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The seminal discovery of ribonuclease P (RNase P) and its catalytic RNA by Sidney Altman has not only revolutionized our understanding of life, but also opened new fields for scientific exploration and investigation. This review focuses on human RNase P and its use as a gene-targeting tool, two topics initiated in Altman's laboratory. We outline early works on human RNase P as a tRNA processing enzyme and comment on its expanding nonconventional functions in molecular networks of transcription, chromatin remodeling, homology-directed repair, and innate immunity. The important implications and insights from these discoveries on the potential use of RNase P as a gene-targeting tool are presented. This multifunctionality calls to a modified structure–function partitioning of domains in human RNase P, as well as its relative ribonucleoprotein, RNase MRP. The role of these two catalysts in innate immunity is of particular interest in molecular evolution, as this dynamic molecular network could have originated and evolved from primordial enzymes and sensors of RNA, including predecessors of these two ribonucleoproteins.

External guide sequences (EGSs) signify the short RNAs that induce ribonuclease P (RNase P), an enzyme responsible for processing the 5′ termini of tRNA, to specifically cleave a target mRNA by forming a precursor tRNA-like complex. Hence, the EGS technology may serve as a potential strategy for gene-targeting therapy. Our previous studies have revealed that engineered EGS variants induced RNase P to efficiently hydrolyze target mRNAs. In the present research, an EGS variant was designed to be complementary to the mRNA coding for human cytomegalovirus (HCMV) major capsid protein (MCP), which is vital to form the viral capsid. In vitro, the EGS variant was about 80-fold more efficient in inducing human RNase P-mediated cleavage of the target mRNA than a natural tRNA-derived EGS. Moreover, the expressed variant and natural tRNA-originated EGSs led to a decrease of MCP expression by 98% and 73%–74% and a decrease of viral growth by about 10,000- and 200-fold in cells infected with HCMV, respectively. These results reveal direct evidence that the engineered EGS variant has higher efficiency in blocking the expression of HCMV genes and viral growth than the natural tRNA-originated EGS. Therefore, our findings imply that the EGS variant can be a potent candidate agent for the treatment of infections caused by HCMV.

Terminal deoxynucleotidyl transferase (TdT) is a unique template-free polymerase that randomly adds multiple deoxyribonucleoside triphosphates (dNTPs) to the 3′-OH terminus of ssDNA. This characteristic makes TdT a versatile enzymatic tool in many fields. Moreover, aberrant TdT expression is a well-recognized biomarker of several leukemic diseases and is related to carcinogenesis. In this study, we developed a facile, rapid, label-free, and convenient assay for TdT detection. TdT-generated poly A tails formed a fluorescent enhancement complex in the presence of coralyne. To achieve a better signal-to-noise ratio, we used potassium thiocyanate (KSCN), instead of other halogen anions (KCl, KBr, KI, NaI) as the quenching agent of dissociate coralyne. Our results demonstrate that this assay is extremely facile, rapid, and label-free; at levels as low as 0.025 U/mL, TdT was distinctly detected within 55 min. And the determination of TdT activity in RBL-2H3 and Reh cells lysates exhibited a good sensing performance, demonstrating its potential applications in biochemical research and clinical diagnosis.

Infection of H5N1 influenza virus causes the highest mortality among all influenza viruses. The mechanisms underlying such high viral pathogenicity are incompletely understood. Here, we report that the H5N1 influenza virus encodes a microRNA-like small RNA, miR-HA-3p, which is processed from a stem loop-containing viral RNA precursor by Argonaute 2, and plays a role in enhancing cytokine production during H5N1 infection. Mechanistic study shows that miR-HA-3p targets poly(rC)-binding protein 2 (PCBP2) and suppresses its expression. Consistent with PCBP2 being an important negative regulator of RIG-I/MAVS-mediated antiviral innate immunity, suppression of PCBP2 expression by miR-HA-3p promotes cytokine production in human macrophages and mice infected with H5N1 virus. We conclude that miR-HA-3p is the first identified influenza virus-encoded microRNA-like functional RNA fragment and a novel virulence factor contributing to H5N1-induced 'cytokine storm' and mortality.

Cytomegalovirus (CMV) infection causes birth defects and life-threatening complications in immunosuppressed patients. Lack of vaccine and need for more effective drugs have driven widespread ongoing therapeutic development efforts against human CMV (HCMV), mostly using murine CMV (MCMV) as the model system for preclinical animal tests. The recent publication (Yu et al., 2017, DOI: 10.1126/science.aam6892) of an atomic model for HCMV capsid with associated tegument protein pp150 has infused impetus for rational design of novel vaccines and drugs, but the absence of high-resolution structural data on MCMV remains a significant knowledge gap in such development efforts. Here, by cryoEM with sub-particle reconstruction method, we have obtained the first atomic structure of MCMV capsid with associated pp150. Surprisingly, the capsid-binding patterns of pp150 differ between HCMV and MCMV despite their highly similar capsid structures. In MCMV, pp150 is absent on triplex Tc and exists as a “Λ”-shaped dimer on other triplexes, leading to only 260 groups of two pp150 subunits per capsid in contrast to 320 groups of three pp150 subunits each in a “Δ”-shaped fortifying configuration. Many more amino acids contribute to pp150-pp150 interactions in MCMV than in HCMV, making MCMV pp150 dimer inflexible thus incompatible to instigate triplex Tc-binding as observed in HCMV. While pp150 is essential in HCMV, our pp150-deletion mutant of MCMV remained viable though with attenuated infectivity and exhibiting defects in retaining viral genome. These results thus invalidate targeting pp150, but lend support to targeting capsid proteins, when using MCMV as a model for HCMV pathogenesis and therapeutic studies., Author summary Cytomegalovirus (CMV) infection is a leading viral cause of birth defects and could be deadly to AIDS patients and organ transplant recipients. Absence of effective vaccines and potent drugs against human CMV (HCMV) infections has motivated animal-based studies, mostly based on the mouse model with murine CMV (MCMV), both for understanding pathogenesis of CMV infections and for developing therapeutic strategies. Distinct from other medically important herpesviruses (those responsible for cold sores, genital herpes, shingles and several human cancers), CMV contains an abundant phosphoprotein, pp150, which is a structurally, immunogenically, and regulatorily important tegument protein and a potential drug target. Here, we used cryoEM with localized reconstruction method to obtain the first atomic structure of MCMV. The structure reveals that the organization patterns of the capsid-associated tegument protein pp150 are different in MCMV and HCMV, despite their highly similar capsid structures. We also show that deleting pp150 did not eliminate MCMV infection in contrast to pp150’s essential role in HCMV infections. Our results have significant implication to the current practice of using mouse infected with MCMV for HCMV therapeutic development.

Antiviral drug development against respiratory syncytial virus (RSV) is urgently needed due to the public health significance of the viral infection. Here, we report the anti-RSV activity of a small molecule, (1S,3R,4R,5R)-3,4- bis{[(E)-3-(3,4-dihydroxyphenyl)prop-2-enoyl]oxy}-1,5-dihydroxycyclohexane-1-carboxylic methyl ester (3,4-DCQAME) or 3,4- O-Dicaffeoylquinic acid methyl ester, which can be isolated from several plants of traditional Chinese medicine. We showed for the first time that compound 3,4-DCQAME potently inhibits RSV entry and infection. In vitro, 3,4-DCQAME can interact with F(ecto), the ectodomain of RSV fusion (F) protein. In cultured cells, the compound can block the interaction of F(ecto) protein with the cellular membrane and inhibit viral fusion during RSV entry, leading to inhibition of viral gene expression and infection. In RSV-infected mice that were treated with 3,4-DCQAME, we observed a reduction of RSV-induced pathologic changes and substantial inhibition of viral infection and growth in the lung tissues. Our results provide the first direct evidence of the anti-RSV activity of 3,4-DCQAME. Furthermore, these results suggest that 3,4-DCQAME represents a promising lead compound for anti-RSV therapeutic development.-Tang, W., Li, M., Liu, Y., Liang, N., Yang, Z., Zhao, Y., Wu, S., Lu, S., Li, Y., Liu, F. Small molecule inhibits respiratory syncytial virus entry and infection by blocking the interaction of the viral fusion protein with the cell membrane.

