Biomedical subjects

Fenyong Liu

Publications and source records attributed to Fenyong Liu.

At least 19 recordsLinked to original sources

PubMed2006

Infection of human cytomegalovirus in cultured human gingival tissue.

BACKGROUND: Human cytomegalovirus (HCMV) infection in the oral cavity plays an important role in its horizontal transmission and in causing viral-associated oral diseases such as gingivitis. However, little is currently known about HCMV pathogenesis in oral mucosa, partially because HCMV infection is primarily limited to human cells and few cultured tissue or animal models are available for studying HCMV infection. RESULTS: In this report, we studied the infection of HCMV in a cultured gingival tissue model (EpiGingival, MatTek Co.) and investigated whether the cultured tissue can be used to study HCMV infection in the oral mucosa. HCMV replicated in tissues that were infected through the apical surface, achieving a titer of at least 300-fold at 10 days postinfection. Moreover, the virus spread from the apical surface to the basal region and reduced the thickness of the stratum coreum at the apical region. Viral proteins IE1, UL44, and UL99 were expressed in infected tissues, a characteristic of HCMV lytic replication in vivo. Studies of a collection of eight viral mutants provide the first direct evidence that a mutant with a deletion of open reading frame US18 is deficient in growth in the tissues, suggesting that HCMV encodes specific determinants for its infection in oral mucosa. Treatment by ganciclovir abolished viral growth in the infected tissues. CONCLUSION: These results suggest that the cultured gingival mucosa can be used as a tissue model for studying HCMV infection and for screening antivirals to block viral replication and transmission in the oral cavity.

Adult↗

PubMed2006

Engineered external guide sequences are highly effective in inducing RNase P for inhibition of gene expression and replication of human cytomegalovirus.

External guide sequences (EGSs), which are RNA molecules derived from natural tRNAs, bind to a target mRNA and render the mRNA susceptible to hydrolysis by RNase P, a tRNA processing enzyme. Using an in vitro selection procedure, we have previously generated EGS variants that efficiently direct human RNase P to cleave a target mRNA in vitro. In this study, a variant was used to target the overlapping region of the mRNAs encoding human cytomegalovirus (HCMV) essential transcription regulatory factors IE1 and IE2. The EGS variant was approximately 25-fold more active in inducing human RNase P to cleave the mRNA in vitro than the EGS derived from a natural tRNA. Moreover, a reduction of 93% in IE1/IE2 gene expression and a reduction of 3000-fold in viral growth were observed in HCMV-infected cells that expressed the variant, while cells expressing the tRNA-derived EGS exhibited a reduction of 80% in IE1/IE2 expression and an inhibition of 150-fold in viral growth. Our results provide the first direct evidence that EGS variant is highly effective in blocking HCMV gene expression and growth and furthermore, demonstrate the feasibility of developing effective EGS RNA variants for anti-HCMV applications by using in vitro selection procedures.

Cells, Cultured↗

PubMed2005

Effective inhibition of human cytomegalovirus gene expression and growth by intracellular expression of external guide sequence RNA.

RNase P complexed with external guide sequence (EGS) represents a novel nucleic-acid-based gene interference approach to modulate gene expression. In this study, a functional EGS RNA was constructed to target the overlapping mRNA region of two human cytomegalovirus (HCMV) capsid proteins, the capsid scaffolding protein (CSP) and assemblin. The EGS RNA was shown to be able to direct human RNase P to cleave the target mRNA sequence efficiently in vitro. A reduction of approximately 75%-80% in the mRNA and protein expression levels of both CSP and assemblin and a reduction of 800-fold in viral growth were observed in human cells that expressed the functional EGS, but not in cells that either did not express the EGS or produced a "disabled" EGS that carried nucleotide mutations that precluded RNase P recognition. The action of the EGS is specific as the RNase P-mediated cleavage only reduces the expression of the CSP and assemblin but not other viral genes examined. Further studies of the antiviral effects of the EGS indicate that the expression of the functional EGS has no effect on HCMV genome replication but blocks viral capsid maturation, consistent with the notion that CSP and assemblin play essential roles in HCMV capsid formation. Our study provides the first direct evidence that EGS RNAs effectively inhibit HCMV gene expression and growth. Moreover, these results demonstrate the utility of EGS RNAs in gene therapy applications, including the treatment of HCMV infection by inhibiting the expression of virus-encoded essential proteins.

