Gene regulation of RNA viruses with uncapped and non-polyadenylated genomic RNA

New roles of RNAs as regulators of gene expression have emerged and expanded in recent years. However, gene regulation by viral RNA in trans is less noted and not well understood. Barley yellow dwarf virus (BYDV) is a positive sense RNA virus with a single genomic RNA (gRNA). As it replicates it generates three subgenomic RNAs (sgRNA). Data in this dissertation show that BYDV sgRNA2 serves as a regulatory RNA to control viral gene expression. In vitro and In vivo, BYDV sgRNA2 inhibits translation of gRNA, but has less or little effect on translation of sgRNA1, respectively. These data support and modify a trans-regulation model proposed previously.;I also report that the 3\u27 cap-independent translation element (3\u27TE) of BYDV functions differently in cis and in trans in plant cells. In cis , the 3\u27TE confers cap-independent translation and increases translation of capped RNAs as well. In trans, the 3\u27TE or the 3\u27TE-containing sgRNA2 serves as a riboregulator to negatively regulate viral replication, most likely via inhibition of translation. Thus a viral subgenomic RNA can perform important regulatory functions instead of acting as a messenger RNA.;RNAs of many important plant and human viruses are translated efficiently in the absence of a 5\u27 cap structure and/or a poly(A) tail. The translation mechanism of the uncapped and non-polyadenylated RNA of Tobacco necrosis virus (TNV) has not been well investigated. Here, I identify a cap-independent translation element (TE) in the 3 \u27 UTR of TNV strain D (TNV-D) that shares many features with BYDV 3\u27TE, even though it is in a different family. TNV-D and other members of genus Necrovirus may initiate translation by a BYDV-like TE-mediated cap-independent translation mechanism.;Finally, I show that sequence at the 3\u27 end of TNV-D RNA functionally mimics a poly(A) tail. A phylogenetically conserved double-stem-loop structure is replaceable by, but cannot substitute for a poly(A) tail. The full-length 3\u27 UTR of TNV-D is sufficient to functionally replace a poly(A) tail. Thus, translation of TNV RNA in plant cells requires both cap-mimic and poly(A) mimic elements. This research provides new insight into our understanding of regulation of gene expression

Stromal Down-Regulation of Macrophage CD4/CCR5 Expression and NF-κB Activation Mediates HIV-1 Non-Permissiveness in Intestinal Macrophages

Tissue macrophages are derived exclusively from blood monocytes, which as monocyte-derived macrophages support HIV-1 replication. However, among human tissue macrophages only intestinal macrophages are non-permissive to HIV-1, suggesting that the unique microenvironment in human intestinal mucosa renders lamina propria macrophages non-permissive to HIV-1. We investigated this hypothesis using blood monocytes and intestinal extracellular matrix (stroma)-conditioned media (S-CM) to model the exposure of newly recruited monocytes and resident macrophages to lamina propria stroma, where the cells take up residence in the intestinal mucosa. Exposure of monocytes to S-CM blocked up-regulation of CD4 and CCR5 expression during monocyte differentiation into macrophages and inhibited productive HIV-1 infection in differentiated macrophages. Importantly, exposure of monocyte-derived macrophages simultaneously to S-CM and HIV-1 also inhibited viral replication, and sorted CD4+ intestinal macrophages, a proportion of which expressed CCR5+, did not support HIV-1 replication, indicating that the non-permissiveness to HIV-1 was not due to reduced receptor expression alone. Consistent with this conclusion, S-CM also potently inhibited replication of HIV-1 pseudotyped with vesicular stomatitis virus glycoprotein, which provides CD4/CCR5-independent entry. Neutralization of TGF-β in S-CM and recombinant TGF-β studies showed that stromal TGF-β inhibited macrophage nuclear translocation of NF-κB and HIV-1 replication. Thus, the profound inability of intestinal macrophages to support productive HIV-1 infection is likely the consequence of microenvironmental down-regulation of macrophage HIV-1 receptor/coreceptor expression and NF-κB activation