Ankylosing spondylitis (AS) is a chronic autoimmune inflammatory disease with severe inflammatory symptoms in the axial skeleton. The cause of ankylosing spondylitis is unknown. TNFAIP3, also named A20, uses ubiquitin-related functions to regulate immune activation, deficiency of which is highly related to autoimmune disease. However, the role of TNFAIP3 in human AS has not been reported. Our objective was to study the role and mechanism of TNFAIP3 in ankylosing spondylitis. TNFAIP3 expression on different types of immunocytes from AS peripheral blood was measured by flow cytometry. In vitro, monocytes were transfected with a TNFAIP3 shRNA lentivirus, and IL6 and IL1B activation was tested using real-time PCR and ELISA. The novel interaction complex TNFAIP3-DEPTOR was determined through GST pull-down, yeast two-hybrid system, confocal microscopy, and co-immunoprecipitation. Transmission electron microscopy, the RFP-GFP-LC3 adenovirus, and LC3 expression were used for autophagy detection. Here, we show that TNFAIP3 expression in AS peripheral blood non-classical monocytes was decreased. In normal monocytes, TNFAIP3 induced autophagy, which restricted inflammasome activation to the early stage of LPS stimulation. Zinc-finger domains of TNFAIP3 were able to interact and stabilize DEPTOR. TNFAIP3 and DEPTOR together rapidly promoted autophagy after LPS treatment to prevent NLRP3 inflammasome formation. Finally, TNFAIP3 and DEPTOR deficiency in AS non-classical monocytes facilitated inflammasome activation. Our study indicates that TNFAIP3-DEPTOR complex-induced early-onset autophagy is vital for immune inhibition in autoimmune disease.

Interferon-γ (IFN-γ) represents one of the most important innate immunity responses in a host to combat infections of many human viruses including human herpesviruses. Human N-myc interactor (Nmi) protein, which has been shown to interact with signal transducer and activator of transcription (STAT) proteins including STAT1, is important for the activation of IFN-γ induced STAT1-dependent transcription of many genes responsible for IFN-γ immune responses. However, no proteins encoded by herpesviruses have been reported to interact with Nmi and inhibit Nmi-mediated activation of IFN-γ immune responses to achieve immune evasion from IFN-γ responses. In this study, we show strong evidence that the UL23 protein of human cytomegalovirus (HCMV), a human herpesvirus, specifically interacts with Nmi. This interaction was identified through a yeast two-hybrid screen and co-immunoprecipitation in human cells. We observed that Nmi, when bound to UL23, was not associated with STAT1, suggesting that UL23 binding of Nmi disrupts the interaction of Nmi with STAT1. In cells overexpressing UL23, we observed (a) significantly reduced levels of Nmi and STAT1 in the nuclei, the sites where these proteins act to induce transcription of IFN-γ stimulated genes, and (b) decreased levels of the induction of the transcription of IFN-γ stimulated genes. UL23-deficient HCMV mutants induced higher transcription of IFN-γ stimulated genes and exhibited lower titers than parental and control revertant viruses expressing functional UL23 in IFN-γ treated cells. Thus, UL23 appears to interact directly with Nmi and inhibit nuclear translocation of Nmi and its associated protein STAT1, leading to a decrease of IFN-γ induced responses and an increase of viral resistance to IFN-γ. Our results further highlight the roles of UL23-Nmi interactions in facilitating viral immune escape from IFN-γ responses and enhancing viral resistance to IFN antiviral effects., Author summary Interferon-γ (IFN-γ) responses are vital for a host to combat infections of many human viruses including human herpesviruses. Upon treatment of IFN-γ, transcription of many genes responsible for IFN-γ immune responses is activated primarily by the signal transducer and activator of transcription (STAT) proteins such as STAT1 protein. Human N-myc interactor (Nmi) protein has been shown to interact with STAT proteins including STAT1 and activate IFN-γ induced STAT-dependent transcription. However, no proteins encoded by herpesviruses have been reported to interact with Nmi and inhibit Nmi-mediated activation of IFN-γ immune responses to achieve immune evasion from IFN-γ responses. In this study, we show strong evidence that the UL23 protein of human cytomegalovirus (HCMV), a human herpesvirus, specifically interacts with Nmi protein. UL23 appears to interact directly with Nmi and inhibit nuclear translocation of Nmi and its associated protein STAT1, leading to a decrease of IFN-γ responses and an increase of viral resistance to IFN-γ. Blocking UL23 expression led to higher transcription of IFN-γ stimulated genes and significant inhibition of viral growth in infected cells. These results suggest that interfering with Nmi function may represent an effective mechanism for a herpesvirus to block Nmi-mediated IFN-γ responses and increase viral resistance to IFN-γ. This also provides a potentially new therapeutic strategy to treat HCMV infection by modulating Nmi activity with blocking the expression of a viral protein.

An engineered RNase P-based ribozyme variant, which was generated using the in vitro selection procedure, was used to target the overlapping mRNA region of two proteins essential for human cytomegalovirus (HCMV) replication: capsid assembly protein (AP) and protease (PR). In vitro studies showed that the generated variant, V718-A, cleaved the target AP mRNA sequence efficiently and its activity was about 60-fold higher than that of wild type ribozyme M1-A. Furthermore, we observed a reduction of 98%–99% in AP/PR expression and an inhibition of 50,000 fold in viral growth in cells with V718-A, while a 75% reduction in AP/PR expression and a 500-fold inhibition in viral growth was found in cells with M1-A. Examination of the antiviral effects of the generated ribozyme on the HCMV replication cycle suggested that viral DNA encapsidation was inhibited and as a consequence, viral capsid assembly was blocked when the expression of AP and PR was inhibited by the ribozyme. Thus, our study indicates that the generated ribozyme variant is highly effective in inhibiting HCMV gene expression and blocking viral replication, and suggests that engineered RNase P ribozyme can be potentially developed as a promising gene-targeting agent for anti-HCMV therapy.

Salmonella can hijack host atypical miRNA processing machinery to cleave its small non-coding RNA into a ~22-nt RNA fragment, Sal-1, which facilitates Salmonella survival in the infected host. The mechanism through which Sal-1 promotes Salmonella survival, however, remains unknown. In the present study, we reported that Sal-1 targets cellular inducible nitric oxide synthase (iNOS) in a miRNA manner, leading to attenuation of host cell iNOS/NO-mediated anti-microbial capacity. First, depletion of Sal-1 in Salmonella-infected epithelial cells significantly increased the iNOS level but not the levels of various inflammatory cytokines. Bioinformatics analysis and mutagenesis strategies were consistent with the identification of mRNA of iNOS as a target of Sal-1 in both human and mice. Second, western blot and immunohistochemical analysis confirmed that Sal-1 suppressed iNOS expression in vitro and in vivo, thus reducing the production of NO. Finally, Sal-1 facilitating Salmonella survival through suppressing iNOS induction was confirmed in mouse model by expressing mutated iNOS that is not targeted by Sal-1 in mice colon. In conclusion, our study provides new insight into the pathogenic mechanism of intracellular bacteria to modulate host innate immune response.

