Publications (10)46.13 Total impact
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Article: Murine cytomegalovirus targets transcription factor ATF4 to exploit the unfolded-protein response.
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ABSTRACT: The unfolded-protein response (UPR), activated by sensor molecules PERK, ATF6, and IRE1 to resolve endoplasmic reticulum (ER) stress, has emerged as a key target for host cells and viruses to control the infection outcomes. The UPR regulates ER protein folding, controls cell fate upon ER stress, and plays an important role in innate immunity. We and others have shown that human cytomegalovirus (HCMV) modulates the UPR. We show here that murine CMV (MCMV), the widely used CMV model for small animal infection, regulated the UPR in a manner similar to that of HCMV. This modulatory ability was triggered by virion entry and enhanced by viral immediate-early and early gene expression. Thus, while vulnerable at early times, MCMV became resistant to exogenous ER stress at late times of infection. MCMV activated the PERK-ATF4 pathway but only induced a subset of representative ATF4 targets at levels somewhat lower than those by the ER stress inducer tunicamycin. Moreover, MCMV induced ER chaperone Bip but actively blocked IRE1-mediated Xbp1(s) protein accumulation. ATF4 depletion severely attenuated viral growth at a low multiplicity of infection by modestly reducing viral DNA synthesis and more pronouncedly inhibiting late gene transcription. Collectively, we show that the UPR is a conserved target of CMVs and identify ATF4, a key UPR component, as a factor critical for MCMV infection. This work sets the stage for using the MCMV model to explore the role of this stress response in CMV biology, particularly during infection of the host, which is difficult to study in HCMV.Journal of Virology 04/2012; 86(12):6712-23. · 5.40 Impact Factor -
Article: The human cytomegalovirus protein pUL38 suppresses endoplasmic reticulum stress-mediated cell death independently of its ability to induce mTORC1 activation.
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ABSTRACT: As obligate intracellular parasites, viruses not only hijack cellular machinery, they also deregulate host stress responses for their infection. Human cytomegalovirus (HCMV) modulates the endoplasmic reticulum (ER) stress response, due at least in part to the viral protein pUL38, and one of the consequences is to maintain the viability of infected cells. Consequently, pUL38-deficient virus induces premature cell death during infection. In addition, pUL38 activates mammalian target of rapamycin complex 1 (mTORC1), which may also antagonize other detrimental cellular stresses (N. J. Moorman et al., Cell Host Microbe 3:253-262, 2008). It remains elusive how pUL38 inhibition of cell death is related to mTORC1 activation. In this study, we defined the interplay of the two pUL38 activities. We constructed a series of pUL38 truncation mutants based on the secondary structure prediction and evolutionary conservation of its sequence. We found that the N-terminal 239 residues of pUL38 were necessary and sufficient to block cell death induced by pUL38-deficient virus or by the ER stress inducer tunicamycin. However, this pUL38 domain was unable to activate mTORC1 when expressed alone. Importantly, small-molecule inhibitors of mTORC1, rapamycin or torin 1, did not compromise pUL38 activity to block cell death in isolation or in virus infection. Expression of a constitutively active variant of an mTORC1 activator, Rheb (Ras homolog enriched in brain), could not prevent cell death induced by pUL38-deficient virus. Collectively, we provide genetic and biochemical evidence that pUL38 prevents ER stress-induced cell death independent of its role in mTORC1 activation.Journal of Virology 06/2011; 85(17):9103-13. · 5.40 Impact Factor -
Article: The human cytomegalovirus gene UL79 is required for the accumulation of late viral transcripts.
