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The use of external guide sequence (EGS) in directing endogenous ribonuclease P (RNase P) for inhibition of viral propagation is described in this chapter, with an emphasis on chemically modified EGSs and their extracellular delivery. Targeting of the mRNA-encoding human cytomegalovirus (HCMV) protease by DNA-based EGSs is presented as an example of how to design chemically modified EGSs for antiviral applications. General information about the EGS-based technology is included, followed by detailed protocols for EGS design, human RNase P purification, in vitro assay of EGS activity, liposome-mediated delivery of chemically modified EGSs and detection of their distribution in cells, and an assay of EGS activity for blocking growth of HCMV in cultured cells.

An in vitro selection procedure for identifying highly efficient RNase P ribozyme (M1GS RNA) variants is presented as a model system for engineering ribozymes to improve their catalytic efficiency. Detailed protocols as well as the rationale for setting up such a system are included, using the mRNA sequence that encodes the thymidine kinase (TK) of herpes simplex virus 1 (HSV 1) as a target substrate of choice. Using the selection system, we have successfully generated M1GS RNA variants that more efficiently cleave the TK mRNA in vitro and more effectively inhibit the TK expression in cultured cells than the ribozyme derived from the wild-type RNase P ribozyme sequence. The in vitro selection system represents a novel and effective approach for engineering highly active RNase P ribozymes that can be used in both basic research and clinical therapeutic settings.

External guide sequences (EGSs) are oligonucleotides that consist of a sequence that is complementary to a target mRNA and recruit intracellular RNase P for specific degradation of the target RNA. Recent studies indicate that increasing the targeting activity of EGSs in directing human RNase P to cleave an mRNA in vitro can lead to better efficacies of the EGSs in inducing RNase P-mediated inhibition of the expression of the target mRNA in cultured cells. This chapter will describe the procedure for the generation of highly functional EGSs by in vitro selection. We also describe protocols for in vitro evaluation of the activity of the EGSs. These methods should provide general guidelines for using in vitro selection for generating highly active EGSs for gene-targeting applications.

A ribozyme (M1GS RNA) constructed from the catalytic RNA subunit of RNase P fromEscherichia coliwas used to target the overlapping region of two human cytomegalovirus (HCMV) mRNAs, which encode for the viral essential protease (PR) and capsid assembly proteins (AP), respectively. The results show a reduction of >80% in the expression levels of PR and AP and an inhibition of ~2000-fold of viral growth in cells that stably expressed the ribozyme. In comparison

Human cytomegalovirus (HCMV), a ubiquitous herpesvirus, causes a lifelong subclinical infection in healthy adults but leads to significant morbidity and mortality in neonates and immunocompromised individuals. Its ability to grow in different cell types is responsible for HCMV-associated diseases, including mental retardation and retinitis, and vascular disorders. To globally assess viral gene function for replication in cells, we determined the genomic sequence of a bacterial artificial chromosome (BAC)-based clone of HCMV Towne strain and used this information to delete each of its 162 unique ORFs and generate a collection of viral mutants. The growth of these mutants in different cultured cells was examined to systematically investigate the necessity of each ORF for replication. Our results showed that 45 ORFs are essential for viral replication in fibroblasts and 117 are nonessential. Some genes were found to be required for viral replication in retinal pigment epithelial cells and microvascular endothelial cells, but not in fibroblasts, indicating their role as tropism factors. Interestingly, several viral mutants grew 10- to 500-fold better than the parental strain in different cell types, suggesting that the deleted ORFs encode replication temperance or repressing functions. Thus, HCMV encodes supportive and suppressive growth regulators for optimizing its replication in human fibroblasts, epithelial, and endothelial cells. Suppression of viral replication by virus-encoded temperance factors represents a novel mechanism for regulating the growth of an animal virus, and may contribute to HCMV's optimal infection of different tissues and successful proliferation among the human population.