We have previously engineered new RNase P-based ribozyme variants with improved in vitro catalytic activity. In this study, we employed a novel engineered variant to target a shared mRNA region of human cytomegalovirus (HCMV) immediate early proteins 1 (IE1) and 2 (IE2), which are essential for the expression of viral early and late genes as well as viral growth. Ribozyme F-R228-IE represents a novel variant that possesses three unique base substitution point mutations at the catalytic domain of RNase P catalytic RNA. Compared to F-M1-IE that is the ribozyme derived from the wild type RNase P catalytic RNA sequence, the functional variant F-R228-IE cleaved the target mRNA sequence in vitro at least 100 times more efficiently. In cultured cells, expression of F-R228-IE resulted in IE1/IE2 expression reduction by 98-99% and in HCMV production reduction by 50,000 folds. In contrast, expression of F-M1-IE resulted in IE1/IE2 expression reduction by less than 80% and in viral production reduction by 200 folds. Studies of the ribozyme-mediated antiviral effects in cultured cells suggest that overall viral early and late gene expression and viral growth were inhibited due to the ribozyme-mediated reduction of HCMV IE1 and IE2 expression. Our results provide direct evidence that engineered RNase P ribozymes, such as F-R228-IE, can serve as a novel class of inhibitors for the treatment and prevention of HCMV infection. Moreover, these results suggest that F-R228-IE, with novel and unique mutations at the catalytic domain to enhance ribozyme activity, can be a candidate for the construction of effective agents for anti-HCMV therapy.

Nucleic acid-based gene interfering approaches, such as those mediated by RNA interference and RNase P-associated external guide sequence (EGS), have emerged as promising antiviral strategies. The RNase P-based technology is unique, because a custom-designed EGS can bind to any complementary mRNA sequence and recruit intracellular RNase P for specific degradation of the target mRNA. In this study, a functional EGS was constructed to target hepatitis B virus (HBV) essential transcripts. Furthermore, an attenuated Salmonella strain was constructed and used for delivery of anti-HBV EGS in cells and in mice. Substantial reduction in the levels of HBV gene expression and viral DNA was detected in cells treated with the Salmonella vector carrying the functional EGS construct. Furthermore, oral inoculation of Salmonella carrying the EGS construct led to an inhibition of ~95% in the levels of HBV gene expression and a reduction of ~200,000-fold in viral DNA level in the livers and sera of the treated mice transfected with a HBV plasmid. Our results suggest that EGSs are effective in inhibiting HBV replication in cultured cells and mammalian livers, and demonstrate the use of Salmonella-mediated delivery of EGS as a promising therapeutic approach for human diseases including HBV infection.

The mechanisms underlying human cytomegalovirus (HCMV) latency remain incompletely understood. Here, we showed that a HCMV-encoded miRNA, miR-UL148D, robustly accumulates during late stages of experimental latent HCMV infection in host cells and promotes HCMV latency by modulating the immediate early response gene 5 (IER5)-cell division cycle 25B (CDC25B) axis in host cells. miR-UL148D inhibited IER5 expression by directly targeting the three-prime untranslated region(3’UTR) of IER5 mRNA and thus rescued CDC25B expression during the establishment of viral latency. Infection with NR-1ΔmiR-UL148D, a derivative of the HCMV clinical strain NR-1 with a miR-UL148D knockout mutation, resulted in sustained induction of IER5 expression but decreased CDC25B expression in host cells. Mechanistically, we further showed that CDC25B plays an important role in suppressing HCMV IE1 and lytic gene transcription by activating cyclin-dependent kinase 1 (CDK-1). Both gain-of-function and lose-of-function assays demonstrated that miR-UL148D promotes HCMV latency by helping maintain CDC25B activity in host cells. These results provide a novel mechanism through which a HCMV miRNA regulates viral latency., Author Summary Human cytomegalovirus (HCMV) is a herpesvirus that is prevalent around the world. Following primary infection, HCMV can persist for the lifetime of a host by establishing a latent infection. While HCMV infection normally causes no clinical symptoms, reactivation of HCMV from latency can cause deadly disease in immunocompromised individuals. HCMV achieves latent infection in hematopoietic progenitor cells by silencing HCMV immediate early (IE) genes, the activation of which serves as the initial step in HCMV replication. HCMV has developed multiple strategies to control the expression of IE genes for latency and reactivation. In the present study, we reported that microRNAs (miRNAs), a class of ~22-nt non-coding nucleotides that post-transcriptionally regulate gene expression, are involved in modulating HCMV latency and reactivation. In particular, we found that HCMV miR-UL148D accumulated in progenitor cells during the establishment of experimental HCMV latency. Furthermore, we identified cellular immediate early response gene 5 (IER5), a p53 target gene, as a novel target of miR-UL148D. Functionally, miR-UL148D efficiently inhibited the up-regulation of IER5 during latent viral infection, maintaining the activity of CDC25B and CDK1 and thus controlling IE1 transcription. In conclusion, our study provides the first evidence that HCMV miR-UL148D facilitates latent viral infection by modulating the IER5-CDC25B axis in host cells.

An external guide sequence (EGS) is a RNA sequence which can interact with a target mRNA to form a tertiary structure like a pre-tRNA and recruit intracellular ribonuclease P (RNase P), a tRNA processing enzyme, to degrade target mRNA. Previously, an in vitro selection procedure has been used by us to engineer new EGSs that are more robust in inducing human RNase P to cleave their targeted mRNAs. In this study, we constructed EGSs from a variant to target the mRNA encoding herpes simplex virus 1 (HSV-1) major transcription regulator ICP4, which is essential for the expression of viral early and late genes and viral growth. The EGS variant induced human RNase P cleavage of ICP4 mRNA sequence 60 times better than the EGS generated from a natural pre-tRNA. A decrease of about 97% and 75% in the level of ICP4 gene expression and an inhibition of about 7,000- and 500-fold in viral growth were observed in HSV infected cells expressing the variant and the pre-tRNA-derived EGS, respectively. This study shows that engineered EGSs can inhibit HSV-1 gene expression and viral growth. Furthermore, these results demonstrate the potential for engineered EGS RNAs to be developed and used as anti-HSV therapeutics.

A functional RNase P ribozyme (M1GS RNA) was constructed to target the overlapping mRNA region of two murine cytomegalovirus (MCMV) capsid proteins essential for viral replication: the assembly protein (mAP) and M80. The customized ribozyme efficiently cleaved the target mRNA sequence in vitro . Moreover, 80% reduction in the expression of mAP and M80 and a 2,000-fold reduction in viral growth were observed in cells expressing the ribozyme. In contrast, there was no significant reduction in viral gene expression and growth in cells that either did not express the ribozyme or produced a “disabled” ribozyme carrying mutations that abolished its catalytic activity. When the ribozyme-expressing constructs were delivered into MCMV-infected SCID mice via a modified “hydrodynamic transfection” procedure, expression of ribozymes was observed in the livers and spleens. Compared with the control animals that did not receive any M1GS constructs or received the disabled ribozyme construct, animals receiving the functional ribozyme construct exhibited a significant reduction of viral gene expression and infection. Viral titers in the spleens, livers, lungs, and salivary glands of the functional ribozyme-treated SCID mice at 21 days after infection were 200- to 2,000-fold lower than those in the control animals. Moreover, survival of the infected animals significantly improved upon receiving the functional ribozyme construct. Our study examines the use of M1GS ribozymes for inhibition of gene expression in animals and demonstrates the utility of RNase P ribozymes for gene targeting applications in vivo .