Gene regulation of RNA viruses with uncapped and non-polyadenylated genomic RNA

New roles of RNAs as regulators of gene expression have emerged and expanded in recent years. However, gene regulation by viral RNA in trans is less noted and not well understood. Barley yellow dwarf virus (BYDV) is a positive sense RNA virus with a single genomic RNA (gRNA). As it replicates it generates three subgenomic RNAs (sgRNA). Data in this dissertation show that BYDV sgRNA2 serves as a regulatory RNA to control viral gene expression. In vitro and In vivo, BYDV sgRNA2 inhibits translation of gRNA, but has less or little effect on translation of sgRNA1, respectively. These data support and modify a trans-regulation model proposed previously.;I also report that the 3' cap-independent translation element (3'TE) of BYDV functions differently in cis and in trans in plant cells. In cis , the 3'TE confers cap-independent translation and increases translation of capped RNAs as well. In trans, the 3'TE or the 3'TE-containing sgRNA2 serves as a riboregulator to negatively regulate viral replication, most likely via inhibition of translation. Thus a viral subgenomic RNA can perform important regulatory functions instead of acting as a messenger RNA.;RNAs of many important plant and human viruses are translated efficiently in the absence of a 5' cap structure and/or a poly(A) tail. The translation mechanism of the uncapped and non-polyadenylated RNA of Tobacco necrosis virus (TNV) has not been well investigated. Here, I identify a cap-independent translation element (TE) in the 3 ' UTR of TNV strain D (TNV-D) that shares many features with BYDV 3'TE, even though it is in a different family. TNV-D and other members of genus Necrovirus may initiate translation by a BYDV-like TE-mediated cap-independent translation mechanism.;Finally, I show that sequence at the 3' end of TNV-D RNA functionally mimics a poly(A) tail. A phylogenetically conserved double-stem-loop structure is replaceable by, but cannot substitute for a poly(A) tail. The full-length 3' UTR of TNV-D is sufficient to functionally replace a poly(A) tail. Thus, translation of TNV RNA in plant cells requires both cap-mimic and poly(A) mimic elements. This research provides new insight into our understanding of regulation of gene expression.</p

The 3′ Untranslated Region of Tobacco Necrosis Virus RNA Contains a Barley Yellow Dwarf Virus-Like Cap-Independent Translation Element

RNAs of many viruses are translated efficiently in the absence of a 5′ cap structure. The tobacco necrosis virus (TNV) genome is an uncapped, nonpolyadenylated RNA whose translation mechanism has not been well investigated. Computational analysis predicted a cap-independent translation element (TE) within the 3′ untranslated region (3′ UTR) of TNV RNA that resembles the TE of barley yellow dwarf virus (BYDV), a luteovirus. Here we report that such a TE does indeed exist in the 3′ UTR of TNV strain D. Like the BYDV TE, the TNV TE (i) functions both in vitro and in vivo, (ii) requires additional sequence for cap-independent translation in vivo, (iii) has a similar secondary structure and the conserved sequence CGGAUCCUGGGAAACAGG, (iv) is inactivated by a four-base duplication in this conserved sequence, (v) can function in the 5′ UTR, and (vi) when located in its natural 3′ location, may form long-distance base pairing with the viral 5′ UTR that is conserved and probably required. The TNV TE differs from the BYDV TE by having only three helical domains instead of four. Similar structures were found in all members of the Necrovirus genus of the Tombusviridae family, except satellite tobacco necrosis virus, which harbors a different 3′ cap-independent translation domain. The presence of the BYDV-like TE in select genera of different families indicates that phylogenetic distribution of TEs does not follow standard viral taxonomic relationships. We propose a new class of cap-independent TE called BYDV-like TE

trans Regulation of Cap-Independent Translation by a Viral Subgenomic RNA

Many positive-strand RNA viruses generate 3′-coterminal subgenomic mRNAs to allow translation of 5′-distal open reading frames. It is unclear how viral genomic and subgenomic mRNAs compete with each other for the cellular translation machinery. Translation of the uncapped Barley yellow dwarf virus genomic RNA (gRNA) and subgenomic RNA1 (sgRNA1) is driven by the powerful cap-independent translation element (BTE) in their 3′ untranslated regions (UTRs). The BTE forms a kissing stem-loop interaction with the 5′ UTR to mediate translation initiation at the 5′ end. Here, using reporter mRNAs that mimic gRNA and sgRNA1, we show that the abundant sgRNA2 inhibits translation of gRNA, but not sgRNA1, in vitro and in vivo. This trans inhibition requires the functional BTE in the 5′ UTR of sgRNA2, but no translation of sgRNA2 itself is detectable. The efficiency of translation of the viral mRNAs in the presence of sgRNA2 is determined by proximity to the mRNA 5′ end of the stem-loop that kisses the 3′ BTE. Thus, the gRNA and sgRNA1 have “tuned” their expression efficiencies via the site in the 5′ UTR to which the 3′ BTE base pairs. We conclude that sgRNA2 is a riboregulator that switches off translation of replication genes from gRNA while permitting translation of structural genes from sgRNA1. These results reveal (i) a new level of control of subgenomic-RNA gene expression, (ii) a new role for a viral subgenomic RNA, and (iii) a new mechanism for RNA-mediated regulation of translation