Animals↗

PubMed2005

Using DNA microarray to study human cytomegalovirus gene expression.

DNA microarray technology has become one of the most widely used tools for functional genomics and is playing an ever increasing role in the study of viral infections and host-pathogen interactions. This paper describes the development of an oligonucleotide microarray representing all the predicted open reading frames of the human cytomegalovirus (HCMV) and an established protocol for simultaneously measuring the expression of all HCMV genes. To evaluate the performance of the HCMV array, human foreskin fibroblasts were either mock infected or infected with the HCMV AD169 or Toledo strains. Hybridizations were performed to determine the level of detection of HCMV transcripts from both the AD169 and Toledo strains and to assess reproducibility within and between slides. Overall, approximately 95% of the predicted HCMV genes produced detectable levels of mRNA, with median signal to noise and signal to background ratios of 41 and 14, respectively. Scatter plots of samples within an array and between two arrays resulted in average linear regressions above 0.95 and 0.9, respectively, indicating that data from the arrays are highly reproducible. In addition, transcripts from genes found in the Toledo strain but not in AD169 were specifically detected.

Cell Line↗

PubMed2005

Dissecting human cytomegalovirus gene function and capsid maturation by ribozyme targeting and electron cryomicroscopy.

Human CMV (HCMV) is the leading viral cause of birth defects and causes one of the most common opportunistic infections among transplant recipients and AIDS patients. Cleavage of internal scaffolding proteins by the viral protease (Pr) occurs during HCMV capsid assembly. To gain insight into the mechanism of HCMV capsid maturation and the roles of the Pr in viral replication, an RNase P ribozyme was engineered to target the Pr mRNA and down-regulate its expression by >99%, generating premature Pr-minus capsids. Furthermore, scaffolding protein processing and DNA encapsidation were inhibited by 99%, and viral growth was reduced by 10,000-fold. 3D structural comparison of the Pr-minus and wild-type B capsids by electron cryomicroscopy, at an unprecedented 12.5-angstroms resolution, unexpectedly revealed that the structures are identical in their overall shape and organization. However, the Pr-minus capsid contains tenuous connections between the scaffold and the capsid shell, whereas the wild-type B capsid has extra densities in its core that may represent the viral Pr. Our findings indicate that cleavage of the scaffolding protein is not associated with the morphological changes that occur during capsid maturation. Instead, the protease appears to be required for DNA encapsidation and the subsequent maturation steps leading to infectious progeny. These results therefore provide key insights into an essential step of HCMV infection using an RNase P ribozyme-based inhibition strategy.

Blotting, Northern↗

PubMed2005

Human cytomegalovirus expresses novel microRNAs during productive viral infection.

MicroRNAs (miRNAs) are a large class of approximately 22-nucleotide non-coding RNAs that facilitate mRNA cleavage and translation repression through the RNA interference pathway. Until recently, miRNAs have been exclusively found in eukaryotic organisms. A non-immunogenic molecule requiring minimal genomic investment, these RNAs may offer an efficient means for viruses to modulate both their own and the host's gene expression during a productive viral infection. In this study we report that human cytomegalovirus (HCMV) expresses miRNAs during its productive lytic infection of four clinically relevant human cell types: fibroblast, endothelial, epithelial and astrocyte cells. The sequences of the miRNAs, expressed from the UL23 and US24 loci of the viral genome, were conserved among all HCMV strains examined and in chimpanzee cytomegalovirus. Furthermore, their expression was detected from both a laboratory-adapted strain and a clinical isolate of HCMV. The conservation of these miRNAs and their expression in different cell types suggests that they represent an evolutionarily primitive feature in the viral genome, and that virus-encoded miRNAs may be more common than previously believed.