Ribonuclease P (RNase P) is a ubiquitous ribonucleoprotein complex responsible for the biosynthesis of tRNA. This enzyme from Escherichia coli contains a catalytic RNA subunit (M1 ribozyme) and a protein subunit (C5 cofactor). M1 ribozyme cleaves an RNA helix that resembles the acceptor stem and T-stem structure of its natural tRNA substrate. When covalently linked with a guide sequence, M1 RNA can be engineered into a sequence-specific endonuclease, M1GS ribozyme, which can cleave any target RNA sequences that base pair with the guide sequence. Recent studies indicate that M1GS ribozymes efficiently cleave the mRNAs of herpes simplex virus 1, human cytomegalovirus, and cancer causing BCR-ABL proteins in vitro and effectively inhibit the expression of these mRNAs in cultured cells. Moreover, RNase P ribozyme variants that are more active than the wild type M1 RNA can be generated using in vitro selection procedures and the selected variants are also more effective in inhibiting gene expression in cultured cells. These results demonstrate that engineered RNase P ribozymes represent a novel class of promising gene-targeting agents for applications in both basic research and clinical therapy. This review discusses the principle underlying M1GS-mediated gene inactivation and methodologies involved in effective M1GS construction, expression in vivo and emerging prospects of this technology for gene therapy.

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

Role of viral genes in modulating T helper 1 (Th1) and T helper 2 (Th2) balance is of principal interest in the study of cytomegalovirus (CMV) immunity. Murine CMV (MCMV) mutants were used to explore a possible mechanism for the ability of virus to induce a predominant Th1 response and to suppress Th2 response by examining the production of Th1 (IFN-γ, IL-2) and Th2 (IL-4, IL-10) cytokines by the splenocytes of mice infected with wild type (WT) and MCMV mutants. Results ( n =6) show that as compared with WT, the MCMV mutant with specific disruption of M43 gene upregulates the production of IL-4 ( P =0.0002) and to a lesser extent IL-10 ( P =0.015) at 14 days post infection. This indicates that M43 gene may play a role in suppressing Th2 (IL-4) production, especially in the later stage of infection. The IL-4 and IL-10 production during infection with M43 mutant occurs in the presence of a strong IFN-γ (Th1) response, overriding the cross-regulatory effects of these cytokines within the Th1/Th2 paradigm and suggesting that the predominant response during CMV infection is still a Th1 type response.

External guide sequences (EGSs) are small RNA molecules that bind to a target mRNA, form a complex resembling the structure of a tRNA, and render the mRNA susceptible to hydrolysis by RNase P, a tRNA processing enzyme. An in vitro selection procedure was used to select EGSs that direct human RNase P to cleave the mRNA encoding thymidine kinase (TK) of herpes simplex virus 1. One of the selected EGSs, TK17, was at least 35 times more active in directing RNase P in cleaving TK mRNA in vitro than the EGS derived from a natural tRNA sequence. TK17, when in complex with the TK mRNA sequence, resembles a portion of tRNA structure and exhibits an enhanced binding affinity to the target mRNA. Moreover, a reduction of 95 and 50% in the TK expression was found in herpes simplex virus 1-infected cells that expressed the selected EGS and the EGS derived from the natural tRNA sequence, respectively. Our study provides direct evidence that EGS molecules isolated by the selection procedure are effective in tissue culture. These results also demonstrate the potential for using the selection procedure as a general approach for the generation of highly effective EGSs for gene-targeting application.

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 (A(86)--C(86)) increases the rate of chemical cleavage while another mutation at nucleotide 205 (G(205)--C(205)) 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.

Salmonella has developed a sophisticated machinery to promote virulence and survival. Here we show that Salmonella can hijack mammalian cell non-classical miRNA processing machinery to further process their non-coding RNAs into miRNA-like RNAs that modulate host cell function. Using Solexa deep sequencing, we detected a panel of 19-24nt Salmonella-derived ncRNA fragments in human colonic epithelial HT-29 cells following Salmonella infection. The fragment with the highest copy number, Sal-1, was validated by qRT-PCR and northern blot analysis. With seven copies in the S. enteritidis genome, Sal-1 has a stem structure-containing precursor with a free energy of ΔG= -26.1 kcal/mol. The processing of the Sal-1 “primary” /“precursor” to the mature Sal-1 in Salmonella-infected cells is Dicer-independent but Argonaute 2-dependent. Functionally, Sal-1 targets the expression of epithelial cell inducible nitric oxide synthase (iNOS) and thus reduces the NO production and bacterial resistance of host cells. Expression or depletion of Sal-1 in mouse colonic epithelium significantly enhanced or reduced Salmonella infection in mice, respectively. Finally, the enhancement of Salmonella infection in mice by Sal-1 was completely abolished by expressing a mutant iNOS isoform that had normal NO generation but could not be targeted by Sal-1. Our results show for the first time that Salmonella can produce functional miRNA-like RNAs, which may represent a novel class of virulence factors.