Ribonuclease P (RNase P) complexed with an external guide sequence (EGS) represents a novel nucleic acid-based gene interference approach to modulate gene expression. This enzyme is a ribonucleoprotein complex for tRNA processing. In Escherichia coli, RNase P contains a catalytic RNA subunit (M1 ribozyme) and a protein subunit (C5 cofactor). EGSs, which are RNAs derived from natural tRNAs, bind to a target mRNA and render the mRNA susceptible to hydrolysis by RNase P and M1 ribozyme. When covalently linked with a guide sequence, M1 can be engineered into a sequence-specific endonuclease, M1GS ribozyme, which cleaves any target RNAs that base pair with the guide sequence. Studies have demonstrated efficient cleavage of mRNAs by M1GS and RNase P complexed with EGSs in vitro. Moreover, highly active M1GS and EGSs were successfully engineered using in vitro selection procedures. EGSs and M1GS ribozymes are effective in blocking gene expression in both bacteria and human cells, and exhibit promising activity for antimicrobial, antiviral, and anticancer applications. In this review, we highlight some recent results using the RNase P-based technology, and offer new insights into the future of using EGS and M1GS RNA as tools for basic research and as gene-targeting agents for clinical applications.

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.

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.

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.

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.

Summary 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.

By using an in vitro selection procedure, we have previously isolated RNase P ribozyme variants that efficiently cleave an mRNA sequence in vitro. In this study, a ribozyme variant was used to target the overlapping region of the mRNAs encoding human cytomegalovirus (HCMV) major transcription regulatory proteins IE1 and IE2. The variant is about 90 times more efficient in cleaving the IE1/IE2 mRNA sequence in vitro than the ribozyme derived from the wild type RNase P ribozyme. Our results provide the first direct evidence that a point mutation at nucleotide position 80 of RNase P catalytic RNA from Escherichia coli (U80--> C80) increases the rate of chemical cleavage, and another mutation at nucleotide position 188 (C188--> U188) enhances substrate binding of the ribozyme. Moreover, the variant is more effective in inhibiting viral IE1 and IE2 expression and growth in HCMV-infected cells than the wild type ribozyme. A reduction of about 99% in the expression level of IE1 and IE2 and a reduction of 10,000-fold in viral growth were observed in cells that expressed the variant. In contrast, a reduction of less than 10% in IE1/IE2 expression and viral growth was observed in cells that either did not express the ribozyme or produced a catalytically inactive ribozyme mutant. Thus, engineered RNase P ribozyme variants are highly effective in inhibiting HCMV gene expression and growth. These results also demonstrate the feasibility of engineering highly effective RNase P ribozymes for gene targeting applications, including anti-HCMV gene therapy.

A sequence-specific ribozyme (M1GS RNA) derived from the catalytic RNA subunit of RNase P from Escherichia coli was used to target the mRNA encoding human cytomegalovirus (HCMV) protease (PR), a viral protein that is responsible for the processing of the viral capsid assembly protein. We showed that the constructed ribozyme cleaved the PR mRNA sequence efficiently in vitro. Moreover, a reduction of about 80% in the expression level of the protease and a reduction of about 100-fold in HCMV growth were observed in cells that expressed the ribozyme stably. In contrast, a reduction of less than 10% in the expression of viral protease and viral growth was observed in cells that either did not express the ribozyme or produced a catalytically inactive ribozyme mutant. Further examination of the antiviral effects of the ribozyme-mediated cleavage of PR mRNA indicates that (1) the proteolytic cleavage of the capsid assembly protein is inhibited significantly, and (2) the packaging of the viral genomic DNA into the CMV capsids is blocked. These observations are consistent with the notion that the protease functions to process the capsid assembly protein and is essential for viral capsid assembly. Moreover, our results indicate that the RNase P ribozyme-mediated cleavage specifically reduces the expression of the protease, but not other viral genes examined. Thus, M1GS ribozyme is highly effective in inhibiting HCMV growth by targeting the PR mRNA and may represent a novel class of general gene-targeting agents for the studies and treatment of infections caused by human viruses, including HCMV.

Ribozymes are promising gene-targeting agents for regulation of gene expression. In our recent studies, RnaseP (M1GS) ribozymes were constructed to target the overlapping region (IE mRNA) of IE1 and IE2 mRNAs of human cytomegalovirus (HCMV) and the mRNA (TK mRNA) coding for thymidine kinase (TK) of herpes simplex virus 1 (HSV-1). Our results indicate that RNase P ribozymes efficiently cleaved the IE mRNA and TK mRNA sequences in vitro. Significant inhibitions (approximately 75-85%) of HCMV IE1/IE2 and HSV-1 TK expression were observed in cells that expressed these ribozymes while a reduction of less than 10% was found in cells that did not express the ribozymes or expressed a disabled one that contained mutations abolishing catalytic activity. Ribozyme variants, which cleaved a TK mRNA sequence in vitro more efficiently than the ribozyme derived from the wildtype RNase P sequence, were selected by an in vitro selection system. When the selected ribozymes were expressed in cultured cells, they were more effective in inhibiting viral IE1/IE2 and TK expression and viral growth than the wildtype ribozyme sequence. Our results provide the first direct evidence that RNase P ribozymes are highly effective in inhibiting HCMV gene expression and growth. Moreover, a selection system was developed for generating novel ribozyme variants that cleave a mRNA substrate efficiently in vitro. These results suggest that M1GS ribozyme-mediated inhibition of expression of viral genes can be used as a new approach for the studies of HCMV gene function and the treatment of HCMV infection.

We have previously employed an in vitro (genetic) selection procedure to select RNase P ribozyme variants for their activity in cleaving a mRNA substrate from a pool of ribozymes containing randomized sequences. In this study, one of the variants was used to target the overlapping region of the mRNAs encoding the major transcription regulatory proteins, IE1 and IE2, of human cytomegalovirus (HCMV). The ribozyme variant exhibited an enhanced substrate binding and rate of chemical cleavage, and was at least 25 times more efficient in cleaving the target mRNA in vitro than the ribozyme derived from the wild-type sequence. Our results provide the first direct evidence that a point mutation at nucleotide 86 of RNase P catalytic RNA from Escherichia coli (A86 → C86) increases the rate of chemical cleavage while another mutation at nucleotide 205 (G205 → C205) enhances substrate binding of the ribozyme. Moreover, the variant was also more effective in inhibiting IE1 and IE2 expression and HCMV growth in cultured cells. A reduction of more than 97 % in IE1 and IE2 expression and a reduction of 3000-fold in viral growth were observed in cells expressing the variant. Thus, RNase P ribozyme variant is highly effective in inhibiting HCMV gene expression and growth. Our results provide the direct evidence that increasing the rate of chemical cleavage and substrate-binding affinity of the ribozymes should lead to an improvement of their anti-HCMV efficacy. Moreover, our data also suggest that highly effective anti-HCMV ribozyme variants can be developed using genetic engineering approaches including in vitro selection.

