Search

SummaryUnderstanding the negative regulators of antiviral immune responses will be critical for advancing immune-modulated antiviral strategies. NLRX1, an NLR protein that negatively regulates innate immunity, was previously identified in an unbiased siRNA screen as required for HIV infection. We find that NLRX1 depletion results in impaired nuclear import of HIV-1 DNA in human monocytic cells. Additionally, NLRX1 was observed to reduce type-I interferon (IFN-I) and cytokines in response to HIV-1 reverse-transcribed DNA. NLRX1 sequesters the DNA-sensing adaptor STING from interaction with TANK-binding kinase 1 (TBK1), which is a requisite for IFN-1 induction in response to DNA. NLRX1-deficient cells generate an amplified STING-dependent host response to cytosolic DNA, c-di-GMP, cGAMP, HIV-1, and DNA viruses. Accordingly, Nlrx1−/− mice infected with DNA viruses exhibit enhanced innate immunity and reduced viral load. Thus, NLRX1 is a negative regulator of the host innate immune response to HIV-1 and DNA viruses.

Rationale The nasopharyngeal (NP) microbiota of newborns and infants plays a key role in modulating airway inflammation and respiratory symptoms during viral infections. Premature (PM) birth modifies the early NP environment and is a major risk factor for severe viral respiratory infections. However, it is currently unknown if the NP microbiota of PM infants is altered relative to full-term (FT) individuals. Objectives To characterize the NP microbiota differences in preterm and FT infants during rhinovirus (RV) infection. Methods We determined the NP microbiota of infants 6 months to ≤2 years of age born FT (n=6) or severely PMResults We observed significant differences in the NP bacterial community of PM versus FT. NP from PM infants had higher within-group dissimilarity (heterogeneity) relative to FT infants. Bacterial composition of NP samples from PM infants showed increased Proteobacteria and decreased in Firmicutes. There were also differences in the major taxonomic groups identified, including Streptococcus, Moraxella, and Haemophilus. Longitudinal data showed that these prematurity-related microbiota features persisted during RV infection. Conclusions PM is associated with NP microbiota changes beyond the neonatal stage. PM infants have an NP microbiota with high heterogeneity relative to FT infants. These prematurity-related microbiota features persisted during RV infection, suggesting that the NP microbiota of PM may play an important role in modulating airway inflammatory and immune responses in this vulnerable group.

Most nuclear-encoded mitochondrial proteins traffic from the cytosol to mitochondria. Some of these proteins localize at mitochondria-associated membranes (MAM), where mitochondria are closely apposed with the endoplasmic reticulum (ER). We have previously shown that the human cytomegalovirus signal-anchored protein known as viral mitochondria-localized inhibitor of apoptosis (vMIA) traffics from the ER to mitochondria and clusters at the outer mitochondrial membrane (OMM). Here, we have examined the host pathways by which vMIA traffics from the ER to mitochondria and clusters at the OMM. By disruption of phosphofurin acidic cluster sorting protein 2 (PACS-2), mitofusins (Mfn1/2), and dynamin related protein 1 (Drp1), we find these conventional pathways for ER to the mitochondria trafficking are dispensable for vMIA trafficking to OMM. Instead, mutations in vMIA that change its hydrophobicity alter its trafficking to mitochondria. Superresolution imaging showed that PACS-2- and Mfn-mediated membrane apposition or hydrophobic interactions alter vMIA’s ability to organize in nanoscale clusters at the OMM. This shows that signal-anchored MAM proteins can make use of hydrophobic interactions independently of conventional ER-mitochondria pathways to traffic from the ER to mitochondria. Further, vMIA hydrophobic interactions and ER-mitochondria contacts facilitate proper organization of vMIA on the OMM.

Podocyte injury has a critical role in the pathogenesis of HIV-associated nephropathy (HIVAN). The HIV-1 transactivator of transcription (Tat), combined with fibroblast growth factor-2 (FGF-2), can induce the dedifferentiation and proliferation of cultured human podocytes. Cellular internalization of Tat requires interactions with heparan sulfate proteoglycans and cholesterol-enriched lipid rafts (LRs). However, the specific distribution of Tat in human podocytes and its ability to associate with LRs have not been documented. Here, we found that Tat is preferentially recruited to LRs in podocytes isolated from children with HIVAN. Furthermore, we identified arginines in the basic domain (RKKRRQRRR) of Tat as essential for (1) targeting Tat to LRs, (2) Tat-mediated increases in the expression of Rho-A and matrix metalloproteinase-9 in LRs, and (3) Tat-mediated enhancement of FGF-2 signaling in human podocytes and HIV-transgenic mouse kidneys and the exacerbation of renal lesions in these mice. Tat carrying alanine substitutions in the basic domain (AKKAAQAAA) remained localized in the cytosol and did not associate with LRs or enhance FGF-2 signaling in cultured podocytes. These results show the specific association of Tat with LRs in podocytes isolated from children with HIVAN, confirm Tat as a regulator of FGF-2 signaling in LRs, and identify the key domain of Tat responsible for promoting these effects and aggravating renal injury in HIV-transgenic mice. Moreover, these results provide a molecular framework for developing novel therapies to improve the clinical outcome of children with HIVAN.

Human cytomegalovirus (HCMV) encodes the UL37 exon 1 protein (pUL37x1), which is the potent viral mitochondrion-localized inhibitor of apoptosis (vMIA), to increase survival of infected cells. HCMV vMIA traffics from the endoplasmic reticulum (ER) to ER subdomains, which are physically linked to mitochondria known as mitochondrion-associated membranes (MAM), and to mitochondria. The antiapoptotic function of vMIA is thought to primarily result from its ability to inhibit Bax-mediated permeabilization of the outer mitochondrial membrane (OMM). Here, we establish that vMIA retargets Bax to the MAM as well as to the OMM from immediate early through late times of infection. However, MAM localization of Bax results in its increased ubiquitination and proteasome-mediated degradation. Surprisingly, HCMV infection does not increase OMM-associated degradation (OMMAD) of Bax, even though the ER and mitochondria are physically connected at the MAM. It was recently found that lipid rafts at the plasma membrane can connect extrinsic and intrinsic apoptotic pathways and can serve as sites of apoptosome assembly. In transfected permissive human fibroblasts, vMIA mediates, through its cholesterol affinity, association of Bax and apoptosome components with MAM lipid rafts. While Bax association with MAM lipid rafts was detected in HCMV-infected cells, association of apoptosome components was not. These results establish that Bax recruitment to the MAM and its MAM-associated degradation (MAMAD) are a newly described antiapoptotic mechanism used by HCMV infection to increase cell survival for its growth.

Airway occlusion in obstructive airway diseases is caused in part by the overproduction of secretory mucin glycoproteins through the up-regulation of mucin (MUC) genes by inflammatory mediators. Some pharmacological agents, including the glucocorticoid dexamethasone (Dex), repress mucin concentrations in lung epithelial cancer cells. Here, we show that Dex reduces the expression of MUC5AC, a major airway mucin gene, in primary differentiated normal human bronchial epithelial (NHBE) cells in a dose-dependent and time-dependent manner, and that the Dex-induced repression is mediated by the glucocorticoid receptor (GR) and two glucocorticoid response elements (GREs) in the MUC5AC promoter. The pre-exposure of cells to RU486, a GR antagonist, and mutations in either the GRE3 or GRE5 cis-sites abolished the Dex-induced repression. Chromatin immunoprecipitation (ChIP) assays showed a rapid temporal recruitment of GR to the GRE3 and GRE5 cis-elements in the MUC5AC promoter in NHBE and in A549 cells. Immunofluorescence showed nuclear colocalization of GR and histone deacetylase–2 (HDAC2) in MUC5AC-expressing NHBE cells. ChIP also showed a rapid temporal recruitment of HDAC2 to the GRE3 and GRE5 cis-elements in the MUC5AC promoter in both cell types. The knockdown of HDAC2 by HDAC2-specific short interfering RNA prevented the Dex-induced repression of MUC5AC in NHBE and A549 cells. These data demonstrate that GR and HDAC2 are recruited to the GRE3 and GRE5 cis-sites in the MUC5AC promoter and mediate the Dex-induced cis repression of MUC5AC gene expression. A better understanding of the mechanisms whereby glucocorticoids repress MUC5AC gene expression may be useful in formulating therapeutic interventions in chronic lung diseases.

