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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.

The precise cell type hosting latent human cytomegalovirus (HCMV) remains elusive. Here, we report that HCMV reprogrammes human haematopoietic progenitor cells (HPCs) into a unique monocyte subset to achieve latency. Unlike conventional monocytes, this monocyte subset possesses higher levels of B7-H4, IL-10 and inducible nitric oxide synthase (iNOS), a longer lifespan and strong immunosuppressive capacity. Cell sorting of peripheral blood from latently infected human donors confirms that only this monocyte subset, representing less than 0.1% of peripheral mononuclear cells, is HCMV genome-positive but immediate-early-negative. Mechanistic studies demonstrate that HCMV promotes the differentiation of HPCs into this monocyte subset by activating cellular signal transducer and activator of transcription 3 (STAT3). In turn, this monocyte subset generates a high level of nitric oxide (NO) to silence HCMV immediate-early transcription and promote viral latency. By contrast, the US28-knockout HCMV mutant, which is incapable of activating STAT3, fails to reprogramme the HPCs and achieve latency. Our findings reveal that via activating the STAT3-iNOS-NO axis, HCMV differentiates human HPCs into a longevous, immunosuppressive monocyte subset for viral latency.

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.

Herpes simplex virus 1 (HSV-1) is a herpesvirus that may cause cold sores or keratitis in healthy or immunocompetent individuals, but can lead to severe and potentially life-threatening complications in immune-immature individuals, such as neonates or immune-compromised patients. Like all other herpesviruses, HSV-1 can engage in lytic infection as well as establish latent infection. Current anti-HSV-1 therapies effectively block viral replication and infection. However, they have little effect on viral latency and cannot completely eliminate viral infection. These issues, along with the emergence of drug-resistant viral strains, pose a need to develop new compounds and novel strategies for the treatment of HSV-1 infection. Genome editing methods represent a promising approach against viral infection by modifying or destroying the genetic material of human viruses. These editing methods include homing endonucleases (HE) and the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR associated protein (Cas) RNA-guided nuclease system. Recent studies have showed that both HE and CRISPR/Cas systems are effective in inhibiting HSV-1 infection in cultured cells in vitro and in mice in vivo. This review, which focuses on recently published progress, suggests that genome editing approaches could be used for eliminating HSV-1 latent and lytic infection and for treating HSV-1 associated diseases.

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.

Cellular receptor-mediated signaling pathways play critical roles during the initial immune response to Human Cytomegalovirus (HCMV) infection. However, the involvement of type-I transmembrane glycoprotein CD147/EMMPRIN (extracellular matrix metalloproteinase inducer) in the antiviral response to HCMV infection is still unknown. Here, we demonstrated the specific knockdown of CD147 significantly decreased HCMV-induced activation of NF-κB and Interferon-beta (IFN-β), which contribute to the cellular antiviral responses. Next, we confirmed that HCMV-encoded miR-US25-1-5p could target the 3′ UTR (Untranslated Region) of CD147 mRNA, and thus facilitate HCMV lytic propagation at a low multiplicity of infection (MOI). The expression and secretion of Cyclophilin A (sCyPA), as a ligand for CD147 and a proinflammatory cytokine, were up-regulated in response to HCMV stimuli. Finally, we confirmed that CD147 mediated HCMV-triggered antiviral signaling via the sCyPA-CD147-ERK (extracellular regulated protein kinases)/NF-κB axis signaling pathway. These findings reveal an important HCMV mechanism for evading antiviral innate immunity through its encoded microRNA by targeting transmembrane glycoprotein CD147, and a potential cause of HCMV inflammatory disorders due to the secretion of proinflammatory cytokine CyPA.

Human cytomegalovirus (HCMV), a herpesvirus, is an important human pathogen that causes asymptomatic infections in healthy or immunocompetent individuals but can lead to severe and potentially life-threatening complications in immune-immature individuals such as neonates or immune-compromised patients such as organ-transplant recipients and HIV-positive individuals. Congenital HCMV infection represents a significant public health issue and poses substantial healthcare and economic burden to society. This virus causes the most common viral congenital infection worldwide, and is the leading non-genetic cause of sensorineural hearing loss in children in developed countries. Congenital HCMV infection is believed to fulfill the criteria of the American College of Medical Genetics to be considered as a condition targeted for a newborn screening program. This is because congenital HCMV infection can be identified during a time (within 2 days after birth) at which it would not ordinarily be detected clinically, and there are demonstrated benefits of early detection, timely intervention, and efficacious treatment of the condition. Recent progresses in developing polymerase chain reaction-based approaches to detect HCMV in samples obtained from newborns have generated much excitement in the field. In this review, we highlight the recent progress in diagnostic techniques that could potentially be used for the detection of HCMV infection in neonates and its direct implications in public health settings for diagnosing congenital HCMV infection.

