)34.5- herpes simplex virus 1: failure of activated RNA-dependent protein kinase to shut off protein synthesis is associated with a deletion in the domain of the alpha47 gene
He B; Chou J; Brandimarti R; Mohr I; Gluzman Y; Roizman B
1997 Aug;71(8):6049-6054, Journal of virology
Earlier studies have shown that infection of human cells by herpes simplex virus 1 (HSV-1) results in the activation of RNA-dependent protein kinase (PKR) but that the alpha subunit of eIF-2 is not phosphorylated and that protein synthesis is unaffected. In the absence of the viral gamma(1)34.5 gene, eIF-2alpha is phosphorylated and protein synthesis is prematurely shut off (J. Chou, J. J. Chen, M. Gross, and B. Roizman, Proc. Natl. Acad. Sci. USA 92:10516-10520, 1995). A second recent paper reported the selection of second-site suppressor mutants characterized by near-wild-type protein synthesis in cells infected with gamma(1)34.5- mutants (I. Mohr and Y. Gluzman, EMBO J. 15:4759-4766, 1996). Here, we report the properties of the spontaneous HSV-1 suppressor mutant Sup-1, which is characterized by spontaneous deletion of 503 bp encompassing the domain of the alpha47 gene and junction with the inverted repeats flanking the unique short (U(S)) sequence of the HSV-1 DNA resulting in the juxtaposition of the alpha47 promoter to the coding domain of the U(S)11 gene. This mutant does not exhibit the shutoff of protein synthesis characteristic of the gamma(1)34.5- virus. Specifically, Sup-1 in SK-N-SH human neuroblastoma cells (i) did not exhibit the function of the alpha47 gene characterized by a reduction in the transport of peptides across the endoplasmic reticulum of permealized cells consistent with the absence of alpha47 gene sequences, (ii) accumulated U(S)11 protein at levels analogous to those of the wild-type parent but the protein was made at earlier times after infection, as would be expected from a change in the promoter, and (iii) activated PKR like that of the parent, gamma(1)34.5- virus, but (iv) did not cause premature shutoff of protein synthesis and therefore was similar to the wild-type parent virus rather than the gamma(1)34.5- virus from which it was derived. We conclude that the mechanism by which Sup-1 blocks the shutoff of protein synthesis associated with phosphorylation of eIF-2alpha by the activated PKR is not readily explainable by a secondary mutation characterized by a deletion
— id: 18113, year: 1997, vol: 71, page: 6049, stat: Journal Article,

A herpesvirus genetic element which affects translation in the absence of the viral GADD34 function
Mohr I; Gluzman Y
1996 Sep 2;15(17):4759-4766, EMBO journal
Novel suppressor variants of conditionally lethal HSV-1 gamma34.5 deletion mutants have been isolated which exhibit restored ability to grow on neoplastic neuronal cells. Deletion of the viral gamma34.5 genes, whose products share functional similarity with the cellular GADD34 gene, renders the virus non-neurovirulent and imposes a block to viral replication in neuronal cells. Protein synthesis ceases at late times post-infection and the translation initiation factor eIF2alpha is phosphorylated by the cellular PKR kinase [Chou et al. (1990) Science, 252, 1262-1266; (1995) Proc. Natl Acad. Sci. USA, 92, 10516-10520]. The suppressor mutants have overcome the translational block imposed by PKR. Multiple, independent isolates all contain rearrangements within a 595 bp element in the HSV-1 genome where the unique short component joins the terminal repeats. This alteration, which affects the production of the viral mRNA and protein from the Us11 and Us12 genes, is both necessary and sufficient to confer the suppressor phenotype on gamma34.5 mutant viruses. HSV-1 thus encodes a specific element which inhibits ongoing protein synthesis in the absence of the viral GADD34-like function. Since this inhibition involves the accumulation of phosphorylated eIF2alpha, the element identified by the suppressor mutations may be a discrete PKR activator. Activation of the PKR kinase thus does not proceed through a general, cellular 'antiviral' sensing mechanism. Instead, the virus deliberately activates PKR and encodes a separate function which selectively prevents the phosphorylation of at least one PKR target, eIF2alpha. The nature of this potential activator element, and how analogous cellular elements could affect PKR pathways which affect growth arrest and differentiation are discussed
— id: 18114, year: 1996, vol: 15, page: 4759, stat: Journal Article,

