Herpes Simplex Virus 1 ICP34.5 Alters Mitochondrial Dynamics in Neurons.

Expression of viral genes and activation of innate antiviral responses during infection result in an increase in reactive oxygen species (ROS) and toxic by-products of energy metabolism which can lead to cell death. The mitochondrion and its associated proteins are crucial regulators of these responses and related pathways such as autophagy and apoptosis. Through a mass spectrometry approach, we have shown that the herpes simplex virus 1 (HSV-1) neurovirulence- and autophagy-modulating protein ICP34.5 interacts with numerous mitochondrion-associated factors. Specifically, we showed that amino acids 68 to 87 of ICP34.5, the domain that binds beclin1 and controls neurovirulence, are necessary for interactions with PGAM5, KEAP1, and other regulators of the antioxidant response, mitochondrial trafficking, and programmed cell death. We further show that while this domain interacts with multiple cellular stress response factors, it does not alter apoptosis or antioxidant gene expression. That said, the attenuated replication of a recombinant virus lacking residues 68 to 87 (termed Δ68-87) in primary human fibroblasts was restored by addition of ferric nitrate. Furthermore, in primary mouse neurons, the perinuclear localization of mitochondria that follows infection with HSV-1 was notably absent following Δ68-87 infection. Through this 20-amino-acid domain, ICP34.5 significantly reduces mitochondrial motility in axons of neurons. We propose the hypothesis that ICP34.5 promotes perinuclear mitochondrial localization by modulating transport of mitochondria through interaction with PGAM5. These data expand upon previous observations of altered mitochondrial dynamics following alphaherpesvirus infections and identify a key determinant of this activity during HSV-1 infections.IMPORTANCE Herpes simplex virus persists lifelong in neurons and can reactivate to cause recurrent lesions in mucosal tissues. A key determinant of virulence is the viral protein ICP34.5, of which residues 68 to 87 significantly contribute to neurovirulence through an unknown mechanism. Our report provides evidence that residues 68 to 87 of ICP34.5 are required for binding mitochondrion-associated factors. These interactions alter mitochondrial dynamics in neurons, thereby facilitating viral replication and pathogenesis.

The US11 Gene of Herpes Simplex Virus 1 Promotes Neuroinvasion and Periocular Replication following Corneal Infection.

Herpes simplex virus 1 (HSV-1) cycles between phases of latency in sensory neurons and replication in mucosal sites. HSV-1 encodes two key proteins that antagonize the shutdown of host translation, US11 through preventing PKR activation and ICP34.5 through mediating dephosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α). While profound attenuation of ICP34.5 deletion mutants has been repeatedly demonstrated, a role for US11 in HSV-1 pathogenesis remains unclear. We therefore generated an HSV-1 strain 17 US11-null virus and examined its properties in vitro and in vivo In U373 glioblastoma cells, US11 cooperated with ICP34.5 to prevent eIF2α phosphorylation late in infection. However, the effect was muted in human corneal epithelial cells (HCLEs), which did not accumulate phosphorylated eIF2α unless both US11 and ICP34.5 were absent. Low levels of phosphorylated eIF2α correlated with continued protein synthesis and with the ability of virus lacking US11 to overcome antiviral immunity in HCLE and U373 cells. Neurovirulence following intracerebral inoculation of mice was not affected by the deletion of US11. In contrast, the time to endpoint criteria following corneal infection was greater for the US11-null virus than for the wild-type virus. Replication in trigeminal ganglia and periocular tissue was promoted by US11, as was periocular disease. The establishment of latency and the frequency of virus reactivation from trigeminal ganglia were unaffected by US11 deletion, although emergence of the US11-null virus occurred with slowed kinetics. Considered together, the data indicate that US11 facilitates the countering of antiviral response of infected cells and promotes the efficient emergence of virus following reactivation.IMPORTANCE Alphaherpesviruses are ubiquitous DNA viruses and include the human pathogens herpes simplex virus 1 (HSV-1) and HSV-2 and are significant causes of ulcerative mucosal sores, infectious blindness, encephalitis, and devastating neonatal disease. Successful primary infection and persistent coexistence with host immune defenses are dependent on the ability of these viruses to counter the antiviral response. HSV-1 and HSV-2 and other primate viruses within the Simplexvirus genus encode US11, an immune antagonist that promotes virus production by preventing shutdown of protein translation. Here we investigated the impact of US11 deletion on HSV-1 growth in vitro and pathogenesis in vivo This work supports a role for US11 in pathogenesis and emergence from latency, elucidating immunomodulation by this medically important cohort of viruses.

