Faculty Bibliography

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

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

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

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

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

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

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

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

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