Background Cystic fibrosis (CF) is an autosomal recessive disease characterized by recurrent lung infections. Studies of the lung microbiome have shown an association between decreasing diversity and progressive disease. 454 pyrosequencing has frequently been used to study the lung microbiome in CF, but will no longer be supported. We sought to identify the benefits and drawbacks of using two state-of-the-art next generation sequencing (NGS) platforms, MiSeq and PacBio RSII, to characterize the CF lung microbiome. Each has its advantages and limitations. Methods Twelve samples of extracted bacterial DNA were sequenced on both MiSeq and PacBio NGS platforms. DNA was amplified for the V4 region of the 16S rRNA gene and libraries were sequenced on the MiSeq sequencing platform, while the full 16S rRNA gene was sequenced on the PacBio RSII sequencing platform. Raw FASTQ files generated by the MiSeq and PacBio platforms were processed in mothur v1.35.1. Results There was extreme discordance in alpha-diversity of the CF lung microbiome when using the two platforms. Because of its depth of coverage, sequencing of the 16S rRNA V4 gene region using MiSeq allowed for the observation of many more operational taxonomic units (OTUs) and higher Chao1 and Shannon indices than the PacBio RSII. Interestingly, several patients in our cohort had Escherichia , an unusual pathogen in CF. Also, likely because of its coverage of the complete 16S rRNA gene, only PacBio RSII was able to identify Burkholderia , an important CF pathogen. Conclusion When comparing microbiome diversity in clinical samples from CF patients using 16S sequences, MiSeq and PacBio NGS platforms may generate different results in microbial community composition and structure. It may be necessary to use different platforms when trying to correctly identify dominant pathogens versus measuring alpha-diversity estimates, and it would be important to use the same platform for comparisons to minimize errors in interpretation.

Background aims Human parainfluenza virus-3 (HPIV) is a common cause of respiratory infection in immunocompromised patients and currently has no effective therapies. Virus-specific T-cell therapy has been successful for the treatment or prevention of viral infections in immunocompromised patients but requires determination of T-cell antigens on targeted viruses. Methods HPIV3-specific T cells were expanded from peripheral blood of healthy donors using a rapid generation protocol targeting four HPIV3 proteins. Immunophenotyping was performed by flow cytometry. Viral specificity was determined by interferon (IFN)-γ ELISpot, intracellular cytokine staining and cytokine measurements from culture supernatants by Luminex assay. Cytotoxic activity was tested by 51 Cr release and CD107a mobilization assays. Virus-specific T cells targeting six viruses were then produced by rapid protocol, and the phenotype of HPIV3-specific T cells was determined by immunomagnetic sorting for IFN-γ-producing cells. Results HPIV3-specific T cells were expanded from 13 healthy donors. HPIV3-specific T cells showed a CD4 + predominance (mean CD4:CD8 ratio 2.89) and demonstrated specificity for multiple HPIV3 antigens. The expanded T cells were polyfunctional based on cytokine production but only had a minor cytotoxic component. T cells targeting six viruses in a single product similarly showed HPIV3 specificity, with a predominant effector memory phenotype (CD3 + /CD45RA – /CCR7 – ) in responder cells. Discussion HPIV3-specific T cells can be produced using a rapid ex vivo protocol from healthy donors and are predominantly CD4 + T cells with Th1 activity. HPIV3 epitopes can also be successfully targeted alongside multiple other viral epitopes in production of six-virus T cells, without loss of HPIV3 specificity. These products may be clinically beneficial to combat HPIV3 infections by adoptive T-cell therapy in immune-compromised patients.

A wide variety of viruses cause significant morbidity and mortality in humans. However, targeted antiviral therapies have been developed for only a subset of these viruses, with the majority of currently licensed antiviral drugs targeting viral entry, replication or exit steps during the viral life cycle. Due to increasing emergence of antiviral drug resistant viruses, the isolation of multiple viral subtypes, and toxicities of existing therapies, there remains an urgent need for the timely development of novel antiviral agents, including those targeting host factors essential for viral replication. This review summarizes viral products that target mitochondria and their effects on common mitochondria regulated pathways. These viral products and the mitochondrial pathways affected by them provide potential novel targets for the rational design of antiviral drugs. Viral products alter oxidative balance, mitochondrial permeability transition pore, mitochondrial membrane potential, electron transport and energy production. Moreover, viruses may cause the Warburg Effect, in which metabolism is reprogrammed to aerobic glycolysis as the main source of energy. Finally, viral products affect proapoptotic and antiapoptotic signaling, as well as mitochondrial innate immune signaling. Because of their importance for the generation of metabolic intermediates and energy as well as cell survival, mitochondrial pathways are targeted by multiple independent viral products. Structural modifications of existing drugs targeted to mitochondrial pathways may lead to the development of novel antiviral drugs with improved efficacy and reduced toxicity.

Neuroblastoma is the most common extracranial solid tumor in children and tumor ganglioside composition has been linked to its biological and clinical behavior. We recently found that high expression of complex gangliosides that are products of the enzyme GM1a/GD1b synthase predicts a more favorable outcome in human neuroblastoma, and others have shown that complex gangliosides such as GD1a inhibit metastasis of murine tumors. To determine how a switch from structurally simple to structurally complex ganglioside expression affects neuroblastoma cell behavior, we engineered IMR32 human neuroblastoma cells, which contain almost exclusively (89%) the simple gangliosides (SG) GM2, GD2, GM3, and GD3, to overexpress the complex gangliosides (CG) GM1, GD1a, GD1b and GT1b, by stable retroviral-mediated transduction of the cDNA encoding GM1a/GD1b synthase. This strikingly altered cellular ganglioside composition without affecting total ganglioside content: There was a 23-fold increase in the ratio of complex to simple gangliosides in GM1a/GD1b synthase-transduced cells (IMR32-CG) vs. wild type (IMR32) or vector-transfected (IMR32-V) cells with essentially no expression of the clinical neuroblastoma marker, GD2, confirming effectiveness of this molecular switch from simple to complex ganglioside synthesis. Probing for consequences of the switch, we found that among functional properties of IMR32-CG cells, cell migration was inhibited and Rho/Rac1 activities were altered, while proliferation kinetics and cell differentiation were unaffected. These findings further implicate cellular ganglioside composition in determining cell migration characteristics of tumor cells. This IMR32 model system should be useful in delineating the impact of ganglioside composition on tumor cell function.

The human cytomegalovirus (HCMV) protein UL37 exon 1 (pUL37x1), also known as viral mitochondrion-localized inhibitor of apoptosis (vMIA), sequentially traffics from the endoplasmic reticulum (ER) through mitochondrion-associated membranes (MAMs) to the outer mitochondrial membrane (OMM), where it robustly inhibits apoptosis. Here, we report the association of pUL37x1/vMIA with internal lipid rafts (LRs) in the ER/MAM. The MAM, which serves as a site for lipid transfer and calcium signaling to mitochondria, is enriched in detergent-resistant membrane (DRM)-forming lipids, including cholesterol and ceramide, which are found in lower concentrations in the bulk ER. Sigma 1 receptor (Sig-1R), a MAM chaperone affecting calcium signaling to mitochondria, is anchored in the MAM by its LR association. Because of its trafficking through the MAM and partial colocalization with Sig-1R, we tested whether pUL37x1/vMIA associates with MAM LRs. Extraction with methyl-β-cyclodextrin (MβCD) removed pUL37x1/vMIA from lysed but not intact cells, indicating its association with internal LRs. Furthermore, the isolation of DRMs from purified intracellular organelles independently verified the localization of pUL37x1/vMIA within ER/MAM LRs. However, pUL37x1/vMIA was not detected in DRMs from mitochondria. pUL37x1/vMIA associated with LRs during all temporal phases of HCMV infection, indicating the likely importance of this location for HCMV growth. Although detected during its sequential trafficking to the OMM, the pUL37x1/vMIA LR association was independent of its mitochondrial targeting signals. Rather, it was dependent upon cholesterol binding. These studies suggest a conserved ability of UL37 proteins to interact with cholesterol and LRs, which is functionally distinguishable from their sequential trafficking to mitochondria.