The cellular DNA polymerase alpha-primase is required for papillomavirus DNA replication and associates with the viral E1 helicase
Park P; Copeland W; Yang L; Wang T; Botchan MR; Mohr IJ
1994 Aug 30;91(18):8700-8704, Proceedings of the National Academy of Sciences of the United States of America
Persistent infection by papillomaviruses involves the maintenance of viral DNA as a nuclear plasmid, the replication of which requires host DNA polymerases. The role of the cellular DNA polymerase alpha-primase holoenzyme was probed by using soluble extracts from rodent cells that replicate bovine papilloma virus 1 and human papilloma virus 6b DNA in the presence of the viral E1 helicase and the E2 transcription factor. Monoclonal antibodies directed against the catalytic 180-kDa subunit of polymerase alpha inhibit DNA synthesis in this system. Addition of purified human polymerase alpha-primase holoenzyme to neutralized extracts restores their DNA synthetic activity. The amino-terminal 424 amino acids of E1 forms a specific protein complex with the p180 polymerase subunit. Immune complexes can be isolated with antibodies directed against E1 that contain a DNA polymerase activity. Moreover, this polymerase activity can be neutralized by anti-polymerase alpha antibodies. Permissivity barriers were not encountered in this in vitro system, as bovine E1 can interface with the murine and human replication apparatus. Although the large tumor antigens encoded by simian virus 40 and polyoma share limited primary sequence homology with the papillomavirus E1 proteins, the organization of functional motifs at the level of primary protein structure is remarkably similar. In addition to their origin-specific DNA-binding activity, each of these helicases may function to help recruit the cellular polymerase alpha-primase complex to the viral replication origin
— id: 18115, year: 1994, vol: 91, page: 8700, stat: Journal Article,

Activation of BPV-1 replication in vitro by the transcription factor E2
Yang L; Li R; Mohr IJ; Clark R; Botchan MR
1991 Oct 17;353(6345):628-632, Nature
Soluble extracts from uninfected murine cells supplemented with purified viral E1 and E2 proteins support the replication of exogenously added papilloma virus DNA. The E2 transactivator stimulates the binding of the E1 replication protein to the minimal origin of replication and activates DNA replication. These results support the concept that transcription factors have a direct role in the initiation of DNA replication in eukaryotes by participating in the assembly of a complex at the origin of replication
— id: 18116, year: 1991, vol: 353, page: 628, stat: Journal Article,

Targeting the E1 replication protein to the papillomavirus origin of replication by complex formation with the E2 transactivator
Mohr IJ; Clark R; Sun S; Androphy EJ; MacPherson P; Botchan MR
1990 Dec 21;250(4988):1694-1699, Science
The mechanism by which transcription factors stimulate DNA replication in eukaryotes is unknown. Bovine papillomavirus DNA synthesis requires the products of the viral E1 gene and the transcriptional activator protein encoded by the E2 gene. Experimental data showed that the 68-kilodalton (kD) E1 protein formed a complex with the 48-kD E2 transcription factor. This complex bound specifically to the viral origin of replication, which contains multiple binding sites for E2. Repressor proteins encoded by the E2 open reading frame failed to complex with E1 suggesting that the 162-amino acid region of E2 that participates in transactivation contained critical determinants for interaction with E1. The physical association between a replication protein and a transcription factor suggests that transcriptional activator proteins may function in targeting replication initiator proteins to their respective origins of replication
— id: 18117, year: 1990, vol: 250, page: 1694, stat: Journal Article,