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.

Salmonella have developed a sophisticated machinery to evade immune clearance and promote survival in the infected cells. Previous studies were mostly focused on either bacteria itself or host cells, the interaction mechanism of host-pathogen awaits further exploration. In the present study, we show that Salmonella can exploit mammalian cell non-classical microRNA processing machinery to further process bacterial small non-coding RNAs into microRNA-like fragments. Sal-1, one such fragment with the highest copy number in the infected cells, is derived from Salmonella 5′-leader of the ribosomal RNA transcript and has a ‘stem’ structure-containing precursor. Processing of Sal-1 precursors to mature Sal-1 is dependent on host cell Argonaute 2 (AGO2) but not Dicer. Functionally, depleting cellular Sal-1 strongly renders the Salmonella bacteria less resistant to the host defenses both in vitro and in vivo. In conclusion, we demonstrate a novel strategy for Salmonella evading the host immune clearance, in which Salmonella produce microRNA-like functional RNA fragments to establish a microenvironment facilitating bacterial survival.

Autologous T cells expressing chimeric antigen receptors (CARs) specific for CD19 have demonstrated remarkable efficacy as therapeutics for B cell malignancies. In the present study, we generated FLAG-tagged CD19-specific CAR-T cells (CD19-FLAG) and compared them to their non-tagged counterparts for their effects on solid and hematological cancer cells

It is widely suspected, yet controversial, that infection plays an etiologic role in the development of acute lymphoblastic leukemia (ALL), the most common childhood cancer and a disease with a confirmed prenatal origin in most cases. We investigated infections at diagnosis and then assessed the timing of infection at birth in children with ALL and age, gender, and ethnicity matched controls to identify potential causal initiating infections. Comprehensive untargeted virome and bacterial analyses of pretreatment bone marrow specimens (n = 127 ALL in comparison with 38 acute myeloid leukemia cases in a comparison group) revealed prevalent cytomegalovirus (CMV) infection at diagnosis in childhood ALL, demonstrating active viral transcription in leukemia blasts as well as intact virions in serum. Screening of newborn blood samples revealed a significantly higher prevalence of in utero CMV infection in ALL cases (n = 268) than healthy controls (n = 270) (odds ratio [OR], 3.71, confidence interval [CI], 1.56-7.92, P = .0016). Risk was more pronounced in Hispanics (OR=5.90, CI=1.89-25.96) than in non-Hispanic whites (OR=2.10 CI= 0.69-7.13). This is the first study to suggest that congenital CMV infection is a risk factor for childhood ALL and is more prominent in Hispanic children. Further investigation of CMV as an etiologic agent for ALL is warranted.