Neuronal Subtype Determines Herpes Simplex Virus 1 Latency-Associated-Transcript Promoter Activity during Latency.

Herpes simplex virus (HSV) latency in neurons remains poorly understood, and the heterogeneity of the sensory nervous system complicates mechanistic studies. In this study, we used primary culture of adult trigeminal ganglion (TG) mouse neurons in microfluidic devices and an in vivo model to examine the subtypes of sensory neurons involved in HSV latency. HSV-infected neurofilament heavy-positive (NefH+) neurons were more likely to express latency-associated transcripts (LATs) than infected neurofilament heavy-negative (NefH-) neurons. This differential expression of the LAT promoter correlated with differences in HSV-1 early infection that manifested as differences in the efficiency with which HSV particles reached the cell body following infection at the distal axon. In vivo, we further identified a specific subset of NefH+ neurons which coexpressed calcitonin gene-related peptide α (NefH+ CGRP+ neurons) as the sensory neuron subpopulation with the highest LAT promoter activity following HSV-1 infection. Finally, an early-phase reactivation assay showed HSV-1 reactivating in NefH+ CGRP+ neurons, although other sensory neuron subpopulations were also involved. Together, these results show that sensory neurons expressing neurofilaments exhibit enhanced LAT promoter activity. We hypothesize that the reduced efficiency of HSV-1 invasion at an early phase of infection may promote efficient establishment of latency in NefH+ neurons due to initiation of the antiviral state preceding arrival of the virus at the neuronal cell body. While the outcome of HSV-1 infection of neurons is determined by a broad variety of factors in vivo, neuronal subtypes are likely to play differential roles in modulating the establishment of latent infection.IMPORTANCE Two pivotal properties of HSV-1 make it a successful pathogen. First, it infects neurons, which are immune privileged. Second, it establishes latency in these neurons. Together, these properties allow HSV to persist for the lifetime of its host. Neurons are diverse and highly organized cells, with specific anatomical, physiological, and molecular characteristics. Previous work has shown that establishment of latency by HSV-1 does not occur equally in all types of neurons. Our results show that the kinetics of HSV infection and the levels of latency-related gene expression differ in certain types of neurons. The neuronal subtype infected by HSV is therefore a critical determinant of the outcome of infection and latency.

Models of Herpes Simplex Virus Latency.

Our current understanding of HSV latency is based on a variety of clinical observations, and in vivo, ex vivo, and in vitro model systems, each with unique advantages and drawbacks. The criteria for authentically modeling HSV latency include the ability to easily manipulate host genetics and biological pathways, as well as mimicking the immune response and viral pathogenesis in human infections. Although realistically modeling HSV latency is necessary when choosing a model, the cost, time requirement, ethical constraints, and reagent availability are also equally important. Presently, there remains a pressing need for in vivo models that more closely recapitulate human HSV infection. While the current in vivo, ex vivo, and in vitro models used to study HSV latency have limitations, they provide further insights that add to our understanding of latency. In vivo models have shed light on natural infection routes and the interplay between the host immune response and the virus during latency, while in vitro models have been invaluable in elucidating molecular pathways involved in latency. Below, we review the relative advantages and disadvantages of current HSV models and highlight insights gained through each.

Ubiquitination and degradation of the hominoid-specific oncoprotein TBC1D3 is mediated by CUL7 E3 ligase.