RNase P ribozyme can be engineered to be a sequence-specific gene-targeting agent with promising application in both basic research and clinical settings. By using an in vitro selection system, we have previously generated RNase P ribozyme variants that have better catalytic activity in cleaving an mRNA sequence than the wild type ribozyme. In this study, one of the variants was used to target the mRNA encoding human cytomegalovirus (HCMV) essential transcription factor immediate-early protein 2 (IE2). The variant was able to cleave IE2 mRNA in vitro 50-fold better than the wild type ribozyme. A reduction of about 98% in IE2 expression and a reduction of 3500-fold in viral production was observed in HCMV-infected cells expressing the variant compared to a 75% reduction in IE2 expression and a 100-fold reduction in viral production in cells expressing the ribozyme derived from the wild type sequence. These results suggest that ribozyme variants that are selected to be highly active in vitro are also more effective in inhibiting the expression of their targets in cultured cells. Our study demonstrates that RNase P ribozyme variants are efficient in reducing HCMV gene expression and growth and are potentially useful for anti-viral therapeutic application.

Using an in vitro selection procedure, we have previously isolated ribonuclease P (RNase P) ribozyme variants that efficiently cleave an mRNA sequence in vitro. In this study, an M1GS RNA variant was used to target the mRNA encoding human herpes simplex virus 1 (HSV-1) major transcription activator ICP4. The variant is about 15 times more efficient in cleaving the ICP4 mRNA sequence in vitro than the ribozyme derived from the wild type RNase P ribozyme. Moreover, the variant is also more effective in inhibiting viral ICP4 expression and growth in HSV-1-infected cells than the wild type ribozyme. A reduction of approximately 90% in the expression level of ICP4 and a reduction of 4000-fold in viral growth were observed in cells that expressed the variant. In contrast, a reduction of10% in the ICP4 expression and viral growth was observed in cells that either did not express the ribozyme or produced a catalytically inactive ribozyme mutant. These results provide direct evidence that RNase P ribozyme variants can be highly effective in inhibiting HSV-1 gene expression and growth and furthermore, demonstrate the feasibility of developing highly effective RNase P ribozyme variants for anti-HSV applications by using in vitro selection procedures.

External guide sequences (EGSs) are oligonucleotides that consist of a sequence complementary to a target mRNA and recruit intracellular RNase P for specific degradation of the target RNA. In this study, DNA-based EGS molecules were chemically synthesized to target the mRNA coding for the protease of human cytomegalovirus (HCMV). The EGS molecules efficiently directed human RNase P to cleave the target mRNA sequence in vitro . When EGSs were exogenously administered into HCMV-infected human foreskin fibroblasts, a reduction of about 80–90% in the expression level of the protease and a reduction of about 300-fold in HCMV growth were observed in the cells that were treated with a functional EGS, but not in cells that were not treated with the EGS or with a “disabled” EGS carrying nucleotide mutations that precluded RNase P recognition. Moreover, packaging of the viral DNA genome into the capsid was blocked in the cells treated with the functional EGS. These results indicate that HCMV protease is essential for viral DNA encapsidation. Moreover, our study provides direct evidence that exogenous administration of a DNA-based EGS can be used as a therapeutic approach for inhibiting gene expression and replication of a human virus.

A sequence-specific ribozyme (M1GS RNA) derived from the catalytic RNA subunit of RNase P from Escherichia coli was used to target the mRNA encoding human herpes simplex virus 1 (HSV-1) major transcription activator, ICP4. A reduction of more than 80 % in the expression level of ICP4 and a reduction of about 1000-fold in viral growth were observed in cells that stably expressed the ribozyme. In contrast, a reduction of less than 10 % in ICP4 expression and viral growth was observed in cells that either did not express the ribozyme or produced a catalytically inactive ribozyme mutant. Thus, M1GS ribozyme is highly effective in inhibiting HSV-1 growth and can be used as a general gene-targeting agent for anti-HSV applications.

Ribonuclease P (RNase P) complexed with external guide sequence (termed as EGS) represents a novel nucleic acid-based gene interference approach to modulate gene expression. In previous studies, by using an in vitro selection procedure, we have successfully generated EGS variants that are complementary to target mRNAs, and these variants exhibit higher efficiency in directing human RNase P to cleave the target mRNAs than those derived from nature RNAs in vitro. This chapter describes the procedure of using engineered EGSs for in vitro trans-cleavage of target viral mRNAs in cultured cells. Detailed information is focused on (1) generation and in vitro cleavage assay of the customized EGS variants and (2) stable expression of EGS and evaluation of its activity in inhibition of viral gene expression and growth in cultured cells. These methods should provide general guidelines for using engineered EGS to direct RNase P-mediated cleavage of target mRNAs in cultured cells.

The complete genome sequence of a human enterovirus 71 strain (EV71/wuhan/3018/2010), which was isolated in Wuhan in 2010, was amplified by a reverse transcription-PCR method and sequenced. Phylogenetic analysis based on the complete genome sequence classified this strain into subgenogroup A.

Controlled regulation of genomic DNA synthesis is a universally conserved process for all herpesviruses, including human cytomegalovirus (HCMV), and plays a key role in viral pathogenesis, such as persistent infections. HCMV UL105 is believed to encode the helicase of the DNA replication machinery that needs to localize in the nuclei, the site of viral DNA synthesis. No host factors that interact with UL105 have been identified. In this study, we show that UL105 specifically interacts with Snapin, a human protein that is predominantly localized in the cytoplasm and associated with cellular vesicles. UL105 was found to interact with Snapin in both the yeast two-hybrid screen and coimmunoprecipitation experiments in HCMV-infected cells. The nuclear and cytoplasmic levels of UL105 were decreased and increased in cells overexpressing Snapin, respectively, while the levels of UL105 in the nuclei and cytoplasm were increased and decreased in cells in which the expression of Snapin was downregulated with anti-Snapin small interfering RNA (siRNA) molecules, respectively. Furthermore, viral DNA synthesis and progeny production were decreased in cells overexpressing Snapin and increased in the anti-Snapin siRNA-treated cells, respectively. Our results provide the first direct evidence to suggest that Snapin interacts with UL105 and alters its cellular distribution, leading to modulation of viral DNA synthesis and progeny production. Our study further suggests that modulation of the cellular distribution of viral helicase by Snapin may represent a possible mechanism for regulating HCMV genomic DNA synthesis, a key step during herpesvirus lytic and persistent infections.

External guide sequences (EGSs) represent a new class of RNA-based gene-targeting agents, consist of a sequence complementary to a target mRNA, and render the target RNA susceptible to degradation by ribonuclease P (RNase P). In this study, EGSs were constructed to target the mRNA encoding human CC-chemokine receptor 5 (CCR5), one of the primary coreceptors for HIV. An EGS RNA, C1, efficiently directed human RNase P to cleave the CCR5 mRNA sequencein vitro. A reduction of about 70% in the expression level of both CCR5 mRNA and protein and an inhibition of more than 50-fold in HIV (R5 strain Ba-L) p24 production were observed in cells that expressed C1. In comparison, a reduction of about 10% in the expression of CCR5 and viral growth was found in cells that either did not express the EGS or produced a “disabled” EGS which carried nucleotide mutations that precluded RNase P recognition. Furthermore, the same C1-expressing cells that were protected from R5 strain Ba-L retained susceptibility to X4 strain IIIB, which uses CXCR4 as the coreceptor instead of CCR5, suggesting that the RNase P-mediated cleavage induced by the EGS is specific for the target CCR5 but not the closely related CXCR4. Our results provide direct evidence that EGS RNAs against CCR5 are effective and specific in blocking HIV infection and growth. These results also demonstrate the feasibility to develop highly effective EGSs for anti-HIV therapy.