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ABSTRACT: In this study, we adopted a conditional protein genetic approach to characterize the role of the human cytomegalovirus (HCMV) gene UL79 during virus infection. We constructed ADddUL79, a recombinant HCMV in which the annotated UL79 open reading frame (ORF) was tagged with the destabilization domain of a highly unstable variant of the human FKBP12 protein (ddFKBP). The ddFKBP domain targets the tagged protein for rapid proteasomal degradation, but the synthetic ligand Shield-1 can stabilize ddFKBP, allowing accumulation of the tagged protein. ADddUL79 failed to replicate without Shield-1, but it grew at wild-type levels with Shield-1 or in human foreskin fibroblasts overexpressing hemagglutinin (HA)-tagged UL79 (HF-UL79HA cells), indicating an essential role of UL79 and the effectiveness of this approach. Without Shield-1, representative immediate-early and early viral proteins as well as viral DNA accumulated normally, but late transcripts and proteins were markedly reduced. UL79 was transcribed with early-late kinetics, which was also regulated via a positive-feedback loop. Using HF-UL79HA cells, we found that the UL79 protein localized to viral replication compartments during HCMV infection. Finally, we created a second UL79 mutant virus (ADinUL79(stop)) in which the UL79 ORF was disrupted by a stop codon mutation and found that ADinUL79(stop) phenocopied ADddUL79 under the destabilizing condition. Taking these results together, we conclude that UL79 acts after viral DNA replication to promote the accumulation of late viral transcripts. Importantly, the comparative analysis of ADddUL79 and ADinUL79(stop) viruses provide additional proof for the power of the protein stability-based conditional approach to dissect the role of viral factors in HCMV biology.Journal of Virology 03/2011; 85(10):4841-52. · 5.40 Impact Factor -
Article: Human cytomegalovirus protein pUL117 targets the mini-chromosome maintenance complex and suppresses cellular DNA synthesis.
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ABSTRACT: Modulation of host DNA synthesis is essential for many viruses to establish productive infections and contributes to viral diseases. Human cytomegalovirus (HCMV), a large DNA virus, blocks host DNA synthesis and deregulates cell cycle progression. We report that pUL117, a viral protein that we recently identified, is required for HCMV to block host DNA synthesis. Mutant viruses in which pUL117 was disrupted, either by frame-shift mutation or by a protein destabilization-based approach, failed to block host DNA synthesis at times after 24 hours post infection in human foreskin fibroblasts. Furthermore, pUL117-deficient virus stimulated quiescent fibroblasts to enter S-phase, demonstrating the intrinsic ability of HCMV to promote host DNA synthesis, which was suppressed by pUL117. We examined key proteins known to be involved in inhibition of host DNA synthesis in HCMV infection, and found that many were unlikely involved in the inhibitory activity of pUL117, including geminin, cyclin A, and viral protein IE2, based on their expression patterns. However, the ability of HCMV to delay the accumulation of the mini-chromosome maintenance (MCM) complex proteins, represented by MCM2 and MCM4, and prevent their loading onto chromatin, was compromised in the absence of pUL117. When expressed alone, pUL117 slowed cell proliferation, delayed DNA synthesis, and inhibited MCM accumulation. Knockdown of MCM proteins by siRNA restored the ability of pUL117-deficient virus to block cellular DNA synthesis. Thus, targeting MCM complex is one mechanism pUL117 employs to help block cellular DNA synthesis during HCMV infection. Our finding substantiates an emerging picture that deregulation of MCM is a conserved strategy for many viruses to prevent host DNA synthesis and helps to elucidate the complex strategy used by a large DNA virus to modulate cellular processes to promote infection and pathogenesis.PLoS Pathogens 01/2010; 6(3):e1000814. · 9.13 Impact Factor -
Article: Human cytomegalovirus protein pUL38 induces ATF4 expression, inhibits persistent JNK phosphorylation, and suppresses endoplasmic reticulum stress-induced cell death.