Human cytomegalovirus (HCMV), infects a majority of population and can undergo life-long, latent infection, is a leading cause of opportunistic and congenital disease. HCMV can encode 17 microRNAs (miRNAs) but the roles of these viral miRNAs during viral infection are unclear. Here, we infected human bone marrow and CD34+ hematopoietic progenitor Kasumi-3 cells with HCMV (strain NR-1) and determined the kinetics of cellular levels of all hcmv miRNAs by qRT-PCR. We found that, different from most other hcmv miRNAs which had high level at early days of infection, hcmv-miR-UL148D was highly expressed at the latent infection (10days or longer). Bioinformatics and experimental validation showed that hcmv-miR-UL148D targets the 3’UTR of host cell immediately-early response gene 5 (IER5). Functional assay indicated that downregulation of IER5 by hcmv-miR-UL148D transcriptionally upregulated CDC25B, leading to lower number of Kasumi-3 cells in G2/M phase. As G2/M phase is a cell cycle of large viral replication, decrease the number of cells in G2/M phase by hcmv-miR-UL148D would suppress viral replication and facilitate hcmv entering in latent infection. In contrast, NR1-Δhcmv-miR-UL148D, an hcmv mutant deleting hcmv-miR-UL148D, was lack of ability to target IER5, increase CDC25B, control the cell cycle of host cells, and achieve latent infection. Our results collectively demonstrate that hcmv-miR-UL148D facilitates hcmv latency through modulating host cell IER5-CDC25B axis.

Using a Tn 3 -based transposon mutagenesis approach, we have generated a pool of murine cytomegalovirus (MCMV) mutants. In this study, one of the mutants, RvM27, which contained the transposon sequence at open reading frame M27, was characterized both in tissue culture and in immunocompetent BALB/c mice and immunodeficient SCID mice. Our results suggest that the M27 carboxyl-terminal sequence is dispensable for viral replication in vitro. Compared to the wild-type strain and a rescued virus that restored the M27 region, RvM27 was attenuated in growth in both BALB/c and SCID mice that were intraperitoneally infected with the viruses. Specifically, the titers of RvM27 in the salivary glands, lungs, spleens, livers, and kidneys of the infected SCID mice at 21 days postinfection were 50- to 500-fold lower than those of the wild-type virus and the rescued virus. Moreover, the virulence of the mutant virus appeared to be attenuated, because no deaths occurred among SCID mice infected with RvM27 for up to 37 days postinfection, while all the animals infected with the wild-type and rescued viruses died within 27 days postinfection. Our observations provide the first direct evidence to suggest that a disruption of M27 expression results in reduced viral growth and attenuated viral virulence in vivo in infected animals. Moreover, these results suggest that M27 is a viral determinant required for optimal MCMV growth and virulence in vivo and provide insight into the functions of the M27 homologues found in other animal and human CMVs as well as in other betaherpesviruses.

We have recently generated a pool of murine cytomegalovirus (MCMV) mutants by using a Tn 3 -based transposon mutagenesis approach. In this study, one of the MCMV mutants, RvM43, which contained the transposon inserted in open reading frame M43, was characterized. Our results provide the first direct evidence to suggest that M43 is not essential for viral replication in vitro in NIH 3T3 cells. Moreover, RvM43 exhibited a titer similar to that of the wild-type virus in the lungs, livers, spleens, and kidneys of both BALB/c and SCID mice and was as virulent as the wild-type virus in killing SCID mice that had been intraperitoneally infected with the viruses. In contrast, titers of the mutant virus in the salivary glands of the infected animals at 21 days postinfection were significantly (100 to 1,000-fold) lower than those of the wild-type virus and a rescued virus that restored the M43 region and its expression. Thus, M43 appears to be not essential for viral growth in vivo in the lungs, livers, spleens, and kidneys of infected animals and is also dispensable for virulence in killing SCID mice. Moreover, our results suggest that M43 is an MCMV determinant for growth in the salivary glands. Studies of viral genes required for replication in the salivary glands are important in understanding the mechanism of viral tropism for the salivary glands and shedding in saliva, which is believed to be one of the major routes of CMV transmission among healthy human populations.