Large T-antigen mutants define multiple steps in the initiation of simian virus 40 DNA replication
Mohr IJ; Fairman MP; Stillman B; Gluzman Y
1989 Oct;63(10):4181-4188, Journal of virology
The biochemical activities of a series of transformation-competent, replication-defective large T-antigen point mutants were examined. The assays employed reflect partial reactions required for the in vitro replication of simian virus 40 (SV40) DNA. Mutants which failed to bind specifically to SV40 origin sequences bound efficiently to single-stranded DNA and exhibited nearly wild-type levels of helicase activity. A mutation at proline 522, however, markedly reduced ATPase, helicase, and origin-specific unwinding activities. This mutant bound specifically to the SV40 origin of replication, but under certain conditions it was defective in binding to both single-stranded DNA and the partial duplex helicase substrate. This suggests that additional determinants outside the amino-terminal-specific DNA-binding domain may be involved in nonspecific binding of T antigen to single-stranded DNA and demonstrates that origin-specific DNA binding can be separated from binding to single-stranded DNA. A mutant containing a lesion at residue 224 retained nearly wild-type levels of helicase activity and recognized SV40 origin sequences, yet it failed to function in an origin-specific unwinding assay. This provides evidence that origin recognition and helicase activities are not sufficient for unwinding to occur. The distribution of mutant phenotypes reflects the complex nature of the initiation reaction and the multiplicity of functions provided by large T antigen
— id: 18118, year: 1989, vol: 63, page: 4181, stat: Journal Article,

Production of simian virus 40 large tumor antigen in bacteria: altered DNA-binding specificity and dna-replication activity of underphosphorylated large tumor antigen
Mohr IJ; Gluzman Y; Fairman MP; Strauss M; McVey D; Stillman B; Gerard RD
1989 Sep;86(17):6479-6483, Proceedings of the National Academy of Sciences of the United States of America
A bacterial expression system was used to produce simian virus 40 large tumor antigen (T antigen) in the absence of the extensive posttranslational modifications that occur in mammalian cells. Wild-type T antigen produced in bacteria retained a specific subset of the biochemical activities displayed by its mammalian counterpart. Escherichia coli T antigen functioned as a helicase and bound to DNA fragments containing either site I or the wild-type origin of replication in a manner identical to mammalian T antigen. However, T antigen purified from E. coli did not efficiently bind to site II, an essential cis element within the simian virus 40 origin of replication. It therefore could not unwind origin-containing plasmids or efficiently replicate simian virus 40 DNA in vitro. The ability of protein phosphorylation to modulate the intrinsic preference of full-length T antigen for either site I or site II is discussed
— id: 18119, year: 1989, vol: 86, page: 6479, stat: Journal Article,

Regulation of SV40 DNA replication by phosphorylation of T antigen
Mohr IJ; Stillman B; Gluzman Y
1987 Jan;6(1):153-160, EMBO journal
The role of phosphorylation in regulating the biochemical properties of SV40 large T antigen has been examined. Treatment of purified T antigen with calf intestinal alkaline phosphatase resulted in the removal of 80% of the 32P label. This partially dephosphorylated T antigen displayed an increase in its ability to support DNA replication in vitro. This increase in replication activity was paralleled by an activation of specific DNA binding to site II, a necessary element within the origin of SV40 DNA replication. In contrast, the ATPase activity of dephosphorylated T antigen remained unchanged. These results demonstrate that DNA replication is regulated by phosphorylation of an origin specific DNA binding protein
— id: 18121, year: 1987, vol: 6, page: 153, stat: Journal Article,

Studies on the origin-specific DNA-binding domain of simian virus 40 large T antigen
Strauss M; Argani P; Mohr IJ; Gluzman Y
1987 Oct;61(10):3326-3330, Journal of virology
The origin-specific DNA-binding domain of simian virus 40 large T antigen was analyzed, and its C-terminal boundary was found to be at or before amino acid 259. This does not include the zinc finger structural motif located at amino acids 302 to 320 (J. M. Berg, Science 232:485-486, 1986). Interestingly, N-terminal fragments of 266 and 272 amino acids and larger displayed dramatically reduced origin-binding activity. In addition, the specific DNA-binding properties of truncated proteins purified from both bacterial and mammalian sources were compared. Truncated T antigens from mammalian cells bound specific DNA fragments more efficiently than did their bacterial counterparts. These results implicate posttranslational modification with a role in regulating the DNA-binding activity of large T antigen
— id: 18120, year: 1987, vol: 61, page: 3326, stat: Journal Article,

Studies on the cytoplasmic organization of early Drosophila embryos
Miller KG; Karr TL; Kellogg DR; Mohr IJ; Walter M; Alberts BM
1985 ;50(1):79-90, Cold Spring Harbor symposia on quantitative biology
— id: 18122, year: 1985, vol: 50, page: 79, stat: Journal Article,