Salmonella is a facultative intracellular pathogen that causes a diverse spectrum of diseases, ranging from mild self-limiting gastroenteritis to fatal systemic typhoid fever. The 3 main serovars of Salmonella are Typhi, Typhimurium, and Enteritidis. S. Enteritidis has recently become the most common cause of food poisoning, with a significant number of case reports [1, 2]. Salmonella have evolved sophisticated machinery to alter host cell function that is essential for its virulence capabilities and survival [3]. Therefore, understanding the host-bacterial interactions is central in developing prevention and treatment strategies for the diseases associated with Salmonella infection. To infect the host, Salmonella must not only pass through the upper gastrointestinal tract and the intestinal epithelial barrier, but also overcome attack from both the innate and adaptive immune systems [4]. As one of major components of host innate immunity, macrophages play an essential role not only in host defense against infection by many pathogens, using a compartmentalized dual detection system, but also in the regulation of immune responses and inflammation [5]. The recruitment and activation of macrophages serves as a major mechanism of defense against infection by different intracellular pathogens [6]. Macrophages can be activated by various proinflammatory cytokines. For instance, macrophage colony-stimulating factor (M-CSF; CSF1) can recruit macrophages to the infection site and promote macrophages to phagocytose and kill foreign microorganisms [7]. Thus, regulation of secreted M-CSF levels may contribute to the modulation of macrophage recruitment and activation. Like other intracellular pathogens, Salmonella can use multiple complex and versatile mechanisms, including type III secretion systems (T3SS), to deliver virulence factors into host cells [8]. These virulence factors may re-program host cells and modulate the host immune response. Accumulating evidence suggests that Salmonella species, host cells, and environmental factors compose a complex network that precisely regulates the immune response during persistent Salmonella infection. The regulatory mechanism of this complicate system, however, remains incompletely understood. MicroRNAs (miRNAs) are approximately 22-nucleotide noncoding RNAs that regulate target cellular messenger RNA (mRNA) expression at the posttranscriptional level [9]. In the context of interplay between the immune system and pathogens, miRNAs have a prominent role in the control of host-pathogen interactions [10]. miR-155, miR-142a, miR-223, miR-146, miR-9, and miR-181a have been recently shown to be crucial regulators of innate immunity and inflammatory responses [11]. The role of miRNAs in microbiota-host interactions has also been reported recently, in which Toll-like receptors (TLRs) serve as essential mediators modulating both gut microbiota and miRNAs/mRNAs in humans [12, 13]. Wang et al [14] further showed that miR-155 expression was induced by TLR signals and that the inducible miR-155 promotes type I interferon signaling in antiviral innate immunity. More recently, Schulte et al [15] analyzed the host miRNA response to Salmonella and uncovered the control of major cytokines by the let-7 family. The discovery of the broad function of miRNAs provides a new layer of molecular control of immune functions during pathogen infection. The present study is the first to show that virulent Salmonella can actively manipulate the host cell immune response by upregulating miR-128 levels in intestinal epithelial cells, which, in turn, decreases epithelia-secreted M-CSF and impairs M-CSF–mediated macrophage recruitment. Moreover, we found that delivery of anti–miR-128 antisense oligonucleotide (ASO) by polyethylenimine (PEI) significantly reduced the level of Salmonella infection. Our study identified a new miR-128–targeting M-CSF mechanism in modulating the interaction between Salmonella and the intestinal epithelia.

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.

The efficacy of interferon α (IFNα) therapy for chronic hepatitis B (CHB) patients is about 40% and often associates with adverse side-effects, thus identification of an easy accessible biomarker that can predict the outcome of IFNα treatment for individual CHB patients would be greatly helpful. Recent reports by us and others show that microRNAs encoded by human cytomegalovirus (HCMV) were readily detected in human serum and can interfere with lymphocyte responses required by IFNα therapeutic effect. We thus postulate that differential expression profile of serum HCMV miRNAs in CHB patients may serve as indicator to predict the efficacy of IFNα treatment for CHB patients. Blood was drawn from 56 individual CHB patients prior to IFNα treatment. By quantifying 13 HCMV miRNAs in serum samples, we found that the levels of HCMV-miR-US4-1 and HCMV-miR-UL-148D were significantly higher in IFNα-responsive group than in IFNα-non-responsive group. In a prospective study of 96 new CHB patients, serum level of HCMV-miR-US4-1 alone classified those who were and were not responsive to IFN-α treatment with correct rate of 84.00% and 71.74%, respectively. In conclusion, our results demonstrate that serum HCMV-miR-US4-1 can serve as a novel biomarker for predicting the outcome of IFNα treatment in CHB patients.

Neuronal diseases, including retinal disorders, stroke, Alzheimer's disease, Parkinson's disease and spinal cord injury, affect a large number of people worldwide and cause heavy social and economic burdens. Although many efforts have been made by scientists and clinicians to develop novel drug and healthcare strategies, few of them received satisfactory outcomes to date. Lycium barbarum is a traditional homology of medicine and food in Chinese medicine, with the capability to nourish the eyes, liver and kidneys. Recent studies have also explored its powerful neuro-protective effects on a number of neuronal diseases. In the current review, we collected key recent findings regarding the neuro-protective effects and mechanisms of L. barbarum derivatives, primarily its polysaccharide (LBP) , in some common diseases of the nervous system. A comprehensive comparison with currently available drugs has also been discussed. In general, LBP is a promising neuronal protector with potent ameliorative effects on key pathological events, such as oxidative stress, inflammation, apoptosis and cell death with minimal side effects.