Human cytomegalovirus (HCMV) UL37 proteins traffic sequentially from the endoplasmic reticulum (ER) to the mitochondria. In transiently transfected cells, UL37 proteins traffic into the mitochondrion-associated membranes (MAM), the site of contact between the ER and mitochondria. In HCMV-infected cells, the predominant UL37 exon 1 protein, pUL37x1, trafficked into the ER, the MAM, and the mitochondria. Surprisingly, a component of the MAM calcium signaling junction complex, cytosolic Grp75, was increasingly enriched in heavy MAM from HCMV-infected cells. These studies show the first documented case of a herpesvirus protein, HCMV pUL37x1, trafficking into the MAM during permissive infection and HCMV-induced alteration of the MAM protein composition.

Human cytomegalovirus UL37 antiapoptotic proteins, including the predominant UL37 exon 1 protein (pUL37x1), traffic sequentially from the endoplasmic reticulum (ER) through the mitochondrion-associated membrane compartment to the mitochondrial outer membrane (OMM), where they inactivate the proapoptotic activity of Bax. We found that widespread mitochondrial distribution occurs within 1 h of pUL37x1 synthesis. The pUL37x1 mitochondrial targeting signal (MTS) spans its first antiapoptotic domain (residues 5 to 34) and consists of a weak hydrophobicity leader (MTSα) and proximal downstream residues (MTSβ). This MTS arrangement of a hydrophobic leader and downstream proximal basic residues is similar to that of the translocase of the OMM 20, Tom20. We examined whether the UL37 MTS functions analogously to Tom20 leader. Surprisingly, lowered hydropathy of the UL37x1 MTSα, predicted to block ER translocation, still allowed dual targeting of mutant to the ER and OMM. However, increased hydropathy of the MTS leader caused exclusion of the UL37x1 high-hydropathy mutant from mitochondrial import. Conversely, UL37 MTSα replacement with the Tom20 leader did not retarget pUL37x1 exclusively to the OMM; rather, the UL37x1-Tom20 chimera retained dual trafficking. Moreover, replacement of the UL37 MTSβ basic residues did not reduce OMM import. Ablation of the MTSα posttranslational modification site or of the downstream MTS proline-rich domain (PRD) increased mitochondrial import. Our results suggest that pUL37x1 sequential ER to mitochondrial trafficking requires a weakly hydrophobic leader and is regulated by MTSβ sequences. Thus, HCMV pUL37x1 uses a mitochondrial importation pathway that is genetically distinguishable from that of known OMM proteins.

Biologically active membrane gangliosides, expressed and released by many human tumors, are hypothesized to significantly impact tumor progression. Lack of a model of complete and specific tumor ganglioside depletion in vivo, however, has hampered elucidation of their role. Here, we report the creation of a novel, stable, genetically induced tumor cell system resulting in specific and complete blockade of ganglioside synthesis. Wild-type (WT) and GM3 synthase/GM2 synthase double knockout (DKO) murine embryonic fibroblasts were transformed using amphotropic retrovirus-transduced oncogenes (pBABE-c-Myc(T58A)+H-RasG12V). The transformed cells, WT(t) and DKO(t) respectively, evidenced comparable integrated copy numbers and oncogene expression. Ganglioside synthesis was completely blocked in the DKO(t) cells, importantly without triggering an alternate pathway of ganglioside synthesis. Ganglioside depletion (to

Alternative splicing and polyadenylation of human cytomegalovirus (HCMV) immediate-early (IE) pre-mRNAs are temporally regulated and rely on cellular RNA-processing factors. This study examined the location and abundance of essential RNA-processing factors, which affect alternative processing of UL37 IE pre-mRNAs, during HCMV infection. Serine/threonine protein kinase 1 (SRPK1) phosphorylates serine/arginine-rich proteins, necessary for pre-spliceosome commitment. It was found that HCMV infection progressively increased the abundance of cytoplasmic SRPK1, which is regulated by subcellular partitioning. The essential polyadenylation factor CstF-64 was similarly increased in abundance, albeit in the nucleus, proximal to and within viral replication compartments (VRCs). In contrast, the location of polypyrimidine tract-binding protein (PTB), known to adversely affect splicing of HCMV major IE RNAs, was temporally regulated during infection. PTB co-localized with CstF-64 in the nucleus at IE times. By early times, PTB was detected in punctate cytoplasmic sites of some infected cells. At late times, PTB relocalized to the nucleus, where it was notably excluded from HCMV VRCs. Moreover, HCMV infection induced the formation of nucleolar stress structures, fibrillarin-containing caps, in close proximity to its VRCs. PTB exclusion from HCMV VRCs required HCMV DNA synthesis and/or late gene expression, whereas the regulation of SRPK1 subcellular distribution did not. Taken together, these results indicated that HCMV increasingly regulates the subcellular distribution and abundance of essential RNA-processing factors, thereby altering their ability to affect the processing of viral pre-mRNAs. These results further suggest that HCMV infection selectively induces sorting of nucleolar and nucleoplasmic components.

By exploiting host cell machineries, viruses provide powerful tools for gaining insight into cellular pathways. Proteins from two unrelated viruses, human CMV (HCMV) and HCV, are documented to traffic sequentially from the ER into mitochondria, probably through the mitochondria-associated membrane (MAM) compartment. The MAM are sites of ER-mitochondrial contact enabling the direct transfer of membrane bound lipids and the generation of high calcium (Ca2+) microdomains for mitochondria signalling and responses to cellular stress. Both HCV core protein and HCMV UL37 proteins are associated with Ca2+ regulation and apoptotic signals. Trafficking of viral proteins to the MAM may allow viruses to manipulate a variety of fundamental cellular processes, which converge at the MAM, including Ca2+ signalling, lipid synthesis and transfer, bioenergetics, metabolic flow, and apoptosis. Because of their distinct topologies and targeted MAM sub-domains, mitochondrial trafficking (albeit it through the MAM) of the HCMV and HCV proteins predictably involves alternative pathways and, hence, distinct targeting signals. Indeed, we found that multiple cellular and viral proteins, which target the MAM, showed no apparent consensus primary targeting sequences. Nonetheless, these viral proteins provide us with valuable tools to access the poorly characterised MAM compartment, to define its cellular constituents and describe how virus infection alters these to its own end. Furthermore, because proper trafficking of viral proteins is necessary for their function, discovering the requirements for MAM to mitochondrial trafficking of essential viral proteins may provide novel targets for the rational design of anti-viral drugs.

Increasingly mechanistic virology studies require dependable and sensitive methods for isolating purified organelles containing functional cellular sub-domains. The mitochondrial network is, in part, closely apposed to the endoplasmic reticulum (ER). The mitochondria-associated membrane (MAM) fraction provides direct physical contact between the ER and mitochondria. Characterization of the dual localization and trafficking of human cytomegalovirus (HCMV) UL37 proteins required establishing protocols in which the ER and mitochondria could be reliably separated. Because of its documented role in lipid and ceramide transfer from the ER to mitochondria, a method to purify MAM from infected cells was also developed. Two robust procedures were developed to efficiently isolate mitochondria, ER, and MAM fractions while providing substantial protein yields from HCMV-infected primary fibroblasts and from transfected HeLa cells. Furthermore, this unit includes protocols for isolation of detergent resistant membranes from subcellular fractions as well as techniques that allow visualization of the mitochondrial network disruption that occurs in permissively infected cells by their optimal resolution in Percoll gradients.