RNase P from Escherichia coli is a tRNA-processing enzyme and consists of a catalytic RNA subunit (M1 RNA) and a protein component (C5 protein). M1GS, a gene-targeting ribozyme derived from M1, can cleave a herpes simplex virus 1 mRNA efficiently in vitro and inhibit its expression effectively in viral-infected cells. In this study, the effects of C5 on the interactions between a M1GS ribozyme and a model mRNA substrate were investigated by site-specific UV crosslink mapping. In the presence of the protein cofactor, the ribozyme regions crosslinked to the substrate sequence 3′ immediately to the cleavage site were similar to those found in the absence of C5. Meanwhile, some of the ribozyme regions (e.g. P12 and J11/12) that were crosslinked to the leader sequence 5′ immediately to the cleavage site in the presence of C5 were different from those regions (e.g. P3 and P4) found in the absence of the protein cofactor and were not among those that are believed to interact with a tRNA. Understanding how C5 affects the specific interactions between the ribozyme and its target mRNA may facilitate the development of gene-targeting ribozymes that function effectively in vivo, in the presence of cellular proteins.

A sequence-specific ribozyme (M1GS RNA) derived from the catalytic RNA subunit of RNase P from Escherichia coli was used to target the overlapping exon 3 region of the mRNAs encoding the major transcription regulatory proteins IE1 and IE2 of human cytomegalovirus. A reduction of more than 80% in the expression levels of IE1 and IE2 and a reduction of about 150-fold in viral growth were observed in human cells that stably expressed the ribozyme. In contrast, a reduction of less than 10% in the IE1/IE2 expression and viral growth was observed in cells that either did not express the ribozyme or produced a “disabled” ribozyme that carried mutations that abolished its catalytic activity. Examination of the expression of several other viral early and late genes in the cells that expressed the M1GS ribozyme further revealed an overall reduction of at least 80% in their expression. These results are consistent with the notion that the antiviral effects in these cells are due to the fact that the ribozyme specifically inhibits the expression of IE1 and IE2 and, consequently, abolishes the expression of viral early and late genes as well as viral growth. Our study is the first, to our knowledge, to use M1GS ribozyme for inhibiting human cytomegalovirus replication and demonstrates the utility of this ribozyme for antiviral applications.

An in vitro selection procedure was used to select RNase P ribozyme variants that efficiently cleaved the sequence of the mRNA encoding thymidine kinase of herpes simplex virus 1. Of the 45 selected variants sequenced, 25 ribozymes carried a common mutation at nucleotides 224 and 225 of RNase P catalytic RNA from Escherichia coli (G(224)G(225) --> AA). These selected ribozymes exhibited at least 10 times higher cleavage efficiency (k(cat)/K(m)) than that derived from the wild type ribozyme. Our results suggest that the mutated A(224)A(225) are in close proximity to the substrate and enhance substrate binding of the ribozyme. When these ribozyme variants were expressed in herpes simplex virus 1-infected cells, the levels of thymidine kinase mRNA and protein were reduced by 95-99%. Our study provides the first direct evidence that RNase P ribozyme variants isolated by the selection procedure can be used for the construction of gene-targeting ribozymes that are highly effective in tissue culture. These results demonstrate the potential for using RNase P ribozymes as gene-targeting agents against any mRNA sequences, and using the selection procedure as a general approach for the engineering of RNase P ribozymes.