Base Sequence↗

PubMed2005

Three-dimensional localization of the smallest capsid protein in the human cytomegalovirus capsid.

The smallest capsid proteins (SCPs) of the human herpesviruses differ substantially in size and sequence and are thought to impart some unique aspects of infection to their respective viruses. We used electron cryomicroscopy and antibody labeling to show that the 8-kDa SCP of human cytomegalovirus is attached only to major capsid protein subunits of the hexons, not the pentons. Thus, the SCPs of different herpesviruses illustrate that a protein can evolve significantly in sequence, structure, and function, while preserving its role in the architecture of the virus by binding to a specific partner in a specific oligomeric state.

Amino Acid Sequence↗

PubMed2005

Two gamma interferon-activated site-like elements in the human cytomegalovirus major immediate-early promoter/enhancer are important for viral replication.

Human cytomegalovirus (HCMV) infection directly initiates a signal transduction pathway that leads to activation of a large number of cellular interferon-stimulated genes (ISGs). Our previous studies demonstrated that two interferon response elements, the interferon-stimulated response element and gamma interferon-activated site (GAS), in the ISG promoters serve as HCMV response sites (VRS). Interestingly, two GAS-like VRS elements (VRS1) were also present in the HCMV major immediate-early promoter-enhancer (MIEP/E). In this study, the importance of these VRS elements in viral replication was investigated. We demonstrate that the expression of the major IE genes, IE1 and IE2, is interferon inducible. To understand the biological significance of this signal transduction pathway in HCMV major IE expression, the two VRS1 in the MIEP/E were mutated. Mutant HCMVs in which the VRS elements were deleted or that contained point mutations grew dramatically more slowly than wild-type virus at a low multiplicity of infection (MOI). Insertion of wild-type VRS1 into the mutant viral genome rescued the slow growth phenotype. Furthermore, the expression levels of major IE RNAs and proteins were greatly reduced during infection with the VRS mutants at a low MOI. HCMV microarray analysis indicated that infection of host cells with the VRS mutant virus resulted in a global reduction in the expression of viral genes. Collectively, these data demonstrate that the two VRS elements in the MIEP/E are necessary for efficient viral gene expression and replication. This study suggests that although the HCMV-initiated signal transduction pathway results in induction of cellular antiviral genes, it also functions to stimulate viral major IE gene expression. This might be a new viral strategy in which the pathway is used to regulate gene expression and play a role in reactivation.

Base Sequence↗

PubMed2005

Genetic analysis of cytomegalovirus by shuttle mutagenesis.

The genomes of Herpesviridae family members are among the largest of all viruses and therefore present a formidable challenge in understanding the roles of every gene in replication or pathogenesis. For example, murine cytomegalovirus (MCMV) has a genome of 230 kb that encodes more than 170 genes, many of which have unknown functions. Many techniques for the genetic analysis of a herpesvirus have been developed over the past two decades. One such procedure involves the use of a shuttle mutagenesis system, and it has successfully generated a pool of MCMV mutants that contained an engineered Tn3-type transposon inserted within their genome. The process of shuttle mutagenesis involves the construction of a genomic fragment library, transposon mutagenesis of the library, and generation of virus mutants through homologous recombination. This chapter details the methodologies required for implementing a Tn3-based shuttle mutagenesis system for construction of a mutant virus library.

Cytomegalovirus↗

PubMed2004

The cytomegalovirus m155 gene product subverts natural killer cell antiviral protection by disruption of H60-NKG2D interactions.

Natural killer (NK) cells are an important early mediator of host immunity to murine cytomegalovirus (MCMV) infection. However, MCMV has evolved mechanisms to elude recognition and clearance by NK cells. We have identified an MCMV immune evasion protein that impairs NKG2D-mediated NK cell antiviral activity. Infection of BALB/c 3T3 cells with the Smith strain of MCMV resulted in strong down-regulation of H60, a high affinity ligand for NKG2D, from the surface of virus-infected cells. The MCMV m155 protein specifically down-regulated H60 without affecting expression of the other known NKG2D ligands, RAE-1 and MULT-1. Treatment with the proteasome inhibitors lactacystin or epoxomicin reversed m155 down-regulation of H60. An MCMV mutant virus lacking m155 was severely attenuated in BALB/c mice; however, treatment with neutralizing anti-NKG2D monoclonal antibody or with NK-depleting anti-asialo GM1 antisera restored virulence of the mutant virus. Thus, down-regulation of H60 by m155 is a powerful mechanism of inhibiting NKG2D-mediated antiviral function.