The endoplasmic reticulum (ER) membrane is closely apposed to the outer mitochondrial membrane (OMM), which facilitates communication between these organelles. These contacts, known as mitochondria-associated membranes (MAM), facilitate calcium signaling, lipid transfer, as well as antiviral and stress responses. How cellular proteins traffic to the MAM, are distributed therein, and interact with ER and mitochondrial proteins are subject of great interest. The human cytomegalovirus UL37 exon 1 protein or viral mitochondria-localized inhibitor of apoptosis (vMIA) is crucial for viral growth. Upon synthesis at the ER, vMIA traffics to the MAM and OMM, where it reprograms the organization and function of these compartments. vMIA significantly changes the abundance of cellular proteins at the MAM and OMM, including proteins that regulate calcium homeostasis and cell death. Through the use of superresolution imaging, we have shown that vMIA is distributed at the OMM in nanometer scale clusters. This is similar to the clusters reported for the mitochondrial calcium channel, VDAC, as well as electron transport chain, translocase of the OMM complex, and mitochondrial inner membrane organizing system components. Thus, aside from addressing how vMIA targets the MAM and regulates survival of infected cells, biochemical studies and superresolution imaging of vMIA offer insights into the formation, organization, and functioning of MAM. Here, we discuss these insights into trafficking, function, and organization of vMIA at the MAM and OMM and discuss how the use of superresolution imaging is contributing to the study of the formation and trafficking of viruses.

The human cytomegalovirus (HCMV) UL37 glycoprotein (gpUL37) is internally cleaved and its products divergently traffic to mitochondria or are retained in the secretory pathway. To define the requirements for gpUL37 cleavage, residues −1 and −3 of the consensus endoplasmic reticulum (ER) signal peptidase I site within exon 3 (UL37x3) were replaced by bulky tyrosines (gpUL37 cleavage site mutant I). Internal cleavage of this UL37x3 mutant was inhibited, verifying usage of the consensus site at amino acids (aa) 193/194. The full-length mitochondrial species of gpUL37 cleavage site mutant I was N glycosylated and endoglycosidase H sensitive, indicating that ER translocation and processing took place prior to its mitochondrial importation. Moreover, these results suggest that internal cleavage of gpUL37 is not necessary for its N glycosylation. Partial deletion or disruption of the UL37 hydrophobic core immediately upstream of the cleavage site resulted in decreased protein abundance, suggesting that the UL37x3 hydrophobic α-helix contributes to either correct folding or stability of gpUL37. Insertion of the UL37x3 hydrophobic core and cleavage site into pUL37M, a splice variant of gpUL37 which lacks these sequences and is neither proteolytically cleaved nor N glycosylated, resulted in its internal cleavage and N glycosylation. Its NH2-terminal fragment, pUL37M-NH2, was detected more abundantly in mitochondria, while its N-glycosylated C-terminal fragment, gpUL37M-COOH, was detected predominantly in the ER in a manner analogous to that of gpUL37 cleavage products. These results indicate that UL37x3 aa 178 to 205 are prerequisite for gpUL37 internal cleavage and alter UL37 protein topology allowing N glycosylation of its C-terminal sequences. In contrast, the NH2-terminal UL37x1 hydrophobic leader, present in pUL37x1, pUL37M, and gpUL37, is not cleaved from mature UL37 protein, retaining a membrane anchor for UL37 isoforms during trafficking. Taken together, these results suggest that HCMV gpUL37 undergoes sequential trafficking, during which it is ER translocated, processed, and then mitochondrially imported.

The human immunodeficiency virus type 1 (HIV-1) potent transactivator Tat protein mediates pleiotropic effects on various cell functions. Posttranslational modification of Tat affects its activity during viral transcription. Tat binds to TAR and subsequently becomes acetylated on lysine residues by histone acetyltransferases. Novel protein-protein interaction domains on acetylated Tat are then established, which are necessary for both sustained transcriptional activation of the HIV-1 promoter and viral transcription elongation. In this study, we investigated the identity of proteins that preferentially bound acetylated Tat. Using a proteomic approach, we identified a number of proteins that preferentially bound AcTat, among which p32, a cofactor of splicing factor ASF/SF-2, was identified. We found that p32 was recruited to the HIV-1 genome, suggesting a mechanism by which acetylation of Tat may inhibit HIV-1 splicing needed for the production of full-length transcripts. Using Tat from different clades, harboring a different number of acetylation sites, as well as Tat mutated at lysine residues, we demonstrated that Tat acetylation affected splicing in vivo. Finally, using confocal microscopy, we found that p32 and Tat colocalize in vivo in HIV-1-infected cells.

Background Human cytomegalovirus (HCMV) replication in epithelial cells is crucial for its pathogenesis. To date, HCMV gene expression has been primarily studied in human foreskin fibroblasts (HFFs), although their importance for HCMV pathogenesis remains unclear. Primary retinal pigment epithelial (RPE) cells are permissive for HCMV. Objectives Our objectives were to determine the production of alternatively processed HCMV major immediate-early (MIE) and UL37 RNAs and their essential products in infected, terminally differentiated immortalized RPE (hTERT-RPE) cells. Study design hTERT-RPE cells were studied because of their notable similarities with primary RPE cells, and because they overcome key limitations of primary cells. hTERT-RPE cells were terminally differentiated in vitro and infected with HCMV. Total RNA or cell proteins were analyzed at various times post-infection. Results We show for the first time that HCMV-infected, differentiated hTERT-RPE cells produce IE1, IE2, UL37 exon 1 (UL37x1) and UL37 alternatively spliced RNAs, albeit with abundances and kinetics distinct from those observed in HCMV-infected HFFs. IE1-72 was produced in HCMV-infected, differentiated hTERT-RPEs within 24 h post-infection (hpi); whereas, IE2-86 and pUL37x1 were produced within 72 hpi. IE2-86 was detected after IE1-72 even though its transcript appeared first. Early/late (pp65) and late (pp28) proteins were produced within 96–120 hpi. Conclusions The temporal cascade of HCMV gene expression was observed in infected, differentiated hTERT-RPE cells. Moreover, HCMV IE RNAs are alternatively and accurately processed in differentiated hTERT-RPE cells. However, the delayed temporal expression suggests further regulation of HCMV gene expression at post-transcriptional/translational levels in differentiated hTERT-RPE cells.

Alternative processing of human cytomegalovirus (HCMV) UL37 pre-mRNA predominantly produces the unspliced UL37 exon 1 (UL37x1) RNA and multiple, lower abundance, alternatively spliced UL37 RNAs. The relative abundance of UL37x1 unspliced RNA is surprising because it requires the favoured use of a polyadenylation signal within UL37 intron 1, just upstream of the UL37 exon 2 (UL37x2) acceptor. Here, it was shown that a downstream element (DSE) in UL37x2 strongly enhanced processing at the UL37x1 polyadenylation site, but did not influence UL37x1-x2 splicing. There was a potential binding site (UCUU) for polypyrimidine tract-binding protein (PTB) at the UL37x1 polyadenylation/cleavage site and its mutation to UGGG reduced both polyadenylation and splicing of UL37x1-x2 minigene pre-mRNA, suggesting a role in both RNA processing events. To determine whether lytic HCMV infection altered the balance of RNA processing factors, which bind to UL37 pre-mRNA cis elements, these were investigated in permissively infected primary and immortalized human diploid fibroblasts (HFFs) and epithelial cells. Induction of polyadenylation factors in HCMV-infected, serum-starved (G(0)) HFFs was also investigated. Permissive HCMV infection consistently increased, albeit with different kinetics, the abundance of cleavage stimulation factor 64 (CstF-64) and PTB, and altered hypo-phosphorylated SF2 in different cell types. Moreover, the preponderance of UL37x1 RNA increased during infection and correlated with CstF-64 induction, whereas the complexity of the lower abundance UL37 spliced RNAs transiently increased following reduction of hypo-phosphorylated SF2. Collectively, multiple UL37 RNA polyadenylation cis elements and induced cellular factors in HCMV-infected cells strongly favoured the production of UL37x1 unspliced RNA.