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ABSTRACT: The endoplasmic reticulum (ER) is a key organelle involved in sensing and responding to stressful conditions, including those resulting from infection of viruses, such as human cytomegalovirus (HCMV). Three signaling pathways collectively termed the unfolded protein response (UPR) are activated to resolve ER stress, but they will also lead to cell death if the stress cannot be alleviated. HCMV is able to modulate the UPR to promote its infection. The specific viral factors involved in such HCMV-mediated modulation, however, were unknown. We previously showed that HCMV protein pUL38 was required to maintain the viability of infected cells, and it blocked cell death induced by thapsigargin. Here, we report that pUL38 is an HCMV-encoded regulator to modulate the UPR. In infection, pUL38 allowed HCMV to upregulate phosphorylation of PKR-like ER kinase (PERK) and the alpha subunit of eukaryotic initiation factor 2 (eIF-2alpha), as well as induce robust accumulation of activating transcriptional factor 4 (ATF4), key components of the PERK pathway. pUL38 also allowed the virus to suppress persistent phosphorylation of c-Jun N-terminal kinase (JNK), which was induced by the inositol-requiring enzyme 1 pathway. In isolation, pUL38 overexpression elevated eIF-2alpha phosphorylation, induced ATF4 accumulation, limited JNK phosphorylation, and suppressed cell death induced by both thapsigargin and tunicamycin, two drugs that induce ER stress by different mechanisms. Importantly, ATF4 overexpression and JNK inhibition significantly reduced cell death in pUL38-deficient virus infection. Thus, pUL38 targets ATF4 expression and JNK activation, and this activity appears to be critical for protecting cells from ER stress induced by HCMV infection.Journal of Virology 03/2009; 83(8):3463-74. · 5.40 Impact Factor -
Article: The full-length protein encoded by human cytomegalovirus gene UL117 is required for the proper maturation of viral replication compartments.
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ABSTRACT: Previously, two large-scale mutagenic analyses showed that mutations in the human cytomegalovirus (HCMV) gene UL117 resulted in a defect in virus growth in fibroblasts. Early transcriptional analyses have revealed several mRNAs from the UL119-UL115 region; however, specific transcripts encoding UL117-related proteins have not been identified. In this study, we identified two novel transcripts arising from the UL117 gene locus, and we reported that the UL117 open reading frame encoded the full-length protein pUL117 (45 kDa) and the shorter isoform pUL117.5 (35 kDa) as the result of translation initiation at alternative in-frame ATGs. Both proteins were expressed with early kinetics, but pUL117 accumulated at a lower abundance relative to that of pUL117.5. During HCMV infection, both proteins localized predominantly to the nucleus, and the major fraction of pUL117 localized in viral nuclear replication compartments. We constructed mutant HCMV viruses in which the entire UL117 coding sequence was deleted or the expression of pUL117 was specifically abrogated. The growth of mutant viruses was significantly attenuated, indicating that pUL117 was required for efficient virus infection in fibroblasts. Cells infected with the pUL117-deficient mutant virus accumulated representative viral immediate-early proteins and early proteins normally. In the absence of pUL117, the accumulation of replicating viral DNA was reduced by no more than twofold at early times and was indistinguishable from that of the wild type at 72 h postinfection. Strikingly, there was a 12- to 24-h delay in the development of nuclear replication compartments and a marked delay in the expression of late viral proteins. We conclude that pUL117 acts to promote the development of nuclear replication compartments to facilitate viral growth.Journal of Virology 05/2008; 82(7):3452-65. · 5.40 Impact Factor -
Article: RNA interference against hepatitis B virus with endoribonuclease-prepared siRNA despite of the target sequence variations.
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ABSTRACT: RNA interference (RNAi) has proven to be very powerful in inhibiting hepatitis B virus (HBV) replication by cell culture and mouse model studies. We have previously reported that endoribonuclease-prepared short interfering RNAs (esiRNAs) were able to inhibit HBV replication more efficiently than synthesized siRNAs. Here we tested the hypothesis that esiRNAs are able to inhibit gene expression with limited mutations within the target region. Target sequences with different similarities to esiHBVP (esiRNA targeting the DNA polymerase and S antigen of Hepatitis B virus) were amplified and cloned into the 3' untranslated region of HBsAg, respectively. When the obtained expression vectors were co-transfected with esiHBVP into CHO cells, HBsAg expression was suppressed with same efficiency regardless of the target sequence similarities. In HepG2 cells, esiHP9 based on one of the amplified sequence that sharing 87% similarity to the target region suppressed HBsAg expression effectively and dose dependently. In vivo experiment showed that a single dose of 5 microg esiHP9 was able to reduce HBsAg and HBeAg level in the mouse sera by 88 and 77% despite of its 87% similarity to the target sequence, which was as good as esiHBVP that is 100% similar to the target sequence. All the data suggest that esiRNA can tolerate limited target sequence variations without losing its inhibitory capacity. It would be very helpful to suppress virus replication by RNAi despite of their high mutation rate.Virus Research 06/2007; 126(1-2):172-8. · 2.94 Impact Factor -
Article: EsiRNAs inhibit Hepatitis B virus replication in mice model more efficiently than synthesized siRNAs.
