Identification of binary interactions between human cytomegalovirus virion proteins

Human cytomegalovirus (HCMV) virions are composed of a DNA-containing nucleocapsid surrounded by a tegument layer and host-derived lipid envelope studded with virally encoded glycoproteins. These complex virions are estimated to be composed of more than 50 viral proteins. Assembly of HCMV virions is poorly understood, especially with respect to acquisition of the tegument; however, it is thought to involve the stepwise addition of virion components through protein-protein interactions. We sought to identify interactions among HCMV virion proteins using yeast two-hybrid analysis. Using 33 known capsid and tegument proteins, we tested 1,089 pairwise combinations for binary interaction in the two-hybrid assay. We identified 24 interactions among HCMV virion proteins, including 13 novel interactions among tegument proteins and one novel interaction between capsid proteins. Several of these novel interactions were confirmed by coimmunoprecipitation of protein complexes from transfected cells. In addition, we demonstrate three of these interactions in the context of HCMV infection. This study reveals several new protein-protein interactions among HCMV tegument proteins, some of which are likely important for HCMV replication and pathogenesis.     

Replication of wild-type and mutant human cytomegalovirus in life-extended human diploid fibroblasts

A cDNA encoding the catalytic subunit of human telomerase was used to generate life-extended derivatives of primary human diploid fibroblasts. The life-extended cells supported efficient human cytomegalovirus (HCMV) replication. A subclone of the life-extended cells was generated containing the HCMV UL82 gene and used to isolate and propagate a virus that exhibited a profound growth defect after infection at a low input multiplicity.     

The human cytomegalovirus (HCMV) tegument protein UL94 is essential for secondary envelopment of HCMV virions

Human cytomegalovirus (HCMV) virions are structurally complex, and the mechanisms by which they are assembled are poorly understood. However, several tegument proteins are known to be essential for proper particle assembly and maturation. Despite intense investigation, the function of many tegument proteins remains unknown. The HCMV UL94 gene is conserved among all herpesviruses and encodes a virion protein of unknown function. We demonstrate here that UL94 is a tegument protein that is expressed with true-late kinetics and localizes to the viral assembly complex during infection. To elucidate the function of UL94, we constructed a UL94-null mutant, designated UL94stop. This mutant is completely defective for replication, demonstrating that UL94 is essential. Phenotypic analysis of the UL94stop mutant shows that in the absence of UL94, viral gene expression and genome synthesis occur at wild-type levels. However, analysis of the localization of viral proteins to the cytoplasmic assembly complex shows that the essential tegument protein UL99 (pp28) exhibits aberrant localization in cells infected with the UL94stop mutant. Finally, we show that there is a complete block in secondary envelopment in the absence of UL94. Taken together, our data suggest that UL94 functions late in infection to direct UL99 to the assembly complex, thereby facilitating secondary envelopment of virions.     

Cutting edge: SIV Nef protein utilizes both leucine- and tyrosine-based protein sorting pathways for down-regulation of CD4.

The Nef protein is unique to primate lentiviruses and is closely linked to accelerated pathogenesis in both human and monkey hosts. Nef acts to down-regulate CD4 and MHC class I, two receptors important for immune function. A recent report demonstrated the presence of two tyrosine motifs in SIV Nef that contribute to its ability to down-regulate CD4 and to associate with clathrin adaptors. These tyrosine motifs are not present in HIV-1 Nef, which instead utilizes a leucine-based motif for its down-regulation of CD4. We now report that SIV Nef also contains a conserved leucine-based motif that contributes to CD4 down-regulation, functions to stimulate internalization, and contributes to the association of SIV Nef with clathrin adaptors AP-1 and AP-2. These results demonstrate that SIV Nef differs from HIV-1 Nef by its ability to use two parallel pathways of the protein-sorting machinery based on either tyrosine or leucine motifs.     

Human cytomegalovirus IE86 attenuates virus- and tumor necrosis factor alpha-induced NFkappaB-dependent gene expression

Human cytomegalovirus (HCMV) infection regulates a number of genes involved in the host antiviral response. We have previously reported that HCMV attenuates the expression of beta interferon (IFN-beta) and a number of proinflammatory chemokines, and this attenuation is mediated by the HCMV immediate-early protein IE86. The present study seeks to identify the mechanism by which IE86 blocks IFN-beta expression. We demonstrate that the induction of IFN-beta during HCMV infection requires the activation of both the IRF-3 and the NFkappaB pathways. Therefore, IE86 may target either pathway to block IFN-beta expression. Our results show that IE86 does not block IRF-3 phosphorylation, dimerization, nuclear translocation, or target gene expression. However, using gel shift analysis, we demonstrate that IE86 efficiently inhibits virus-induced binding of NFkappaB to the IFN-beta promoter, resulting in attenuation of IFN-beta and NFkappaB-dependent gene expression. Furthermore, IE86 expression inhibits tumor necrosis factor alpha-induced NFkappaB DNA binding and target gene expression. Together, these results identify IE86 as a NFkappaB antagonist, which results in the suppression of NFkappaB-dependent cytokine and chemokine gene expression.     