Animals↗

PubMed2004

RNase P ribozyme inhibits cytomegalovirus replication by blocking the expression of viral capsid proteins.

By linking a guide sequence to the catalytic RNA subunit of RNase P (M1 RNA), we constructed a functional ribozyme (M1GS RNA) that targets the overlapping mRNA region of two human cytomegalovirus (HCMV) capsid proteins, the capsid scaffolding protein (CSP) and assemblin, which are essential for viral capsid formation. The ribozyme efficiently cleaved the target mRNA sequence in vitro. Moreover, a reduction of >85% in the expression of CSP and assemblin and a reduction of 4000-fold in viral growth were observed in the HCMV-infected cells that expressed the functional ribozyme. In contrast, there was no significant reduction in viral gene expression and growth in virus-infected cells that either did not express the ribozyme or produced a 'disabled' ribozyme carrying mutations that abolished its catalytic activity. Characterization of the effects of the ribozyme on the HCMV lytic replication cycle further indicates that the expression of the functional ribozyme specifically inhibits the expression of CSP and assemblin, and consequently blocks viral capsid formation and growth. Our results provide the direct evidence that RNase P ribozymes can be used as an effective gene-targeting agent for antiviral applications, including abolishing HCMV growth by blocking the expression of the virus-encoded capsid proteins.

Animals↗

PubMed2004

Effective inhibition of Rta expression and lytic replication of Kaposi's sarcoma-associated herpesvirus by human RNase P.

Ribonuclease P (RNase P) complexed with external guide sequence (EGS) represents a nucleic acid-based gene interference approach to knock-down gene expression. Unlike other strategies, such as antisense oligonucleotides, ribozymes, and RNA interference, the RNase P-based technology is unique because a custom-designed EGS molecule can bind to any complementary mRNA sequence and recruit intracellular RNase P for specific degradation of the target mRNA. In this study, we demonstrate that the RNase P-based strategy is effective in blocking gene expression and growth of Kaposi's sarcoma (KS)-associated herpesvirus (KSHV), the causative agent of the leading AIDS-associated neoplasms, such as KS and primary-effusion lymphoma. We constructed 2'-O-methyl-modified EGS molecules that target the mRNA encoding KSHV immediate-early transcription activator Rta, and we administered them directly to human primary-effusion lymphoma cells infected with KSHV. A reduction of 90% in Rta expression and a reduction of approximately 150-fold in viral growth were observed in cells treated with a functional EGS. In contrast, a reduction of <10% in the Rta expression and viral growth was found in cells that were either not treated with an EGS or that were treated with a disabled EGS containing mutations that preclude recognition by RNase P. Our study provides direct evidence that EGSs are highly effective in inhibiting KSHV gene expression and growth. Exogenous administration of chemically modified EGSs in inducing RNase P-mediated cleavage represents an approach for inhibiting specific gene expression and for treating human diseases, including KSHV-associated tumors.

Antiviral Agents↗

PubMed2004

Engineered RNase P ribozymes increase their cleavage activities and efficacies in inhibiting viral gene expression in cells by enhancing the rate of cleavage and binding of the target mRNA.