The human cytomegalovirus UL37 gene encodes at least three isoforms, which share N-terminal UL37 exon 1 (UL37x1) sequences. UL37 proteins traffic dually into the endoplasmic reticulum (ER) and to mitochondria. Trafficking of the UL37 glycoprotein (gpUL37) in relation to its post-translational processing was investigated. gpUL37 is internally cleaved in the ER and its products traffic differentially. Its C-terminal fragment (UL37COOH) is ER-localized and N-glycosylated. Unlike conventional ER signal sequences, its N-terminal () fragment is stable and traffics to mitochondria. Inhibition of N-glycosylation did not block pUL37 cleavage and dramatically decreased the levels of but not of UL37COOH. pUL37M, which differs from gpUL37 by the lack of residues 178–262 and hence the UL37x3 consensus signal peptidase cleavage site, traffics into the ER and mitochondria, but is neither cleaved nor N-glycosylated. This finding of a relationship between ER processing and mitochondrial importation of UL37 proteins is unique for herpesvirus proteins.

The human cytomegalovirus (HCMV) UL37 immediate-early (IE) gene minimally encodes three protein isoforms that share NH2-terminal sequences. The predominant UL37 isoform detected during HCMV infection was the UL37 exon 1 protein (pUL37x1), which was produced from IE and, more abundantly, through late times of infection. pUL37x1 was localized in both the endoplasmic reticulum (ER) and mitochondria in infected cells. To determine which UL37x1 NH2-terminal residues serve as ER and mitochondrial targeting signals, we examined the subcellular localization of two deletion mutants. pUL37x1Δ2–23, which lacks the hydrophobic leader, is neither translocated into the ER nor imported mitochondrially; conversely, pUL37x1Δ23–34, lacking the juxtaposed basic residues, was translocated into the ER but only imported weakly into mitochondria. These studies show for the first time the temporal production and localization of pUL37x1 during HCMV infection. The trafficking patterns of mutants suggest that the pUL37x1 targeting signal to ER and mitochondria is bipartite.

The human cytomegalovirus (HCMV) UL36-38 immediate early (IE) locus encodes proteins required for its growth. The UL37 promoter drives production of an unspliced and several alternatively spliced RNAs. The UL37 exon 1 (UL37x1) unspliced RNA is abundant from IE to late times of HCMV infection, whereas the UL37 spliced RNAs are markedly less abundant. Production of the UL37x1 unspliced RNA requires polyadenylation (PA) at nucleotide 50998, which lies within intron 1, upstream of the UL37 exon 2 (UL37x2) acceptor. The physical proximity of its cis elements suggests steric hindrance between PA and splicing machineries for UL37 pre-mRNA. To test this possibility, we generated site-specific mutants in Target 1 PA and RNA splicing cis elements and compared the PA and splicing efficiencies of mutant RNAs with those of wild-type RNA. The mutually exclusive processing events of UL37x1 PA and UL37x1-UL37x2 splicing have been accurately recapitulated in transfected permissive human fibroblasts (HFFs) expressing a Target 1 minigene RNA, which contains the required splicing and PA cis elements. Two mutants in the invariant PA signal dramatically decreased UL37x1 PA as expected and, concomitantly, increased the efficiency of UL37x1-UL37x2 RNA splicing. Consistent with these results, changes to consensus UL37x1 donor and UL37x2 acceptor sites increased the efficiency of UL37x1-UL37x2 RNA splicing but decreased the efficiency of UL37x1 PA. Moreover, HCMV infection of HFFs increased the abundance of the PA cleavage stimulatory factor CstF-64, the potent splicing suppressor PTB, and the hypophosphorylated form of the splicing factor SF2 at 4 h postinfection. Induction of these factors further favors production of the UL37x1 unspliced RNA over that of the spliced RNAs. Taken together, these results suggest that there is a convergence in UL37 RNA regulation by cis elements and cellular proteins which favors production of the UL37x1 unspliced RNA during HCMV infection at the posttranscriptional level.

Differentially processed human cytomegalovirus (HCMV) UL37 RNAs encode biologically significant proteins. Due to the recent discovery of alternative UL37 exon 3 (UL37x3) splice donors, permissively infected cells were thoroughly examined for additional alternatively spliced UL37 RNAs. Newly described donors within UL37 exon 1 (nt 52520) and intron 1 (nt 52209) as well as UL37x3 di (nt 50770) and dii (nt 50782) were differentially spliced to known downstream UL37 acceptors. The alternatively spliced UL37S, UL37L, UL37di and UL37dii RNAs predictably encode proteins of 83, 163, 217 and 213 residues, respectively, which share UL37x1 N-terminal sequences but differ downstream in their C termini. Moreover, temporal expression of the alternatively spliced UL37 RNAs differs during HCMV infection. The complexity of UL37 pre-mRNA processing is evidenced by the detection of 11 UL37 spliced and unspliced UL37x1 RNAs in HCMV-infected cells. Based upon these data, a revised HCMV UL37 gene map is presented, which incorporates all RNA species detected during permissive infection.

The human cytomegalovirus (HCMV) UL36-38 immediate early (IE) locus encodes proteins required for virus growth. The UL37 IE promoter drives production of differentially spliced and unspliced RNAs. To study their post-transcriptional processing, we generated target minigenes encoding each UL37 RNA splicing substrate. Target 1 RNA, spanning UL37 exon 1 (x1) donor and 2 (x2) acceptor as well as adjacent intronic sequences, but not the UL38 gene, accurately reproduced UL37 x1/x2 RNA splicing in transfected permissive cells. Surprisingly, deletion of distal intronic sequences nt -82 to -143 from the UL37x2 acceptor resulted in aberrant splicing to an upstream non-consensus exonic donor. Target 1 RNAs carry the UL37x1 polyadenylation (PA) signal and site as well as a downstream SV40 early PA signal. Both the UL37x1 and SV40 PA signals are used in wild-type target 1 RNAs but inhibited in UL37x1 PA signal mutants. Alternative RNA splicing of UL37 exons 2 to 3 or 3A as well as exons 3 to 4, observed in HCMV mature UL37 and UL36 spliced RNAs, is accurately reproduced with target minigene RNAs carrying the corresponding UL37 exonic and intronic sequences. Moreover, alternative splicing using two novel UL37 exon 3 consensus splice donors (di and dii) was found in target and in HCMV-infected cell RNA. These results demonstrate that: (i) target minigene RNAs accurately recapitulate the processing of UL37 IE RNAs in the HCMV-infected cell; (ii) precise UL37x1 donor selection is modulated by 3'-distal UL37 intronic sequences; and (iii) UL37 exon 3 contains multiple alternative consensus splice donors.

Microbial infections are the most common cause of death in humans. Type I interferon (IFN-I) is a key element bridging the host innate and adaptive immune response against infections. Delineating the molecular regulation network of IFN-I signaling is critical for developing novel antiviral strategy and benefiting rational therapy. Using an unbiased siRNA screen, we find NLRX1, one nucleotide-binding leucine-rich-repeat-containing protein, is a host factor that promotes an early step of HIV-1 infection. NLRX1 suppresses type-I interferon (IFN-I) and cytokines in response to HIV-1 reverse-transcribed DNA and enhances the nuclear import of HIV-1 DNA. In addition to HIV, NLRX1 also reduces STING-dependent host response to cytosolic DNA, c-di-GMP, cGAMP, and DNA virus. Mechanistically, NLRX1 associates with STING in mitochondria-associated ER membranes, and prevents STING recruiting TBK1 and activating downstream interferon signaling. By using purified recombinant proteins, we found NLRX1 interacts directly with STING. Furthermore, DNA virus infected Nlrx1−/− mice exhibited enhanced innate immunity and reduced morbidity and viral load. In summary, these findings reveal that NLRX1 is a checkpoint protein for DNA sensing adaptor STING and may represent a novel precision target for anti-viral therapy.