Ribonuclease-resistant RNA molecules that bind to infectious human cytomegalovirus (HCMV) were isolated in vitro from a pool of randomized sequences after 16 cycles of selection and amplification. The two ligands (L13 and L19) characterized exhibited high HCMV-binding affinity in vitro and effectively inhibited viral infection in tissue culture. Their antiviral activity was also specific as they only reacted with two different strains of HCMV but not with the related herpes simplex virus 1 and human cells. These two ligands appeared to function as antivirals by blocking viral entry. Ultraviolet (UV) crosslinking studies suggested that L13 and L19 bind to HCMV essential glycoproteins B and H, respectively. Thus, RNA ligands that bind to different surface antigens of HCMV can be simultaneously isolated by the selection procedure. Our study demonstrates the feasibility of using these RNA ligands as a research tool to identify viral proteins required for infectivity and as an antiviral agent to block viral infection.

RNase P ribozyme cleaves an RNA helix that resembles the acceptor stem and T-stem structure of its natural ptRNA substrate. When covalently linked with a guide sequence, the ribozyme can function as a sequence-specific endonuclease and cleave any target RNA sequences that base pair with the guide sequence. Using a site-directed ultraviolet (UV) cross-linking approach, we have mapped the regions of the ribozyme that are in close proximity to a substrate that contains the mRNA sequence encoding thymidine kinase of human herpes simplex virus 1. Our data suggest that the cleavage site of the mRNA substrate is positioned at the same regions of the ribozyme that bind to the cleavage site of a ptRNA. The mRNA-binding domains include regions that interact with the acceptor stem and T-stem and in addition, regions that are unique and not in close contact with a ptRNA. Identification of the mRNA-binding site provides a foundation to study how RNase P ribozymes achieve their sequence specificity and facilitates the development of gene-targeting ribozymes.

External guide sequences (EGSs) are small RNA molecules which consist of a sequence complementary to a target mRNA and render the target RNA susceptible to degradation by ribonuclease P (RNase P). EGSs were designed to target the mRNA encoding thymidine kinase (TK) of herpes simplex virus 1 for degradation. These EGSs were shown to be able to direct human RNase P to cleave the TK mRNA sequence efficiently in vitro. A reduction of about 80% in the expression level of both TK mRNA and protein was observed in human cells that steadily expressed an EGS, but not in cells that either did not express the EGS or produced a "disabled" EGS which carried a single nucleotide mutation that precluded RNase P recognition. Thus, EGSs may represent novel gene-targeting agents for inhibition of gene expression and antiviral activity.

External guide sequences (EGSs) are RNA molecules that consist of a sequence complementary to a target mRNA and recruit intracellular ribonuclease P (RNase P), a tRNA processing enzyme, for specific degradation of the target mRNA. We have previously used an in vitro selection procedure to generate EGS variants that efficiently induce 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 S mRNA, pre-S/L mRNA, and pregenomic RNA (pgRNA) of hepatitis B virus (HBV), which are essential for viral replication and infection. The EGS variant was about 50-fold more efficient in inducing human RNase P to cleave the mRNA in vitro than the EGS derived from a natural tRNA. Following Salmonella -mediated gene delivery, the EGSs were expressed in cultured HBV-carrying cells. A reduction of about 97% and 75% in the level of HBV RNAs and proteins and an inhibition of about 6,000- and 130-fold in the levels of capsid-associated HBV DNA were observed in cells treated with Salmonella vectors carrying the expression cassette for the variant and the tRNA-derived EGS, respectively. Our study provides direct evidence that the EGS variant is more effective in blocking HBV gene expression and DNA replication than the tRNA-derived EGS. Furthermore, these results demonstrate the feasibility of developing Salmonella -mediated gene delivery of highly active EGS RNA variants as a novel approach for gene-targeting applications such as anti-HBV therapy.

The catalytic RNA subunit (M1 RNA) of RNase P from Escherichia coli has been converted to an endoribonuclease that specifically cleaves the mRNA that encodes thymidine kinase (TK) of herpes simplex virus 1 (HSV-1). Covalent attachment to the 3' end of M1 RNA of a sequence complementary to TK mRNA results in very efficient cleavage of the target RNA in vitro. This reaction can be stimulated by proteins extracted from both E. coli and HeLa cells. When mouse cells in culture that express the novel RNA construct are infected with HSV-1, the levels of both TK mRNA and protein are reduced by approximately 80% as compared with cells that either do not express the novel RNA construct or express constructs with certain deletions that are known to abolish the catalytic activity of M1 RNA.