Expression of the hominoid-specific TBC1D3 oncoprotein enhances growth factor receptor signaling and subsequently promotes cellular proliferation and survival. Here we report that TBC1D3 is degraded in response to growth factor signaling, suggesting that TBC1D3 expression is regulated by a growth factor-driven negative feedback loop. To gain a better understanding of how TBC1D3 is regulated, we studied the effects of growth factor receptor signaling on TBC1D3 post-translational processing and turnover. Using a yeast two-hybrid screen, we identified CUL7, the scaffolding subunit of the CUL7 E3 ligase complex, as a TBC1D3-interacting protein. We show that CUL7 E3 ligase ubiquitinates TBC1D3 in response to serum stimulation. Moreover, TBC1D3 recruits F-box 8 (Fbw8), the substrate recognition domain of CUL7 E3 ligase, in pull-down experiments and in an in vitro assay. Importantly, alkaline phosphatase treatment of TBC1D3 suppresses its ability to recruit Fbw8, indicating that TBC1D3 phosphorylation is critical for its ubiquitination and degradation. We conclude that serum- and growth factor-stimulated TBC1D3 ubiquitination and degradation are regulated by its interaction with CUL7-Fbw8.

Between a rock and a dry place: the water-stressed moss.

The earliest land plants faced a suite of abiotic stresses largely unknown to their aquatic algal ancestors. The descendants of these plants evolved two general mechanisms for survival in the relatively arid aerial environment. While the vascular plants or 'tracheophytes' developed tissue specializations to transport and retain water, the other main lineages of land plants, the bryophytes, retained a simple, nonvascular morphology. The bryophytes--mosses, hornworts, and liverworts--continually undergo a co-equilibration of their water content with the surrounding environment and rely to a great extent on intrinsic cellular mechanisms to mitigate damage due to water stress. This short review will focus on the cellular and molecular responses to dehydration and rehydration in mosses, and offer insights into general plant responses to water stress.

The hominoid-specific oncogene TBC1D3 activates Ras and modulates epidermal growth factor receptor signaling and trafficking.

Hominoid- and human-specific genes may have evolved to modulate signaling pathways of a higher order of complexity. TBC1D3 is a hominoid-specific oncogene encoded by a cluster of eight paralogs on chromosome 17. Initial work indicates that TBC1D3 is widely expressed in human tissues ( Hodzic, D., Kong, C., Wainszelbaum, M. J., Charron, A. J., Su, X., and Stahl, P. D. (2006) Genomics 88, 731-736 ). In this study, we show that TBC1D3 expression has a powerful effect on cell proliferation that is further enhanced by epidermal growth factor (EGF) in both human and mouse cell lines. EGF activation of the Erk and protein kinase B/Akt pathways is enhanced, both in amplitude and duration, by TBC1D3 expression, whereas RNA interference silencing of TBC1D3 suppresses the activation. Light microscopy and Western blot experiments demonstrate that increased signaling in response to EGF is coupled with a significant delay in EGF receptor (EGFR) trafficking and degradation, which significantly extends the life span of EGFR. Moreover, TBC1D3 suppresses polyubiquitination of the EGFR and the recruitment of c-Cbl. Using the Ras binding domain of Raf1 to monitor GTP-Ras we show that TBC1D3 expression enhances Ras activation in quiescent cells, which is further increased by EGF treatment. We speculate that TBC1D3 may alter Ras GTP loading. We conclude that the expression of TBC1D3 generates a delay in EGFR degradation, a decrease in ubiquitination, and a failure to recruit adapter proteins that ultimately dysregulate EGFR signal transduction and enhance cell proliferation. Altered growth factor receptor trafficking and GTP-Ras turnover may be sites where recently evolved genes such as TBC1D3 selectively modulate signaling in hominoids and humans.

TBC1D3, a hominoid oncoprotein, is encoded by a cluster of paralogues located on chromosome 17q12.

TBC1D3 is a member of the TBC1 domain family of proteins that stimulates the intrinsic GTPase activity of RAB5A, an essential actor in early endosome trafficking. Oncogenic properties of TBC1D3 have been demonstrated previously both in vitro and in mouse models. Although the oncogenic mechanism of TBC1D3 has yet to be elucidated, the TBC1D3 locus (chromosome 17q12) is amplified in 15% of primary prostate tumors. Here, we describe eight highly related TBC1D3 paralogues located within that genomic region, potentially encoding six variant TBC1D3 proteins. We found that human tissues display specific transcription patterns of these paralogues. Furthermore, that pattern was altered in several primary prostate tumors in comparison to healthy prostate tissues. Potential TBC1D3 oncogenic mechanisms are discussed in light of these results.