To the Editor: Rhinovirus wheezing illnesses during early childhood are strongly linked with development of asthma later in life [1]. Indeed, rhinovirus infection in the first 3 years of life is associated with an almost 10-fold increase in risk for asthma at age 6 years [1]. The exact mechanism by which rhinovirus elicits a pro-asthmatic propensity in young children is largely unknown, but is purportedly related to a viral-induced T-helper cell (Th)2 airway inflammatory response [2]. Interestingly, the discovery of interactions between epithelial innate immunity and adaptive allergic responses has unveiled new potential links between rhinovirus and asthma. Of particular interest is the epithelial-derived cytokine thymic stromal lymphopoietin (TSLP), which is considered a “master Th2 cytokine” because it primes the differentiation of naive T0 cells into Th2 lymphocytes via activation of antigen presenting cells [3]. TSLP is induced by rhinovirus infection or by exposure to double stranded (ds)RNA (viral surrogate) in the lungs of allergic mice [4], and in human bronchial epithelial cells (HBEC) [5]. Together, these data suggest that TSLP may be the missing link between innate antiviral epithelial immunity and the Th2 immune response characteristic of asthma. This cross-sectional preliminary study aimed to investigate whether rhinovirus infections that occur naturally during the first 3 years of life are associated with elevated airway TSLP levels and enhanced Th2 responses, which may potentially facilitate the establishment of rhinovirus-induced pro-asthmatic changes during early childhood. We measured nasal airway TSLP, Th2 cytokines and antiviral responses in nasal washes obtained from newborns, infants and toddlers (≤3 years) with PCR-confirmed acute …

Chronic airway diseases are characterized by inflammation and mucus overproduction. The MUC5AC mucin gene is upregulated by the proinflammatory cytokine interleukin-1 β (IL-1β) via activation of cAMP response element-binding protein (CREB) in the NCI-H292 cancer cell line and nuclear factor-κB (NF-κB) in the HBE1 transformed cell line, with each transcription factor binding to a cognate cis site in the proximal or distal region, respectively, of the MUC5AC promoter. We utilized primary differentiated human bronchial epithelial (HBE) and A549 lung adenocarcinoma cells to further investigate the contributions of CREB and NF-κB subunits to the IL-1β-induced upregulation of MUC5AC. Data show that ligand binding of IL-1β to the IL-1β receptor is required to increase MUC5AC mRNA abundance. Chromatin immunoprecipitation analyses show direct binding of CREB to the previously identified cAMP response element site and binding of p65 and p50 subunits to a novel NF-κB site in a mucin-regulatory domain in the proximal promoter and to a previously identified NF-κB site in the distal promoter. P50 binds to both NF-κB sites at 1 h following IL-1β exposure, but is replaced at 2 h by p65 in A549 cells and by a p50/p65 heterodimer in HBE cells. Thus IL-1β activates multiple domains in the MUC5AC promoter but exhibits some cell-specific responses, highlighting the complexity of MUC5AC transcriptional regulation. Data show that dexamethasone, a glucocorticoid that transcriptionally represses MUC5AC gene expression under constitutive conditions, also represses IL-1β-mediated upregulation of MUC5AC gene expression. A further understanding of mechanisms mediating MUC5AC regulation should lead to a honing of therapeutic approaches for the treatment of mucus overproduction in inflammatory lung diseases.

The human cytomegalovirus (HCMV) viral mitochondria-localized inhibitor of apoptosis (vMIA) protein, traffics to mitochondria-associated membranes (MAM), where the endoplasmic reticulum (ER) contacts the outer mitochondrial membrane (OMM). vMIA association with the MAM has not been visualized by imaging. Here, we have visualized this by using a combination of confocal and superresolution imaging. Deconvolution of confocal microscopy images shows vMIA localizes away from mitochondrial matrix at the Mitochondria-ER interface. By gated stimulated emission depletion (GSTED) imaging, we show that along this interface vMIA is distributed in clusters. Through multicolor, multifocal structured illumination microscopy (MSIM), we find vMIA clusters localize away from MitoTracker Red, indicating its OMM localization. GSTED and MSIM imaging show vMIA exists in clusters of ~100–150 nm, which is consistent with the cluster size determined by Photoactivated Localization Microscopy (PALM). With these diverse superresolution approaches, we have imaged the clustered distribution of vMIA at the OMM adjacent to the ER. Our findings directly compare the relative advantages of each of these superresolution imaging modalities for imaging components of the MAM and sub-mitochondrial compartments. These studies establish the ability of superresolution imaging to provide valuable insight into viral protein location, particularly in the sub-mitochondrial compartments, and into their clustered organization.

Background Thymic stromal lymphoproetin (TSLP) is a cytokine secreted by the airway epithelium in response to respiratory viruses and it is known to promote allergic Th2 responses in asthma. This study investigated whether virally-induced secretion of TSLP is directional in nature (apical vs. basolateral) and/or if there are TSLP-mediated effects occurring at both sides of the bronchial epithelial barrier in the asthmatic state. Methods Primary human bronchial epithelial cells (HBEC) from control (n = 3) and asthmatic (n = 3) donors were differentiated into polarized respiratory tract epithelium under air-liquid interface (ALI) conditions and treated apically with dsRNA (viral surrogate) or TSLP. Sub-epithelial effects of TSLP were examined in human airway smooth muscle cells (HASMC) from normal (n = 3) and asthmatic (n = 3) donors. Clinical experiments examined nasal airway secretions obtained from asthmatic children during naturally occurring rhinovirus-induced exacerbations (n = 20) vs. non-asthmatic uninfected controls (n = 20). Protein levels of TSLP, CCL11/eotaxin-1, CCL17/TARC, CCL22/MDC, TNF-α and CXCL8 were determined with a multiplex magnetic bead assay. Results Our data demonstrate that: 1) Asthmatic HBEC exhibit an exaggerated apical, but not basal, secretion of TSLP after dsRNA exposure; 2) TSLP exposure induces unidirectional (apical) secretion of CCL11/eotaxin-1 in asthmatic HBEC and enhanced CCL11/eotaxin-1 secretion in asthmatic HASMC; 3) Rhinovirus-induced asthma exacerbations in children are associated with in vivo airway secretion of TSLP and CCL11/eotaxin-1. Conclusions There are virally-induced TSLP-driven secretory immune responses at both sides of the bronchial epithelial barrier characterized by enhanced CCL11/eotaxin-1 secretion in asthmatic airways. These results suggest a new model of TSLP-mediated eosinophilic responses in the asthmatic airway during viral-induced exacerbations.

The human cytomegalovirus UL37 exon 1 gene encodes the immediate early protein pUL37x1 that has antiapoptotic and regulatory activities. Deletion mutagenesis analysis of the open reading frame of UL37x1 identified two domains that are necessary and sufficient for its antiapoptotic activity. These domains are confined within the segments between amino acids 5 to 34, and 118 to 147, respectively. The first domain provides the targeting of the protein to mitochondria. Direct PCR sequencing of UL37 exon 1 amplified from 26 primary strains of human cytomegalovirus demonstrated that the promoter, polyadenylation signal, and the two segments of pUL37x1 required for its antiapoptotic function were invariant in all sequenced strains and identical to those in AD169 pUL37x1. In total, UL37 exon 1 varies between 0.0 and 1.6% at the nucleotide level from strain AD169. Only 11 amino acids were found to vary in one or more viral strains, and these variations occurred only in the domains of pUL37x1 dispensable for its antiapoptotic function. We infer from this remarkable conservation of pUL37x1 in primary strains that this protein and, probably, its antiapoptotic function are required for productive replication of human cytomegalovirus in humans.

The human cytomegalovirus (HCMV) UL36–38 immediate-early (IE) locus encodes the UL37 exon 1 (pUL37x1) and UL37 (gpUL37) regulatory proteins, which have anti-apoptotic activities. pUL37x1 shares its entire sequence, including a hydrophobic leader and an acidic domain, with the exception of one residue, with the amino terminus of gpUL37. gpUL37 has, in addition, uniqueN-linked glycosylation, transmembrane and cytosolic domains. A rabbit polyvalent antiserum was generated against residues 27–40 in the shared amino-terminal domain and a mouse polyvalent antiserum was generated against the full-length protein to study trafficking of individual UL37 proteins in human cells that transiently expressed gpUL37 or pUL37x1. Co-localization studies by confocal laser scanning microscopy detected trafficking of gpUL37 and pUL37x1 from the endoplasmic reticulum to the Golgi apparatus in permissive U373 cells and in human diploid fibroblasts (HFF). Trafficking of gpUL37 to the cellular plasma membrane was detected in unfixed HFF cells. FLAG-tagged gpUL37 trafficked similarly through the secretory apparatus to the plasma membrane. By using confocal microscopy and immunoblotting of fractionated cells, gpUL37 and pUL37x1 were found to co-localize with mitochondria in human cells. This unconventional dual trafficking pattern through the secretory apparatus and to mitochondria is novel for herpesvirus IE regulatory proteins.