The herpes simplex virus 1 UL26 open reading frame encodes a protease which cleaves a small carboxyl-terminal peptide of itself and its substrate encoded by an overlapping, 3'-coterminal transcriptional unit, designated UL26.5. The translational product of UL26.5 is infected-cell protein 35c,d (ICP35c,d) (F. Liu and B. Roizman, J. Virol. 65:206-212, 1991; F. Liu and B. Roizman, J. Virol. 65:5149-5156, 1991). The protease activity maps at the amino terminus of UL26 translation product designated Pra. Cleavage of Pra to remove the carboxyl-terminal 25 amino acids converts the protein to Prb (F. Liu and B. Roizman, Proc. Natl. Acad. Sci. USA 89:2076-2080, 1992). Other studies reported a second, amino-terminus-proximal cleavage in UL26 gene products made in Escherichia coli (I. C. Deckman, M. Hagen, and P. J. McCann III, J. Virol 66:7362-7367, 1992; C. L. DiIanni, D. A. Drier, I. C. Deckman, P.J. McCann III, F. Liu, B. Roizman, R. J. Colonno, and M. G. Cordingley, J. Biol. Chem., 368:2048-2051, 1993). We report the following results. (i) The amino-terminus-proximal cleavage of UL26 protein in eukaryotic cells generates two polypeptides, an apparent M(r)-25,000 amino-terminal polypeptide designated Prn and a carboxyl-terminal polypeptide which corresponds in electrophoretic mobility to ICP35a. Cleavage of the carboxyl-terminal 25 amino acids by the UL26 protease converted ICP35a to ICP35b. (ii) Replacement of Ala-247-Ser-248 with Arg-Pro precluded the amino-terminus-proximal cleavage. (iii) Prn, the amino-terminal product of the cleavage reaction at amino acid 247 functions as a protease. (iv) Additional amino acid substitutions in the putative domain of the protease yielded results consistent with the hypothesis that UL26 encodes a serine protease. (v) The domain of the UL26 protein whose modification confers the formation of double bands for all products (Pra, Prb, ICP35a, ICP35b, ICP35c,d, and ICP35e,f) except Prn maps in the domain shared by UL26 and UL26.5, between codons 307 and 417.

Background The type III secretion systems (T3SSs) encoded by Salmonella pathogenicity island 1 and 2 (SPI-1 and SPI-2) are important for invasion of epithelial cells during development of Salmonella-associated enterocolitis and for replication in macrophages during systemic infection, respectively. In vitro studies have previously revealed hierarchical transport of different SPI-1 factors and ordered synergistic/antagonistic relationships between these proteins during Salmonella entry. These results suggest that the level and timing of the expression of these proteins dictate the consequences of bacterial infection and pathogenesis. However, the expression of these proteins has not been extensively studied in vivo, especially during the later stages of salmonellosis when the infection is established. Results In this study, we have constructed bacterial strains that contain a FLAG epitope inserted in frame to SPI-1 genes prgI, sipA, sipB, sopE2, spaO, and sptP, and investigated the expression of the tagged proteins both in vitro and in vivo during murine salmonellosis. The tagged Salmonella strains were inoculated intraperitoneally or intragastrically into mice and recovered from various organs. Our results provide direct evidence that PrgI and SipB are expressed in Salmonella colonizing the spleen and cecum of the infected animals at early and late stages of infection. Furthermore, this study demonstrates that the SpaO protein is expressed preferably in Salmonella colonizing the cecum but not the spleen and that SptP is expressed preferably in Salmonella colonizing the spleen but not the cecum. Conclusion These results suggest that Salmonella may express different SPI-1 proteins when they colonize specific tissues and that differential expression of these proteins may be important for tissue-specific aspects of bacterial pathogenesis such as gastroenterititis in the cecum and systemic infection in the spleen.