Diagnosis of hepatitis B virus (HBV)-positive hepatocellular carcinoma (HCC), particularly HCC independent of cirrhosis etiology, presents a great challenge because of a lack of biomarkers. Here we test the hypothesis that expression profiles of microRNAs (miRNAs) in serum can serve as biomarkers for diagnosis of HBV infection and HBV-positive HCC. We recruited 513 subjects (210 controls and 135 HBV-, 48 hepatitis C virus (HCV)-, and 120 HCC-affected individuals) and employed a strategy of initial screening by Solexa sequencing followed by validation with TaqMan probe-based quantitative reverse transcription-PCR assay. First, because of a close link between chronic hepatitis B and HCC, we compared miRNA expression profiles in HBV serum with that in control serum and successfully obtained 13 miRNAs that were differentially expressed in HBV serum. This 13-miRNA–based biomarker accurately discriminated not only HBV cases from controls and HCV cases, but also HBV-positive HCC cases from control and HBV cases. Second, we directly compared miRNA expressions in HCC serum with those in controls and identified 6 miRNAs that were significantly upregulated in HCC samples. Interestingly, 2 of these miRNAs, miR-375 and miR-92a, were also identified by our first approach as HBV specific. When we employed 3 of these miRNAs (miR-25, miR-375, and let-7f) as biomarkers, we could clearly separate HCC cases from controls, and miR-375 alone had an ROC of 0.96 (specificity: 96%; sensitivity: 100%) in HCC prediction. In conclusion, our study demonstrates for the first time that serum miRNA profiles can serve as novel and noninvasive biomarkers for HBV infection and HBV-positive HCC diagnosis. Cancer Res; 70(23); 9798–807. ©2010 AACR.

The CRISPR/Cas9 system has been applied in the genome editing and disruption of latent infections for herpesviruses such as the herpes simplex virus, Epstein–Barr virus, cytomegalovirus, and Kaposi’s sarcoma-associated herpesvirus. CRISPR/Cas9-directed mutagenesis can introduce similar types of mutations to the viral genome as can bacterial artificial chromosome recombination engineering, which maintains and reconstitutes the viral genome successfully. The cleavage mediated by CRISPR/Cas9 enables the manipulation of disease-associated viral strains with unprecedented efficiency and precision. Additionally, current therapies for herpesvirus productive and latent infections are limited in efficacy and cannot eradicate viruses. CRISPR/Cas9 is potentially adapted for antiviral treatment by specifically targeting viral genomes during latent infections. This review, which focuses on recently published progress, suggests that the CRISPR/Cas9 system is not only a useful tool for basic virology research, but also a promising strategy for the control and prevention of herpesvirus latent infections.

A fundamental challenge in gene therapy is to develop approaches for delivering nucleic acid-based gene interfering agents, such as small interfering RNAs and ribozymes, to the appropriate cells in a way that is tissue/cell specific, efficient, and safe. Using human cytomegalovirus (HCMV) infection of differentiated macrophages as the model, we showed that Salmonella can efficiently deliver RNase P-based ribozyme sequence in specific human cells, leading to substantial ribozyme expression and effective inhibition of viral infection. We constructed a functional RNase P ribozyme (M1GS RNA) that targets the overlapping mRNA region of two HCMV capsid proteins, the capsid scaffolding protein (CSP) and assemblin, which are essential for viral capsid formation. Substantial expression of ribozymes was observed in human differentiated macrophages that were treated with attenuated Salmonella strains carrying the ribozyme sequence constructs. A reduction of 87–90% in viral CSP expression and a reduction of about 5,000-fold in viral growth were observed in cells that were treated with Salmonella carrying the sequence of the functional ribozyme but not with those carrying the sequence of a control ribozyme that contained mutations abolishing the catalytic activity. To our knowledge, this study showed for the first time that ribozymes expressed following targeted gene transfer with Salmonella -based vectors are highly active and specific in blocking viral infection. Moreover, these results demonstrate the feasibility to develop Salmonella -mediated gene transfer of RNase P ribozymes as an effective approach for gene-targeting applications.

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.

Effector proteins encoded by Salmonella pathogenicity islands play a key role in promoting bacterial intracellular survival, colonization, and pathogenesis. In this study, we investigated the function of the virulence-associated effector SrfA (SsrAB regulated factor) both in macrophages in vitro and in infected mice in vivo. SrfA was secreted into the cytoplasm during S. Typhimurium infection and disassociated IL-1R–associated kinase-1 (IRAK-1) from the IRAK-1–Toll interacting protein (Tollip) complex by interacting with Tollip. The released IRAK-1 was phosphorylated and subsequently activated the NF-κB signaling pathway, which enhanced the LPS-induced expression of inflammatory cytokines, such as IL-8, IL-1β, and TNF-α. The coupling of ubiquitin to endoplasmic reticulum degradation aa 183–219 domain of Tollip is the binding region for SrfA, and both the MDaa207–226 and CTaa357–377 regions of SrfA mediate binding to Tollip and NF-κB signaling activation. Deletion of SrfA in S. Typhimurium had no notable effects on its replication but impaired the induction of NF-κB activation in infected macrophages. The mice infected with srfA-deficient bacteria exhibited a decreased inflammatory response and an increased survival rate compared with those infected with wild-type S. Typhimurium. We conclude that SrfA is a novel Salmonella virulence effector that helps modulate host inflammatory responses by promoting NF-κB signaling activation.