The human cytomegalovirus (HCMV) UL37 immediate-early gene is predicted to encode a type I membrane-bound glycoprotein, gpUL37. Following expression of the UL37 open reading frame in vitro, its signals for translocation and N-glycosylation were recognized by microsomal enzymes. Its orientation in the microsomes is that of a type I protein. gpUL37 produced in HCMV-infected human cells was selectively immunoprecipitated by rabbit polyvalent antiserum generated against the predicted unique domains of the UL37 open reading frame and migrated as an 83- to 85-kDa protein. Tunicamycin treatment, which inhibits N-glycosylation, increased the rate of migration of the UL37 protein to 68 kDa, verifying its modification by N-glycosylation in HCMV-infected cells. Consistent with this observation, gpUL37 was found to be resistant to digestion with either endoglycosidase F or H but sensitive to peptide N-glycosidase F digestion. These results suggested that gpUL37 is N-glycosylated and processed in both the endoplasmic reticulum (ER) and the Golgi apparatus. Direct demonstration of passage of gpUL37 through the ER and the Golgi was obtained by confocal microscopy. gpUL37 colocalized with protein disulfide isomerase, a protein resident in the ER, and with a Golgi protein. Subcellular fractionation of HCMV-infected cells demonstrated that gpUL37 is an integral membrane protein. Taken together, our results demonstrate that the HCMV gpUL37 immediate-early regulatory protein is a type I integral membrane N-glycoprotein which traffics through the ER and the Golgi network.

The human cytomegalovirus (HCMV) major immediate-early promoter (MIEP) is one of the first promoters to activate upon infection. To examine HCMV MIEP tissue-specific expression, transgenic mice were established containing the lacZ gene regulated by the MIEP (nucleotides -670 to +54). In the transgenic mice, lacZ expression was demonstrated in 19 of 29 tissues tested by histochemical and immunochemical analyses. These tissues included brain, eye, spinal cord, esophagus, stomach, pancreas, kidney, bladder, testis, ovary, spleen, salivary gland, thymus, bone marrow, skin, cartilage, and cardiac, striated and smooth muscles. Although expression was observed in multiple organs, promoter activity was restricted to specific cell types. The cell types which demonstrated HCMV MIEP expression included retinal cells of the eye, ductile cells of the salivary gland, exocrine cells of the pancreas, mucosal cells of the stomach and intestine, neuronal cells of the brain, muscle fibers, thecal cells of the corpus luteum, and Leydig and sperm cells of the testis. These observations indicate that the HCMV MIEP is not a pan-specific promoter and that the majority of expressing tissues correlate with tissues naturally infected by the virus in the human host.

The major immediate-early promoter (MIEP) of human, cytomegalovirus (HCMV) constitutes a primary genetic switch for viral activation. In this study, regulation of the enhancer-containing segment (nucleotides -670 to +54) of the HCMV MIEP attached to the 1acZ reporter gene was examined in the developing embryos of transgenic mice to identify temporal and tissue-specific expression. We find that the transgene reporter is first detected as a dorsal stripe of expression in the neural folds of embryos at day 8.5 postcoitum (p.c.). A broad expression pattern is exhibited in embryos at day 9.5 p.c. This pattern becomes more restricted by day 10.5 p.c. as organogenesis progresses. By day 14.5 p.c., prominent expression is observed in a subpopulation of central nervous system cells and spinal ganglia, endothelial cells, muscle, skin, thyroid, parathyroid, kidney, lung, liver, and gut cells, and the pancreas and submandibular and pituitary glands. This distribution pattern is discussed in relation to human congenital HCMV infection. These results suggest that the transcriptional activity of the HCMV MIEP may determine in part, the ability of the virus to specifically target developing fetal tissues in utero.

Human cytomegalovirus (HCMV) encodes multiple regulatory proteins at immediate early (IE) times of infection. Ancillary IE proteins are encoded by the UL36-38, UL115-119, TRS1/IRS1 and US3 loci. In contrast to the major IE nuclear proteins, several of the ancillary IE proteins are type I integral membrane N-glycoproteins. Nonetheless, all of the ancillary proteins examined to date have the ability to regulate nuclear gene expression and to interact cooperatively. Significantly, products from the UL36-38 and TRS1/IRSI IE loci as well as products from the MIE locus are required for HCMV ori-Lyt DNA replication. Moreover, the products of the UL36 and UL37 IE genes are essential for HCMV growth in human cells. Finally, one ancillary IE glycoprotein, gpUS3, is known to have a nonregulatory function; that is, gpUS3 binds and retains major histocompatibility complex class I heavy chains in the endoplasmic reticulum, thereby inhibiting antigen presentation. Thus, the functional presence of multiple IE proteins is required during HCMV replication both in vitro and in vivo to orchestrate necessary events for HCMV replication as well as for the survival of the infected host cell.

Congenital human cytomegalovirus (HCMV) infection can cause severe brain abnormalities. Apoptotic HCMV-infected brain cells have been detected in a congenitally infected infant. In biologically relevant human neural precursor cells (hNPCs), cultured in physiological oxygen tensions, HCMV infection (m.o.i. of 1 or 3) induced cell death within 3 days post-infection (p.i.) and increased thereafter. Surprisingly, its known anti-apoptotic genes, including the potent UL37 exon 1 protein (pUL37x1) or viral mitochondria-localized inhibitor of apoptosis (vMIA), which protects infected human fibroblasts (HFFs) from apoptosis and from caspase-independent, mitochondrial serine protease-mediated cell death, were expressed by 2 days p.i. Consistent with this finding, an HCMV UL37x1 mutant, BADsubstitutionUL37x1 (BADsubUL37x1) induced cell death in hNPCs (m.o.i. = 1) to level which were indistinguishable from parental virus (BADwild-type)-infected hNPCs. Surprisingly, although BADsubUL37x1 is growth defective in permissive HFFs, it produced infectious progeny in hNPCs with similar kinetics and to levels comparable to BADwild-type-infected hNPCs (m.o.i. = 1). While delayed at a lower multiplicity (m.o.i. = 0.3), the BADsubUL37x1 mutant reached similar levels to revertant within 12 days, in contrast to its phenotype in HFFs. The inability of pUL37x1/vMIA to protect hNPCs from HCMV-induced cell death did not result from impaired trafficking as pUL37x1/vMIA trafficked efficiently to mitochondria in transfected hNPCs and in HCMV-infected hNPCs. These results establish that pUL37x1/vMIA, although protective in permissive HFFs, does not protect HCMV-infected hNPCs from cell death under physiologically relevant oxygen tensions. They further suggest that pUL37x1/vMIA is not essential for HCMV growth in hNPCs and has different cell type-specific roles.