Background The highly pathogenic avian influenza H5N1 virus can infect a variety of animals and continually poses a threat to animal and human health. While many genotypes of H5N1 virus can be found in chicken, few are associated with the infection of mammals. Characterization of the genotypes of viral strains in animal populations is important to understand the distribution of different viral strains in various hosts. This also facilitates the surveillance and detection of possible emergence of highly pathogenic strains of specific genotypes from unknown hosts or hosts that have not been previously reported to carry these genotypes. Methodology/Principal Findings Two H5N1 isolates were obtained from lung samples of two raccoon dogs that had died from respiratory disease in China. Pathogenicity experiments showed that the isolates were highly pathogenic to chicken. To characterize the genotypes of these viruses, their genomic sequences were determined and analyzed. The genetic contents of these isolates are virtually identical and they may come from the same progenitor virus. Phylogenetic analysis indicated that the isolates were genetically closely related to genotype V H5N1 virus, which was first isolated in China in 2003, and were distinct from the dominant virus genotypes (e.g. genotype Z) of recent years. The isolates also contain a multibasic amino acid motif at their HA cleavage sites and have an E residue at position 627 of the PB2 protein similar to the previously-identified avian viruses. Conclusions/Significance This is the first report that genotype V H5N1 virus is found to be associated with a mammalian host. Our results strongly suggest that genotype V H5N1 virus has the ability to cross species barriers to infect mammalian animals. These findings further highlight the risk that avian influenza H5N1 virus poses to mammals and humans, which may be infected by specific genotypes that are not known to infect these hosts.

The herpes simplex virus 1 (HSV-1) genome specifies an abundant capsid protein which in denaturing gels forms multiple bands designated family 35 proteins (D.K. Braun, B. Roizman, and L. Pereira, J. Virol. 49:142-153, 1984). Nucleotide-sequencing studies have assigned the coding sequences of these proteins to the open reading frame UL26 (D.J. McGeoch, M.A. Dalrymple, A.J. Davidson, A. Dolan, M.C. Frame, D. McNab, L.J. Perry, J.E. Scott, and P. Taylor, J. Gen. Virol. 69:1531-1574, 1988). IN studies reported here, a series of plasmid constructs containing deletions or insertions of an alpha 4 promoter or of a sequence encoding a cytomegalovirus epitope reacting with a mouse monoclonal antibody revealed the following: the open reading frame previously designated UL26 encodes two proteins which share amino acid sequences, and each coding domain is contained in its own transcriptional unit that terminates at a common, unique poly(A) site. On the basis of the transcription initiation site (+1), it was predicted that the UL26 open reading frame encodes a protein of 635 amino acids, and a protein with an apparent molecular weight of approximately 75,000 has been identified. The second transcriptional unit, designated UL26.5, predicted to specify a protein of 329 amino acids, encodes the family 35 proteins; it is transcribed by an mRNA which initiates at approximately nucleotide +1000 of the UL26 transcription initiation site and is translated from the methionine initiation codon located at position +1099 of the UL26 transcriptional unit. The DNA fragment comprising the sequences downstream of the HpaI cleavage site (+832 of UL26) contains both the promoter and the coding sequence of family 35 proteins and is both competent and efficient in expressing the proteins in transfected cells superinfected with HSV-1 or HSV-2.

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

It has been discovered that any RNA can be targeted for cleavage by RNAase P from eukaryotic cells, for example, human RNAase P, using a suitably designed oligoribonucleotide ("external guide sequence", or EGS) to form a hybrid with the target RNA, thereby creating a substrate for cleavage by RNAase P in vitro. The EGS hydrogen bonds to the targeted RNA to form a partial tRNA like structure including the aminoacyl acceptor stem, the T stem and loop, and part of the D stem. The most efficient EGS with human RNAase P is the EGS in which the anticodon stem and loop was deleted. Modifications can also be made within the T-loop. Methods are also disclosed to randomly select and to express a suitable EGS in vivo to make a selected RNA a target for cleavage by the host cell RNAase P, thus preventing expression of the function of the target RNA. The methods and compositions should be useful to prevent the expression of disease-causing genes in vivo.