Using an in vitro selection procedure, we have previously isolated RNase P ribozyme variants that efficiently cleave an mRNA sequence in vitro. In this study, a variant was used to target the HIV RNA sequence in the tat region. The variant cleaved the tat RNA sequence in vitro about 20 times more efficiently than the wild type ribozyme. Our results provide the first direct evidence that combined mutations at nucleotide 83 and 340 of RNase P catalytic RNA from Escherichia coli (G(83) -> U(83) and G(340) -> A(340)) increase the overall efficiency of the ribozyme in cleaving an HIV RNA sequence. Moreover, the variant is more effective in reducing HIV-1 p24 expression and intracellular viral RNA level in cells than the wild type ribozyme. A reduction of about 90% in viral RNA level and a reduction of 150 fold in viral growth were observed in cells that expressed the variant, while a reduction of less than 10% was observed in cells that either did not express the ribozyme or produced a catalytically inactive ribozyme mutant. Thus, engineered ribozyme variants are effective in inhibiting HIV infection. These results also demonstrate the potential of engineering RNase P ribozymes for anti-HIV application.

Genomic DNA replication is a universal and essential process for all herpesvirus including human cytomegalovirus (HCMV). HCMV UL70 protein, which is believed to encode the primase activity of the viral DNA replication machinery and is highly conserved among herpesviruses, needs to be localized in the nucleus, the site of viral DNA synthesis. No host factors that facilitate the nuclear import of UL70 have been reported. In this study, we provided the first direct evidence that UL70 specifically interacts with a highly conserved and ubiquitously expressed member of the heat shock protein Hsp40/DNAJ family, DNAJB6, which is expressed as two isoforms, a and b, as a result of alternative splicing. The interaction of UL70 with a common region of DNAJB6a and b was identified by both a two hybrid screen in yeast and coimmunoprecipitation in human cells. In transfected cells, UL70 was primarily co-localized with DNAJB6a in the nuclei and with DNAJB6b in the cytoplasm, respectively. The nuclear import of UL70 was increased in cells in which DNAJB6a was up-regulated or DNAJB6b was down-regulated, and was reduced in cells in which DNAJB6a was down-regulated or DNAJB6b was up-regulated. Furthermore, the level of viral DNA synthesis and progeny production was increased in cells in which DNAJB6a was up-regulated or DNAJB6b was down-regulated, and was reduced in cells in which DNAJB6a was down-regulated or DNAJB6b was up-regulated. Thus, DNAJB6a and b appear to enhance the nuclear import and cytoplasmic accumulation of UL70, respectively. Our results also suggest that the relative expression levels of DNAJB6 isoforms may play a key role in regulating the cellular localization of UL70, leading to modulation of HCMV DNA synthesis and lytic infection., Author Summary Genomic DNA replication is highly conserved across all herpesviruses including human cytomegalovirus (HCMV) and is the target for most of the current FDA-approved anti-herpes therapeutic agents. Little is known about how UL70, which is believed to encode the primase activity of the viral DNA replication machinery and is essential for genomic replication, is imported to the nuclei, the site of viral DNA synthesis. In this study, we demonstrated that the HCMV primase interacts with a highly conserved and ubiquitously expressed chaperone protein DNAJB6 that belongs to the heat shock protein 40 (Hsp40) family. As a result of alternative splicing, DNAJB6 is expressed as two isoforms, a and b. While DNAJB6b promotes cytoplasmic accumulation of the viral primase, DNAJB6a enhances its nuclear distribution, representing the first example of a cellular factor involved in facilitating nuclear import of a herpesvirus primase. Our study suggests that the relative expression level of DNAJB6 isoforms may represent a novel mechanism for modulating HCMV lytic replication by regulating the cellular localization of the viral primase. Furthermore, our results raise the possibility of developing new strategies for treating herpesvirus replication by modulating the cellular distribution of the primase with altered expression of a cellular protein.

External guide sequences (EGSs) are RNA molecules that can bind to a target mRNA and direct ribonuclease P (RNase P), a tRNA processing enzyme, for specific cleavage of the target mRNA. 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, we constructed EGSs from a variant to target the overlapping region of the mRNAs coding for human cytomegalovirus (HCMV) capsid scaffolding protein (CSP) and assemblin, which are essential for viral capsid formation. The EGS variant was about 40-fold more active in directing human RNase P to cleave the mRNA in vitro than the EGS derived from a natural tRNA. Moreover, a reduction of about 98% and 75% in CSP/assemblin gene expression and a reduction of 7000- and 250-fold in viral growth were observed in HCMV-infected cells that expressed the variant and the tRNA-derived EGS, respectively. Our study shows that the EGS variant is more effective in blocking HCMV gene expression and growth than the tRNA-derived EGS. Moreover, these results demonstrate the utility of highly active EGS RNA variants in gene targeting applications including anti-HCMV therapy.

Ribonuclease P complexed with external guide sequence (EGS) bound to mRNA represents a unique nucleic acid-based gene interference approach for modulation of gene expression. Compared with other strategies, such as RNA interference, the EGS-based technology is unique because a custom-designed EGS molecule can hybridize with any mRNA and recruit intracellular ribonuclease P for specific degradation of the target mRNA. It has not been reported whether the EGS-based technology can modulate gene expression in mice. In this study, a functional EGS was constructed to target the mRNA encoding the protease (mPR) of murine cytomegalovirus (MCMV), which is essential for viral replication. Furthermore, a unique attenuated strain of Salmonella was generated for gene delivery of EGS in cultured cells and in mice. Efficient expression of EGS was observed in cultured cells treated with the generated Salmonella vector carrying constructs with the EGS expression cassette. Moreover, a significant reduction in mPR expression and viral growth was found in MCMV-infected cells treated with Salmonella carrying the construct with the functional EGS sequence. When MCMV-infected mice were orally treated with Salmonella carrying EGS expression cassettes, viral gene expression and growth in various organs of these animals were reduced and animal survival improved. Our study suggests that EGS RNAs, when expressed following Salmonella -mediated gene transfer, effectively inhibit viral gene expression and infection in mice. Furthermore, these results demonstrate the feasibility of developing Salmonella -mediated delivery of EGS as a unique approach for treatment that reduces viral diseases in vivo.

Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus that causes birth defects in newborns and life-threatening complications in immunocompromised individuals. Among all human herpesviruses, HCMV contains a much larger dsDNA genome within a similarly-sized capsid compared to the others, and it was proposed to require pp150, a tegument protein only found in cytomegaloviruses, to stabilize its genome-containing capsid. However, little is known about how pp150 interacts with the underlying capsid. Moreover, the smallest capsid protein (SCP), while dispensable in herpes simplex virus type 1, was shown to play essential, yet undefined, role in HCMV infection. Here, by cryo electron microscopy (cryoEM), we determine three-dimensional structures of HCMV capsid (no pp150) and virion (with pp150) at sub-nanometer resolution. Comparison of these two structures reveals that each pp150 tegument density is composed of two helix bundles connected by a long central helix. Correlation between the resolved helices and sequence-based secondary structure prediction maps the tegument density to the N-terminal half of pp150. The structures also show that SCP mediates interactions between the capsid and pp150 at the upper helix bundle of pp150. Consistent with this structural observation, ribozyme inhibition of SCP expression in HCMV-infected cells impairs the formation of DNA-containing viral particles and reduces viral yield by 10,000 fold. By cryoEM reconstruction of the resulting “SCP-deficient” viral particles, we further demonstrate that SCP is required for pp150 functionally binding to the capsid. Together, our structural and biochemical results point to a mechanism whereby SCP recruits pp150 to stabilize genome-containing capsid for the production of infectious HCMV virion., Author Summary Human cytomegalovirus (HCMV) causes birth defects in newborns and life-threatening complications in immunocompromised individuals, such as AIDS patients and organ transplant recipients. The smallest capsid protein (SCP) – only 8 kDa molecular mass as compared to the 155 kDa major capsid protein – has been demonstrated to be essential for HCMV growth, but is dispensable in herpes simplex virus type 1. These seemingly contradictory observations have been a paradox. Here, we solve this paradox by high resolution cryo electron microscopy (cryoEM), in conjunction with functional studies using ribozyme inhibition. Our structural comparisons of HCMV virion and capsid reveal molecular interactions at the secondary structure level and suggest that SCP might contribute to capsid binding of pp150, an essential, cytomegalovirus-specific tegument protein. SCP-deficient particles generated by ribozyme inhibition of SCP-expression in HCMV-infected cells show no pp150 tegument density, demonstrating that SCP is required for the functional binding of pp150 to the capsid. Our results suggest that SCP recruits pp150 to stabilize the HCMV nucleocapsid to enable encapsidation of the genome, which is more densely packaged in HCMV than in other herpesviruses. Overall, this study not only resolves the above paradox, but also illustrates the passive acquisition of a new, essential function by SCP in the production of infectious HCMV virions.

Regulated gene expression and progeny production are essential for persistent and chronic infection by human pathogens, such as hepatitis B virus (HBV), which affects >400 million people worldwide and is a major cause of liver disease. In this study, we provide the first direct evidence that a liver-specific microRNA, miR-122, binds to a highly conserved HBV pregenomic RNA sequence via base-pairing interactions and inhibits HBV gene expression and replication. The miR-122 target sequence is located at the coding region of the mRNA for the viral polymerase and the 3′ untranslated region of the mRNA for the core protein. In cultured cells, HBV gene expression and replication reduces with increased expression of miR-122, and the expression of miR-122 decreases in the presence of HBV infection and replication. Furthermore, analyses of clinical samples demonstrated an inverse linear correlation in vivo between the miR-122 level and the viral loads in the peripheral blood mononuclear cells of HBV-positive patients. Our results suggest that miR-122 may down-regulate HBV replication by binding to the viral target sequence, contributing to the persistent/chronic infection of HBV, and that HBV-induced modulation of miR-122 expression may represent a mechanism to facilitate viral pathogenesis.—Chen, Y., Shen, A., Rider, P. J., Yu, Y., Wu, K., Mu, Y., Hao, Q, Liu, Y., Gong, H., Zhu, Y., Liu, F., Wu, J. A liver-specific microRNA binds to a highly conserved RNA sequence of hepatitis B virus and negatively regulates viral gene expression and replication.

Genomic DNA synthesis is a universally conserved process for all herpesviruses, including human cytomegalovirus (HCMV). HCMV UL70 is believed to encode the primase of the DNA replication machinery, a function which requires localization in the nucleus, the site of viral DNA synthesis. No host factors that interact with UL70 have been reported. In this study, we provide the first direct evidence that UL70 specifically interacts with Snapin, a human protein that is predominantly localized in the cytoplasm and is associated with cellular vesicles. The interaction between UL70 and Snapin was identified in both the two-hybrid screen in yeast and coimmunoprecipitation in human cells. The nuclear import of UL70 was decreased in cells overexpressing Snapin and increased in cells in which the expression of Snapin was downregulated with anti-Snapin small interfering RNA (siRNA) molecules, respectively. Furthermore, viral DNA synthesis and progeny production were decreased in cells overexpressing Snapin and increased in the anti-Snapin siRNA-treated cells, respectively. In contrast, no significant difference in the nuclear level of UL70, viral DNA synthesis, and progeny production was found among the parental cells and cells that either expressed a control empty vector or were treated with control siRNA molecules that did not recognize any viral or cellular transcripts. Our results suggest that Snapin may play a key role in regulating the cellular localization of UL70 in HCMV, leading to modulation of viral DNA synthesis and progeny production.

Small non-coding RNAs (sRNAs) that act as regulators of gene expression have been identified in all kingdoms of life, including microRNA (miRNA) and small interfering RNA (siRNA) in eukaryotic cells. Numerous sRNAs identified in Salmonella are encoded by genes located at Salmonella pathogenicity islands (SPIs) that are commonly found in pathogenic strains. Whether these sRNAs are important for Salmonella pathogenesis and virulence in animals has not been reported. In this study, we provide the first direct evidence that a pathogenicity island-encoded sRNA, IsrM, is important for Salmonella invasion of epithelial cells, intracellular replication inside macrophages, and virulence and colonization in mice. IsrM RNA is expressed in vitro under conditions resembling those during infection in the gastrointestinal tract. Furthermore, IsrM is found to be differentially expressed in vivo, with higher expression in the ileum than in the spleen. IsrM targets the mRNAs coding for SopA, a SPI-1 effector, and HilE, a global regulator of the expression of SPI-1 proteins, which are major virulence factors essential for bacterial invasion. Mutations in IsrM result in disregulation of expression of HilE and SopA, as well as other SPI-1 genes whose expression is regulated by HilE. Salmonella with deletion of isrM is defective in bacteria invasion of epithelial cells and intracellular replication/survival in macrophages. Moreover, Salmonella with mutations in isrM is attenuated in killing animals and defective in growth in the ileum and spleen in mice. Our study has shown that IsrM sRNA functions as a pathogenicity island-encoded sRNA directly involved in Salmonella pathogenesis in animals. Our results also suggest that sRNAs may represent a distinct class of virulence factors that are important for bacterial infection in vivo., Author Summary Regulated expression of virulence factors is essential for infection by human pathogens such as Salmonella. Small non-coding RNAs (sRNAs) that act as regulators of gene expression have been identified in all kingdoms of life, and many sRNAs in Salmonella are encoded by genes located at Salmonella pathogenicity islands commonly found in pathogenic strains. In this study, we demonstrated that a pathogenicity island-encoded sRNA directly targets the expression of both a global regulator of virulence genes as well as a specific virulence factor critical for Salmonella pathogenesis. The sRNA is important for Salmonella invasion of epithelial cells, replication inside macrophages, and virulence/colonization in mice, representing the first example of a pathogenicity island-encoded sRNA that is directly involved in Salmonella pathogenesis in vivo. Our study suggests that sRNA may function as a distinct class of virulence factors that significantly contribute to bacterial infection in vivo. Furthermore, our results raise the possibility of developing new strategies against bacterial infection by preventing the expression of regulatory sRNAs.

Herpes simplex virus type-1 (HSV-1) encodes a protease responsible for proteolytic processing of the virus assembly protein, ICP35 (infected cell protein 35). The coding region of ICP35 is contained within the gene that encodes the protease, and ICP35 shares amino acid identity with the carboxyl-terminal 329 amino acids of the protease. The HSV-1 protease was expressed in Escherichia coli as a fusion protein containing a unique epitope and the protein A Fc binding domain at its carboxyl terminus. The fusion protease underwent autoproteolytic cleavage at two distinct sites. The size of the cleavage products containing the carboxyl-terminal epitope mapped one cleavage site near the carboxyl terminus of the protease corresponding to the proteolytic processing site of ICP35, and the second site proximal to the amino terminus consistent with previous data. The carboxyl-terminal autoproteolytic cleavage products were partially purified on an IgG affinity column by virtue of the protein A Fc binding domain and subjected to direct amino-terminal sequence analysis. Protein sequencing revealed that cleavage occurs between the Ala and Ser residues at amino acids 610/611 and 247/248 of the HSV-1 protease. The flanking sequences share homology with each other and are highly conserved in homologous proteases of other herpes viruses.