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ABSTRACT: RNA interference (RNAi) has been proved to be a promising strategy to combat Hepatitis B virus (HBV) infection by way of cell culture and animal model studies. In this work, esiRNAs (endoribonuclease-prepared siRNAs) targeting all of the four open reading frames (ORFs) of HBV genome were prepared. In vitro experiment showed that esiHBVP suppressed HBsAg expression most effectively. Its capacity to suppress HBV replication in vivo was then tested. A single dose of 1 microg esiHBVP was able to reduce HBsAg and HBeAg level in the mouse serum by 90 and 89% one day after injection, while the same amount of chemically synthesized siRNA only reduced that by 33 and 45%. Immunostaining of HBcAg showed that esiHBVP inhibited HBcAg expression more potently than chemically synthesized siRNA. Quantification of HBV DNA in the mouse serum showed 1 microg eiHBVP treatment reduced serum HBV DNA copy number to 18% that of the untreated control, while 1 microg siRNA treatment only reduced that to 63%. In conclusion, the data presented here proved that esiRNA is much more efficient in suppressing HBV replication than chemically synthesized siRNA, and it might be a better therapeutic agent to fight against HBV infection.Virus Research 07/2006; 118(1-2):150-5. · 2.94 Impact Factor -
Article: esiRNAs purified with chromatography suppress homologous gene expression with high efficiency and specificity.
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ABSTRACT: Many preclinical studies have shown RNA interference (RNAi) as a new promising way to treat various human diseases including cancer and virus infection and there is an increasing demand for the large-scale preparation of short interfering RNAs (siRNAs) at low cost. Data are accumulating to show that endoribonuclease-prepared siRNAs (esiRNAs) are superior to chemically synthesized siRNAs in terms of expense, efficiency, and specificity. Yet all procedures available for esiRNA purification were designed to produce small amount of siRNAs for laboratory use. In this article, a new method of purification of esiRNAs based on ion exchange chromatography and size exclusion chromatography is reported. The esiRNAs prepared with this method are shown here to be of high purity and specifically suppress homologous gene expression without activating interferon response and with higher efficiency than chemically synthesized siRNAs. We can expect that the new method can be scaled up easily to provide large quantities of esiRNAs to meet the requirement of preclinical and clinical studies.Molecular Biotechnology 12/2005; 31(3):203-9. · 2.17 Impact Factor -
Article: Expression and purification of the carboxyl terminus domain of Schizosaccharomyces pombe dicer in Escherichia coli.
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ABSTRACT: The carboxyl terminus domain of Schizosaccharomyces pombe dicer (yDicerC) was expressed in Escherichia coli as an MBP-fusion protein (MBP-yDicerC). When the E. coli strain was cultured and induced at 25 degrees C, the MBP-yDicerC was partly expressed in the soluble fraction. It was then purified by two step affinity chromatography with amylose resin and Ni-NTA His Bind(R) resin. The purified MBP-yDicerC showed double-strand RNA digestion activity. siRNA-like products about 22-nt in length were generated.Protein and Peptide Letters 06/2005; 12(4):311-4. · 1.94 Impact Factor
Top co-authors
Top Journals
Institutions
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2008–2012
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Washington University in St. Louis
- Department of Molecular Microbiology
Saint Louis, MO, USA
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2005–2007
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Fudan University
- Department of Biochemistry
Shanghai, Shanghai Shi, China
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