Tau deletion impairs intracellular β-amyloid-42 clearance and leads to more extracellular plaque deposition in gene transfer models

Background

Tau is an axonal protein that binds to and regulates microtubule function. Hyper-phosphorylation of Tau reduces its binding to microtubules and it is associated with β-amyloid deposition in Alzheimer’s disease. Paradoxically, Tau reduction may prevent β-amyloid pathology, raising the possibility that Tau mediates intracellular Aβ clearance. The current studies investigated the role of Tau in autophagic and proteasomal intracellular Aβ1-42 clearance and the subsequent effect on plaque deposition.

Results

Tau deletion impaired Aβ clearance via autophagy, but not the proteasome, while introduction of wild type human Tau into Tau^?/? mice partially restored autophagic clearance of Aβ1-42, suggesting that exogenous Tau expression can support autophagic Aβ1-42 clearance. Tau deletion impaired autophagic flux and resulted in Aβ1-42 accumulation in pre-lysosomal autophagic vacuoles, affecting Aβ1-42 deposition into the lysosome. This autophagic defect was associated with decreased intracellular Aβ1-42 and increased plaque load in Tau^?/? mice, which displayed less cell death. Nilotinib, an Abl tyrosine kinase inhibitor that promotes autophagic clearance mechanisms, reduced Aβ1-42 only when exogenous human Tau was expressed in Tau^?/? mice.

Conclusions

These studies demonstrate that Tau deletion affects intracellular Aβ1-42 clearance, leading to extracellular plaque.

     

Glial tumor necrosis factor alpha (TNFα) generates metaplastic inhibition of spinal learning

Injury-induced overexpression of tumor necrosis factor alpha (TNFα) in the spinal cord can induce chronic neuroinflammation and excitotoxicity that ultimately undermines functional recovery. Here we investigate how TNFα might also act to upset spinal function by modulating spinal plasticity. Using a model of instrumental learning in the injured spinal cord, we have previously shown that peripheral intermittent stimulation can produce a plastic change in spinal plasticity (metaplasticity), resulting in the prolonged inhibition of spinal learning. We hypothesized that spinal metaplasticity may be mediated by TNFα. We found that intermittent stimulation increased protein levels in the spinal cord. Using intrathecal pharmacological manipulations, we showed TNFα to be both necessary and sufficient for the long-term inhibition of a spinal instrumental learning task. These effects were found to be dependent on glial production of TNFα and involved downstream alterations in calcium-permeable AMPA receptors. These findings suggest a crucial role for glial TNFα in undermining spinal learning, and demonstrate the therapeutic potential of inhibiting TNFα activity to rescue and restore adaptive spinal plasticity to the injured spinal cord. TNFα modulation represents a novel therapeutic target for improving rehabilitation after spinal cord injury.     

Interaction between the Human Cytomegalovirus Tegument Proteins UL94 and UL99 Is Essential for Virus Replication

Human cytomegalovirus (HCMV) virions are structurally complex, and the mechanisms by which they are assembled are poorly understood, especially with respect to the cytoplasmic phase of assembly, during which the majority of the tegument is acquired and final envelopment occurs. These processes occur at a unique cytoplasmic structure called the assembly complex, which is formed through a reorganization of the cellular secretory apparatus. The HCMV tegument protein UL99 (pp28) is essential for viral replication at the stage of secondary envelopment. We previously demonstrated that UL99 interacts with the essential tegument protein UL94 in infected cells as well as in the absence of other viral proteins. Here we show that UL94 and UL99 alter each other's localization and that UL99 stabilizes UL94 in a binding-dependent manner. We have mapped the interaction between UL94 and UL99 to identify the amino acids of each protein that are required for their interaction. Mutation of these amino acids in the context of the viral genome demonstrates that HCMV is completely defective for replication in the absence of the interaction between UL94 and UL99. Further, we demonstrate that in the absence of their interaction, both UL94 and UL99 exhibit aberrant localization and do not accumulate at the assembly complex during infection. Taken together, our data suggest that the interaction between UL94 and UL99 is essential for the proper localization of each protein to the assembly complex and thus for the production of infectious virus.