Ribonuclease P (RNase P) complexed with external guide sequence (EGS) represents a novel nucleic acid-based gene interference approach to modulate gene expression. We have previously used an in vitro selection procedure to generate 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 mRNA encoding the protease of human cytomegalovirus (HCMV), which is essential for viral capsid formation and replication. The EGS variant was about 35-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 95% in the expression of the protease and a reduction of 4,000-fold in viral growth were observed in HCMV-infected cells that expressed the EGS variant, whereas a reduction of 80% in the protease expression and an inhibition of 150-fold in viral growth were detected in cells that expressed the EGS derived from a natural tRNA sequence. No significant reduction in viral protease expression or viral growth was observed in cells that either did not express an EGS or produced a "disabled" EGS, which carried nucleotide mutations that precluded RNase P recognition. Our results provide direct evidence that engineered EGS variant is highly effective in blocking HCMV expression and growth by targeting the viral protease. Furthermore, these results demonstrate the utility of engineered EGS RNAs in gene targeting applications, including the inhibition of HCMV infection by blocking the expression of virus-encoded essential proteins.

The use of RNase P ribozyme (M1GS catalytic RNA) for inhibition of murine cytomegalovirus (MCMV) propagation in mice is described in this chapter. General information about RNase P based technology is included and followed by detailed protocols focused on (1) construction and in vitro cleavage assay of the customized M1GS ribozyme, (2) stable expression of the M1GS RNA and evaluation of its activity in inhibition of viral gene expression and growth in cultured cells, and (3) investigation of M1GS-mediated inhibition of viral infection and pathogenesis in animals. Using these methods, we have successfully constructed catalytic M1-1 RNA against the MCMV assembly protein (mAP) and M80 mRNA. Our recent study has demonstrated that an 80% reduction in the expression of mAP and M80 and a 2,000-fold reduction in viral growth were observed in cells expressing the ribozyme. Furthermore, after the functional ribozyme-expressing constructs were delivered into MCMV-infected SCID mice, a significant reduction of viral gene expression and infection was detected, and the survival of the infected animals was significantly improved. Collectively, our data demonstrate the feasibility of the use of RNase P ribozyme for inhibition of viral gene expression in animals and support the utility of RNase P ribozyme for gene-targeting applications in vivo.

The use of RNase P ribozyme (M1GS catalytic RNA) for inhibition of murine cytomegalovirus (MCMV) propagation in mice is described in this chapter. General information about RNase P based technology is included and followed by detailed protocols focused on (1) construction and in vitro cleavage assay of the customized M1GS ribozyme, (2) stable expression of the M1GS RNA and evaluation of its activity in inhibition of viral gene expression and growth in cultured cells, and (3) investigation of M1GS-mediated inhibition of viral infection and pathogenesis in animals. Using these methods, we have successfully constructed catalytic M1-1 RNA against the MCMV assembly protein (mAP) and M80 mRNA. Our recent study has demonstrated that an 80% reduction in the expression of mAP and M80 and a 2,000-fold reduction in viral growth were observed in cells expressing the ribozyme. Furthermore, after the functional ribozyme-expressing constructs were delivered into MCMV-infected SCID mice, a significant reduction of viral gene expression and infection was detected, and the survival of the infected animals was significantly improved. Collectively, our data demonstrate the feasibility of the use of RNase P ribozyme for inhibition of viral gene expression in animals and support the utility of RNase P ribozyme for gene-targeting applications in vivo.

Previous studies have shown that the herpes simplex virus 1 gene UL26 encodes a 635-amino acid protease that cleaves approximately 20 amino acids from the carboxyl terminus of itself and of a 329-amino acid product of the UL26.5 gene. The results of studies with a variety of protease inhibitors showed that the UL26 protease was inhibited by serine protease inhibitors but not by inhibitors of cysteine protease, aspartic acid protease, or metalloprotease. Mutations resulting in amino acid substitutions, deletions, or insertion of stop codons in the gene or of 20-amino acid stretches into the protease have delineated the dispensable domains I and IV at the amino and carboxyl domains of the gene product. The essential carboxyl-proximal domain (III) can be separated from the essential amino-proximal domain (II) by at least 20 amino acids. The amino-proximal domain is the most conserved region among varicella-zoster virus and human cytomegalovirus homologues of UL26. Of the conserved aspartic acid, histidine, or serine amino acids in this domain, only histidine-61 and -148 could not be replaced without impairment of the proteolytic activity.

Background Salmonella enterica, a common food-borne bacterial pathogen, is believed to change its protein expression profile in the presence of different environmental stress such as that caused by the exposure to hydrogen peroxide (H2O2), which can be generated by phagocytes during infection and represents an important antibacterial mechanism of host cells. Among Salmonella proteins, the effectors of Salmonella pathogenicity island 1 and 2 (SPI-1 and SPI-2) are of particular interest since they are expressed during host infection in vivo and are important for invasion of epithelial cells and for replication in organs during systemic infection, respectively. However, the expression profiles of these proteins upon exposure to H2O2 or to host cells in vivo during the established phase of systemic infection have not been extensively studied. Results Using stable isotope labeling coupled with mass spectrometry, we performed quantitative proteomic analysis of Salmonella enterica serovar Enteritidis and identified 76 proteins whose expression is modulated upon exposure to H2O2. SPI-1 effector SipC was expressed about 3-fold higher and SopB was expressed approximately 2-fold lower in the presence of H2O2, while no significant change in the expression of another SPI-1 protein SipA was observed. The relative abundance of SipA, SipC, and SopB was confirmed by Western analyses, validating the accuracy and reproducibility of our approach for quantitative analysis of protein expression. Furthermore, immuno-detection showed substantial expression of SipA and SipC but not SopB in the late phase of infection in macrophages and in the spleen of infected mice. Conclusions We have identified Salmonella proteins whose expression is modulated in the presence of H2O2. Our results also provide the first direct evidence that SipC is highly expressed in the spleen at late stage of salmonellosis in vivo. These results suggest a possible role of SipC and other regulated proteins in supporting survival and replication of Salmonella under oxidative stress and during its systemic infection in vivo.

We report the design and characterization of a microarray with 46 short virus-specific oligonucleotides for detecting influenza A virus of 5 subtypes: H1N1, H1N2, H3N2, H5N1, and H9N2. A unique combination of 3 specific modifications was introduced into the microarray assay: (1) short probes of 19 to 27 nucleotides, (2) simple amplification of full-length hemagglutinin and neuraminidase cDNAs with universal primers, and (3) Klenow-mediated labeling and further amplification of the samples before hybridization. The assay correctly and specifically detected and subtyped 11 different influenza A isolates from human, avian, and swine species representing the 5 subtypes. When tested with 225 clinical samples, 20 were detected to be positive using our microarray-based assay, whereas only 10 were positive by the conventional culture method. The entire analysis was completed within 7 h. Thus, these modifications result in a specific, sensitive, and rapid microarray assay and may be used for constructing microarrays for the detection of all influenza subtypes and strains.