Endoplasmic reticulum-mitochondrial contacts, known as mitochondria-associated membranes, regulate important cellular functions including calcium signaling, bioenergetics, and apoptosis. Human cytomegalovirus is a medically important herpesvirus whose growth increases energy demand and depends upon continued cell survival. To gain insight into how human cytomegalovirus infection affects endoplasmic reticulum-mitochondrial contacts, we undertook quantitative proteomics of mitochondria-associated membranes using differential stable isotope labeling by amino acids in cell culture strategy and liquid chromatography-tandem MS analysis. This is the first reported quantitative proteomic analyses of a suborganelle during permissive human cytomegalovirus infection. Human fibroblasts were uninfected or human cytomegalovirus-infected for 72 h. Heavy mitochondria-associated membranes were isolated from paired unlabeled, uninfected cells and stable isotope labeling by amino acids in cell culture-labeled, infected cells and analyzed by liquid chromatography-tandem MS analysis. The results were verified by a reverse labeling experiment. Human cytomegalovirus infection dramatically altered endoplasmic reticulum-mitochondrial contacts by late times. Notable is the increased abundance of several fundamental networks in the mitochondria-associated membrane fraction of human cytomegalovirus-infected fibroblasts. Chaperones, including HSP60 and BiP, which is required for human cytomegalovirus assembly, were prominently increased at endoplasmic reticulum-mitochondrial contacts after infection. Minimal translational and translocation machineries were also associated with endoplasmic reticulum-mitochondrial contacts and increased after human cytomegalovirus infection as were glucose regulated protein 75 and the voltage dependent anion channel, which can form an endoplasmic reticulum-mitochondrial calcium signaling complex. Surprisingly, mitochondrial metabolic enzymes and cytosolic glycolytic enzymes were confidently detected in the mitochondria-associated membrane fraction and increased therein after infection. Finally, proapoptotic regulatory proteins, including Bax, cytochrome c, and Opa1, were augmented in endoplasmic reticulum-mitochondrial contacts after infection, suggesting attenuation of proapoptotic signaling by their increased presence therein. Together, these results suggest that human cytomegalovirus infection restructures the proteome of endoplasmic reticulum-mitochondrial contacts to bolster protein translation at these junctions, calcium signaling to mitochondria, cell survival, and bioenergetics and, thereby, allow for enhanced progeny production.

By exploiting host cell machineries, viruses provide powerful tools for gaining insight into cellular pathways. Proteins from two unrelated viruses, human CMV (HCMV) and HCV, are documented to traffic sequentially from the ER into mitochondria, probably through the mitochondria-associated membrane (MAM) compartment. The MAM are sites of ER-mitochondrial contact enabling the direct transfer of membrane bound lipids and the generation of high calcium (Ca2+) microdomains for mitochondria signalling and responses to cellular stress. Both HCV core protein and HCMV UL37 proteins are associated with Ca2+ regulation and apoptotic signals. Trafficking of viral proteins to the MAM may allow viruses to manipulate a variety of fundamental cellular processes, which converge at the MAM, including Ca2+ signalling, lipid synthesis and transfer, bioenergetics, metabolic flow, and apoptosis. Because of their distinct topologies and targeted MAM sub-domains, mitochondrial trafficking (albeit it through the MAM) of the HCMV and HCV proteins predictably involves alternative pathways and, hence, distinct targeting signals. Indeed, we found that multiple cellular and viral proteins, which target the MAM, showed no apparent consensus primary targeting sequences. Nonetheless, these viral proteins provide us with valuable tools to access the poorly characterized MAM compartment, to define its cellular constituents and describe how virus infection alters these to its own end. Furthermore, because proper trafficking of viral proteins is necessary for their function, discovering the requirements for MAM to mitochondrial trafficking of essential viral proteins may provide novel targets for the rational design of anti-viral drugs.

During permissive in vitro infection, the human cytomegalovirus (HCMV) UL36-38 and US3 immediate-early (IE) regions give rise to multiple distinct species of RNA in a temporally regulated manner. We have compared the temporally regulated expression of the UL36-38 and US3 regions with that of the well-characterized major IE (MIE) region. Northern (RNA) blot hybridizations with antisense RNA probes were used to examine RNA isolated from infected cells at IE, early, and late times after infection and from cells infected in the presence of anisomycin (used to block de novo viral protein synthesis) or in the presence of phosphonoformate (used to block HCMV DNA synthesis). Different US3 region transcripts were expressed in the cytoplasm during the IE and late phases of infection, with kinetics similar to those of the MIE region. In contrast, various cytoplasmic transcripts from the UL36-38 region were expressed during each of the IE, early, and late phases of infection, including some expressed from IE through late times. The levels of steady-state RNA from the US3 and MIE regions were increased dramatically by infection in the presence of anisomycin, predominantly because of an increase in multiply spliced transcripts. Two of the three UL36-38 IE transcripts were largely unaffected by anisomycin and were expressed abundantly throughout infection, but a third, multiply spliced UL36-38 IE transcript was abundant only during infection in the presence of anisomycin. Nuclear, cytoplasmic, and polysome-associated transcripts from the three IE regions were not significantly different qualitatively or quantitatively. These results suggest that posttranscriptional controls at the levels of nuclear retention or polysome exclusion of transcripts are not operative for the IE region genes. Overall, these results indicate common features of expression of US3, MIE, and UL36-38, in addition to distinctive expression of the UL36-38 region during all temporal phases of expression.

We have characterized the expression of human cytomegalovirus immediate early (IE) genes encoding the UL36-38 open reading frames. Single-stranded RNA hybridization probes, transcribed from UL36–38 cDNAs or smaller exon-specific clones, were used in analyses of RNA isolated during various phases of infection in the presence and absence of inhibitors of protein and HCMV DNA synthesis. There are three IE transcripts that arise by the use of alternative promoters and splicing signals. Different patterns of expression are observed for each of the three IE RNAs during permissive infection. Comparison of nucleotide sequences of UL36–38 IE cDNAs with those of the genomic sequence verified the use of polyadenylation and splice signals. An additional abundant early class RNA, totally encoded within the UL36–38 region, was found to be expressed from a different promoter than the IE RNAs. In vitro transcription and translation of the IE cDNAs were used to express the UL36–38 IE proteins. These studies define the structure of the UL36–38 gene products and demonstrate that expression from the region is both qualitatively and temporally complex.

Increasingly mechanistic virology studies require dependable and sensitive methods for isolating purified organelles containing functional cellular sub-domains. The mitochondrial network is, in part, closely apposed to the endoplasmic reticulum (ER). The mitochondria-associated membrane (MAM) fraction provides direct physical contact between the ER and mitochondria. Characterization of the dual localization and trafficking of human cytomegalovirus (HCMV) UL37 proteins required establishing protocols in which the ER and mitochondria could be reliably separated. Because of its documented role in lipid and ceramide transfer from the ER to mitochondria, a method to purify MAM from infected cells was also developed. Two robust procedures were developed to efficiently isolate mitochondria, ER, and MAM fractions while providing the substantial protein yields from HCMV-infected primary fibroblasts and from transfected HeLa cells. Moreover, this unit includes a protocol that allows visualization of the mitochondria network disruption that occurs in permissively infected cells by their optimal resolution in Percoll gradients.

The human cytomegalovirus (HCMV) UL37 exon 1 protein (pUL37x1), also known as vMIA, is the predominant UL37 isoform during permissive infection. pUL37x1 is a potent antiapoptotic protein, which prevents cytochrome c release from mitochondria. The UL37x1 NH 2 -terminal bipartite localization signal, which remains uncleaved, targets UL37 proteins to the endoplasmic reticulum (ER) and then to mitochondria. Based upon our findings, we hypothesized that pUL37x1 traffics from the ER to mitochondria through direct contacts between the two organelles, provided by mitochondrion-associated membranes (MAMs). To facilitate its identification, we cloned and tagged the human phosphatidylserine synthase 1 (huPSS-1) cDNA, whose mouse homologue localizes almost exclusively in the MAM. Using subcellular fractionation of stable HeLa cell transfectants expressing mEGFP-huPSS-1, we found that HCMV pUL37x1 is present in purified microsomes, mitochondria, and MAM fractions. We further examined the trafficking of the full-length UL37 glycoprotein cleavage products, which divergently traffic either through the secretory apparatus or into mitochondria. Surprisingly, pUL37 NH2 and gpUL37 COOH were both detected in the ER and MAM fraction, even though only pUL37 NH2 is preferentially imported into mitochondria but gpUL37 COOH is not. To determine the sequences required for MAM importation, we examined pUL37x1 mutants that were partially defective for mitochondrial importation. Deletion mutants of the NH 2 -terminal UL37x1 mitochondrial localization signal were reduced in trafficking into the MAM, indicating partial overlap of MAM and mitochondrial targeting signals. Taken together, these results suggest that HCMV UL37 proteins traffic from the ER into the MAM, where they are sorted into either the secretory pathway or to mitochondrial importation.