Engineered RNase P ribozymes are promising gene-targeting agents that can be used in both basic research and clinical applications. We have previously selected ribozyme variants for their activity in cleaving an mRNA substrate from a pool of ribozymes containing randomized sequences. In this study, one of the variants was used to target the mRNA encoding thymidine kinase (TK) of herpes simplex virus 1 (HSV-1). The variant exhibited enhanced cleavage and substrate binding and was at least 30 times more efficient in cleaving TK mRNA in vitro than the ribozyme derived from the wild type sequence. Our results provide the first direct evidence to suggest that a point mutation at nucleotide 95 of RNase P catalytic RNA from Escherichia coli (G(95) --> U(95)) increases the rate of cleavage, whereas another mutation at nucleotide 200 (A(200) --> C(200)) enhances substrate binding of the ribozyme. A reduction of about 99% in TK expression was observed in cells expressing the variant, whereas a 70% reduction was found in cells expressing the ribozyme derived from the wild type sequence. Thus, the RNase P ribozyme variant is highly effective in inhibiting HSV-1 gene expression. Our study demonstrates that ribozyme variants increase their cleavage activity and efficacy in blocking gene expression in cells through enhanced substrate binding and rate of cleavage. These results also provide insights into the mechanism of how RNase P ribozymes efficiently cleave an mRNA substrate and, furthermore, facilitate the development of highly active RNase P ribozymes for gene-targeting applications.

Base Sequence↗

PubMed2004

Identification of essential and non-essential genes of the guinea pig cytomegalovirus (GPCMV) genome via transposome mutagenesis of an infectious BAC clone.

We report application of a transposition methodology that allows the easy characterization and mutation of genes encoded on an infectious bacterial artificial chromosome (BAC) clone. We characterized mutants generated by transposome (Tn) mutagenesis of a BAC clone of guinea pig cytomegalovirus (GPCMV). A pool of Tn mutant GPCMV BACs were screened initially by restriction profile analysis to verify they were full-length, and subsequently GPCMV BAC DNA from individual mutants was transfected onto guinea pig lung fibroblast cells in order to generate virus. Tn GPCMV BAC mutants were classed as either essential or non-essential gene insertions, depending upon their ability to regenerate viable, replication-competent virus. Representative mutants were more fully characterized. Analysis by sequencing the Tn insertion site on the mutated BACs, and by regeneration of virus using transfection of guinea pig fibroblasts (GPL), demonstrated that a recombinant with a Tn insertion in the UL35 homolog gene (GP35) was a non-essential gene for viral replication in tissue culture. A mutant with an insertion in the UL46 homolog (GP46) was nonviable, a phenotype which could be rescued by homologous recombination of BAC DNA with wild-type UL46 sequences, suggesting an essential role of this putative capsid gene in virus replication.

Amino Acid Sequence↗

PubMed2004

Developing RNase P ribozymes for gene-targeting and antiviral therapy.

RNase P, a tRNA processing enzyme, contains both RNA and protein subunits. M1 RNA, the catalytic RNA subunit of RNase P from Escherichia coli, recognizes its target RNA substrate mainly on the basis of its structure and cleaves a double stranded RNA helix at the 5' end that resembles the acceptor stem and T-stem structure of its natural tRNA substrate. Accordingly, a guide sequence (GS) can be covalently attached to the M1 RNA to generate a sequence specific ribozyme, M1GS RNA. M1GS ribozyme can target any mRNA sequence of choice that is complementary to its guide sequence. Recent studies have shown that M1GS ribozymes efficiently cleave the mRNAs of herpes simplex virus 1 and human cytomegalovirus, and the BCR-ABL oncogenic mRNA in vitro and effectively reduce the expression of these mRNAs in cultured cells. Moreover, an in vitro selection scheme has been developed to select for M1 GS ribozyme variants with more efficient catalytic activity in cleaving mRNAs. When expressed in cultured cells, these selected ribozymes also show an enhance ability to inhibit viral gene expression and growth. These recent results demonstrate the feasibility of developing the M1GS ribozyme-based technology as a promising gene targeting approach for basic research and clinical therapeutic application.

Antiviral Agents↗

PubMed2004

Murine cytomegalovirus with a transposon insertional mutation at open reading frame m155 is deficient in growth and virulence in mice.