The 3 Untranslated Region of Tobacco Necrosis Virus RNA Contains a Barley Yellow Dwarf Virus-Like Cap-Independent Translation Element

RNAs of many viruses are translated efficiently in the absence of a 5' cap structure. The tobacco necrosis virus (TNV) genome is an uncapped, nonpolyadenylated RNA whose translation mechanism has not been well investigated. Computational analysis predicted a cap-independent translation element (TE) within the 3' untranslated region (3' UTR) of TNV RNA that resembles the TE of barley yellow dwarf virus (BYDV), a luteovirus. Here we report that such a TE does indeed exist in the 3' UTR of TNV strain D. Like the BYDV TE, the TNV TE (i) functions both in vitro and in vivo, (ii) requires additional sequence for cap-independent translation in vivo, (iii) has a similar secondary structure and the conserved sequence CGGAUCCUGGGAA ACAGG, (iv) is inactivated by a four-base duplication in this conserved sequence, (v) can function in the 5' UTR, and (vi) when located in its natural 3' location, may form long-distance base pairing with the viral 5' UTR that is conserved and probably required. The TNV TE differs from the BYDV TE by having only three helical domains instead of four. Similar structures were found in all members of the Necrovirus genus of the Tombusviridae family, except satellite tobacco necrosis virus, which harbors a different 3' cap-independent translation domain. The presence of the BYDV-like TE in select genera of different families indicates that phylogenetic distribution of TEs does not follow standard viral taxonomic relationships. We propose a new class of cap-independent TE called BYDV-like TE.This article is from Journal of Virology 78 (2004): 4655, doi: 10.1128/JVI.78.9.4655-4664.2004 . Posted with permission.</p

An Agent-Based Threshold Payment Model for Metering Web Services

The Service-Oriented technology is an emerging paradigm for services integration over the Internet. Use of applications as services across heterogeneous hardware and software platforms has raised new challenges. Access to Web services and request to provide a secure and trusted payment model for the Web services in a risky environment such as the Internet is among those challenges that must be addressed. Cryptographic approaches known as “Metering Schemes ” provide some sophisticated solutions for access control to a Web service. This paper proposes an agent-based threshold payment model for metering Web services. The model has taken into account the abilities of software agents in reasoning and problem solving for designing some cryptographic concepts such as threshold schemes

trans Regulation of Cap-Independent Translation by a Viral Subgenomic RNA

Many positive-strand RNA viruses generate 3′-coterminal subgenomic mRNAs to allow translation of 5′-distal open reading frames. It is unclear how viral genomic and subgenomic mRNAs compete with each other for the cellular translation machinery. Translation of the uncapped Barley yellow dwarf virus genomic RNA (gRNA) and subgenomic RNAI (sgRNAI) is driven by the powerful cap-independent translation element (BTE) in their 3′ untranslated regions (UTRs). The BTE forms a kissing stem-loop interaction with the 5′ UTR to mediate translation initiation at the 5′ end. Here, using reporter mRNAs that mimic gRNA and SgRNA1, we show that the abundant sgRNA2 inhibits translation of gRNA, but not sgRNA1, in vitro and in vivo. This trans inhibition requires the functional BTE in the 5′ UTR of sgRNA2, but no translation of sgRNA2 itself is detectable. The efficiency of translation of the viral mRNAs in the presence of sgRNA2 is determined by proximity to the mRNA 5′ end of the stem-loop that kisses the 3′ BTE. Thus, the gRNA and sgRNA1 have "tuned" their expression efficiencies via the site in the 5′ UTR to which the 3′ BTE base pairs. We conclude that sgRNA2 is a riboregulator that switches off translation of replication genes from gRNA while permitting translation of structural genes from sgRNAI. These results reveal (i) a new level of control of subgenomic-RNA gene expression, (ii) a new role for a viral subgenomic RNA, and (iii) a new mechanism for RNA-mediated regulation of translation.This article is from Journal of Virology 80 (2006): 10045, doi: 10.1128/JVI.00991-06. Posted with permission.</p