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.

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-Å 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.

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.

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.

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.

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.

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.

The three-dimensional structure of the intact human cytomegalovirus (HCMV) was determined to 18-Å resolution by electron cryomicroscopy and computer reconstruction. Its capsid shell is composed of pentons, hexons, and triplexes arranged on a T = 16 icosahedral lattice and is identical to that of the B-capsid isolated from host cell nuclei. An icosahedrally ordered tegument layer formed by 960 copies of filamentous density is also visualized, which interacts with the pentons, hexons, and triplexes of the underlying capsid. The observed structural similarities and differences of HCMV with those of herpes simplex virus offer insights into the significance of the different tegument components for their infection processes while maintaining similar capsids.

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.

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.

A quantitative real time reverse-transcription polymerase chain reaction (qRT-PCR) assay with specific primers recommended by the World Health Organization (WHO) has been widely used successfully for detection and monitoring of the pandemic H1N1/2009 influenza A virus. In this study, we report the design and characterization of a novel set of primers to be used in a qRT-PCR assay for detecting the pandemic H1N1/2009 virus. The newly designed primers target three regions that are highly conserved among the hemagglutinin (HA) genes of the pandemic H1N1/2009 viruses and are different from those targeted by the WHO-recommended primers. The qRT-PCR assays with the newly designed primers are highly specific, and as specific as the WHO-recommended primers for detecting pandemic H1N1/2009 viruses and other influenza viruses including influenza B viruses and influenza A viruses of human, swine, and raccoon dog origin. Furthermore, the qRT-PCR assays with the newly designed primers appeared to be at least 10-fold more sensitive than those with the WHO-recommended primers as the detection limits of the assays with our primers and the WHO-recommended primers were 2.5 and 25 copies of target RNA per reaction, respectively. When tested with 83 clinical samples, 32 were detected to be positive using the qRT-PCR assays with our designed primers, while only 25 were positive by the assays with the WHO-recommended primers. These results suggest that the qRT-PCR system with the newly designed primers represent a highly sensitive assay for diagnosis of the pandemic H1N1/2009 virus infection.

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.

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.

An estimated one-third of individuals positive for HIV are also infected with hepatitis C virus (HCV). Chronic infection with HCV can lead to serious liver disease including cirrhosis and hepatocellular carcinoma. Liver-related disease is among the leading causes of death in patients with HIV, and individuals with HIV and HCV co-infection are found to progress more rapidly to serious liver disease than mono-infected individuals. The mechanism by which HIV affects HCV infection in the absence of immunosuppression by HIV is currently unknown. In a recent article published in BMC Immunology, Qu et al. demonstrated that HIV tat is capable of inducing IP-10 expression. Further, they were able to show that HIV tat, when added to cells, was able to enhance the replication of HCV. Importantly, the increase in HCV replication by tat was found to be dependent on IP-10. This work has important implications for understanding the effect HIV has on the outcome of HCV infection in co-infected individuals. The findings of Qu et al. may inform the design of intervention and treatment strategies for co-infected individuals. Please see related article: http://www.biomedcentral.com/1471-2172/13/15.

The methods of using engineered RNase P catalytic RNA (termed as M1GS RNA) for in vitro and in vivo in trans-cleavage of target viral mRNA are described in this chapter. Detailed information is focused on (1) mapping accessible regions of target viral mRNA in infected cells, (2) generation and in vitro cleavage assay of the customized M1GS ribozyme, (3) stable expression of M1GS RNAs and evaluation of its antiviral activity in cultured cells. Using these methods, we have constructed functional M1GS ribozyme that can cleave an overlapping region of the mRNAs coding for the human cytomegalovirus (HCMV) capsid scaffolding protein (CSP) and assemblin in vitro. Further study has demonstrated that, in cultured human cells expressing the functional M1GS ribozyme and infected with HCMV, more than 85% reduction in the expression of CSP and assemblin and a 4,000-fold reduction in viral growth were achieved. Our study provided the direct evidence that the customized M1GS ribozyme can be used as an effective gene-targeting agent for in trans-cleavage of viral genes and inhibition of viral growth in cultured cells.

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.