A pool of murine cytomegalovirus (MCMV) mutants was previously generated by using a Tn3-based transposon mutagenesis approach (X. Zhan, M. Lee, J. Xiao, and F. Liu, J. Virol. 74:7411-7421, 2000). In this study, one of the MCMV mutants, Rvm155, which contained the transposon insertion in open reading frame m155, was characterized in vitro for its replication in tissue culture and in vivo for its growth and virulence in immunodeficient SCID mice. Compared to the wild-type strain and a rescued virus that restored the m155 region, the mutant is significantly deficient in growth in many organs of the infected animals. At 21 days postinfection the titers of Rvm155 in the salivary glands, lungs, spleens, livers, and kidneys of the intraperitoneally infected SCID mice were lower than the titers of the wild-type virus and the rescued virus by 50-, 1,000-, 500-, 100-, and 500-fold, respectively. Moreover, the viral mutant was attenuated in killing the SCID mice, as none of the SCID mice that were intraperitoneally infected with Rvm155 died until 38 days postinfection while all the animals infected with the wild-type and rescued viruses died at 27 days postinfection. Our results provide the first direct evidence that a disruption of m155 expression leads to attenuation of viral virulence and growth in animals. Moreover, these results suggest that m155 is a viral determinant for optimal MCMV growth and virulence in vivo.

Animals↗

PubMed2004

Molecular, biological, and in vivo characterization of the guinea pig cytomegalovirus (CMV) homologs of the human CMV matrix proteins pp71 (UL82) and pp65 (UL83).

We recently identified the genes encoding the guinea pig cytomegalovirus (GPCMV) homologs of the upper and lower matrix proteins of human CMV, pp71 (UL82) and pp65 (UL83), which we designated GP82 and GP83, respectively. Transient-expression studies with a GP82 plasmid demonstrated that the encoded protein targets the nucleus and that the infectivity and plaquing efficiency of cotransfected GPCMV viral DNA was enhanced by GP82. The transactivation function of GP82 was not limited to GPCMV, but was also observed for a heterologous virus, herpes simplex virus type 1 (HSV-1). This was confirmed by its ability to complement the growth of an HSV-1 VP16 transactivation-defective mutant virus in an HSV viral DNA cotransfection assay. Study of a GP82 "knockout" virus (and its attendant rescuant), generated on a GPCMV bacterial artificial chromosome construct, confirmed the essential nature of the gene. Conventional homologous recombination was used to generate a GP83 mutant to examine the role of GP83 in the viral life cycle. Comparison of the one-step growth kinetics of the GP83 mutant (vAM409) and wild-type GPCMV indicated that GP83 protein is not required for viral replication in tissue culture. The role of GP83 in vivo was examined by comparing the pathogenesis of wild-type GPCMV, vAM409, and a control virus, vAM403, in guinea pigs. The vAM409 mutant was significantly attenuated for dissemination in immunocompromised strain 2 guinea pigs, suggesting that the GP83 protein is essential for full pathogenicity in vivo.

Amino Acid Sequence↗

PubMed2004

Intracellular expression of engineered RNase P ribozymes effectively blocks gene expression and replication of human cytomegalovirus.

A ribozyme (M1GS RNA) constructed from the catalytic RNA subunit of RNase P from Escherichia coli was used to target the overlapping region of two human cytomegalovirus (HCMV) mRNAs, which encode for the viral essential protease (PR) and capsid assembly proteins (AP), respectively. The results show a reduction of >80% in the expression levels of PR and AP and an inhibition of approximately 2000-fold of viral growth in cells that stably expressed the ribozyme. In comparison, <10% reduction in the expression of the targets and viral growth was found in cells that either did not express the ribozyme or produced a "disabled" ribozyme carrying mutations that abolished its catalytic activity. Examination of replication of the virus in the ribozyme-expressing cells indicates that packaging of the viral genomic DNA into capsids is blocked, and suggests that the antiviral effects are because the ribozyme specifically inhibits the AP and PR expression and, consequently, abolishes viral capsid formation and growth. Our results show that RNase P ribozymes are highly effective in blocking HCMV growth by targeting the PR and AP mRNAs and demonstrate the feasibility to use these ribozymes in gene therapy for antiviral applications.

Cytomegalovirus↗