Mispolarization of desmosomal proteins and altered intercellular adhesion in autosomal dominant polycystic kidney disease.

Polycystin-1, the product of the major gene mutated in autosomal dominant polycystic kidney disease (ADPKD), has been shown to associate with multiple epithelial cell junctions. Our hypothesis is that polycystin-1 is an important protein for the initial establishment of cell-cell junctions and maturation of the cell and that polycystin-1 localization is dependent on the degree of cell polarization. Using laser-scanning confocal microscopy and two models of cell polarization, polycystin-1 and desmosomes were found to colocalize during the initial establishment of cell-cell contact when junctions were forming. However, colocalization was lost in confluent monolayers. Parallel morphological and biochemical evaluations revealed a profound mispolarization of desmosomal components to both the apical and basolateral domains in primary ADPKD cells and tissue. Studies of the intermediate filament network associated with desmosomes showed that there is a decrease in cytokeratin levels and an abnormal expression of the mesenchymal protein vimentin in the disease. Moreover, we show for the first time that the structural alterations seen in adherens and desmosomal junctions have a functional impact, leaving the ADPKD cells with weakened cell-cell adhesion. In conclusion, in this paper we show that polycystin-1 transiently colocalizes with desmosomes and that desmosomal proteins are mislocalized as a consequence of polycystin-1 mutation.

Host cells: mobilizable lipid resources for the intracellular parasite Toxoplasma gondii.

Successful replication of the intracellular parasite Toxoplasma gondii within its parasitophorous vacuole necessitates a substantial increase in membrane mass. The possible diversion and metabolism of host cell lipids and lipid precursors by Toxoplasma was therefore investigated using radioisotopic and fluorophore-conjugated compounds. Confocal microscopic analyses demonstrated that Toxoplasma is selective with regards to both the acquisition and compartmentalization of host cell lipids. Lipids were compartmentalized into parasite endomembranes and, in some cases, were apparently integrated into the surrounding vacuolar membrane. Additionally, some labels became concentrated in discrete lipid bodies that were biochemically and morphologically distinct from the parasite apical secretory organelles. Thin layer chromatography established that parasites readily scavenged long-chain fatty acids as well as cholesterol, and in certain cases modified the host-derived lipids. When provided with radiolabeled phospholipid precursors, including polar head groups, phosphatidic acid and small fatty acids, intracellular parasites preferentially accrued phosphatidylcholine (PtdCho) over other phospholipids. Moreover, Toxoplasma was found to be competent to synthesize PtdCho from radiolabeled precursors obtained from its environment. Together, these studies underscore the ability of Toxoplasma gondii to divert and use lipid resources from its host, a process that may contribute to the biogenesis of parasite membranes.

Molecular partitioning during host cell penetration by Toxoplasma gondii.

During invasion by Toxoplasma gondii, host cell transmembrane proteins are excluded from the forming parasitophorous vacuole membrane (PVM) by the tight apposition of host and parasite cellular membranes. Previous studies suggested that the basis for the selective partitioning of membrane constituents may be a preference for membrane microdomains, and this hypothesis was herein tested. The partitioning of a diverse group of molecular reporters for raft and nonraft membrane subdomains was monitored during parasite invasion by time-lapse video or confocal microscopy. Unexpectedly, both raft and nonraft lipid probes, as well as both raft and nonraft cytosolic leaflet proteins, flowed unhindered past the host-parasite junction into the PVM. Moreover, neither a raft-associated type 1 transmembrane protein nor its raft-dissociated counterpart accessed the PVM, while a multispanning membrane raft protein readily did so. Considered together with previous data, these studies demonstrate that selective partitioning at the host-parasite interface is a highly complex process, in which raft association favors, but is neither necessary nor sufficient for, inclusion into the T. gondii PVM.