Viral RNA-binding Proteins Research Articles

Abstract 2873: Identification of a new mechanism of microRNA turnover from miR-106a-363 cluster leading to epithelial-to-mesenchymal transition in prostate cancer

Abstract Background: Prostate cancer (PCa) is the second leading cause of cancer mortality in US. The majority of PCa mortality is due to the recurrent of metastatic castration resistant PCa. The acquisition of epithelial-to-mesenchymal transition (EMT) signifies PCa metastasis. In particular, altered microRNAs (miRNAs) expression is closely associated with PCa progression from prostatic intraepithelial neoplasia to metastatic adenocarcinoma. miRNAs are small noncoding RNAs regulating approximately 60% protein-coding genes by post-transcriptional suppression or translational inhibition. miRNA gene expression at post-transcriptional level becomes more complicated when multiple miRNAs derived from the same cluster generate a polycistronic primary transcript, and each individual miRNA displays different expression profile and functional role. miRNA-363 (miR-363) belongs to the miR-106a-363 cluster containing miR-106a, miR-18b, miR-20b, miR-19b-2, miR-92a-2 and miR-363. Unlike the other five miRNAs which are closely resemble to the oncogenic miR-17-92 cluster in their seed sequence and function, miR-363 has been implicated to play a tumor suppressor role in several types of cancer, indicating a different regulatory mechanism of miR-363 from the miR-106a-363 cluster. Interferon-induced tetratricopeptide repeat 5 (IFIT5) is first characterized as a viral RNA binding protein and has been shown to directly bind to cellular tRNA, which partially shared a structural similarity with precursor miRNAs (pre-miRNAs). However, until now, there is no report indicating the RNA recognition role of IFIT5 in miRNA biogenesis machinery. Results: In the present study, miR-363 was clearly identified as a tumor suppressor miRNA by inhibiting EMT in PCa cells via targeting slug/SNAI2. More importantly, we have demonstrated, for the first time, that IFIT5 is able to recognize a unique structure at the 5′ end of precursor miR-363, which facilitates pre-miR-363 degradation by the recruitment of a 5′-3′exoribonuclease, XRN1. Meanwhile, we have also shown that the significant elevation of IFIT5 is detected in several PCa cells undergone EMT leading to highly metastatic potential. In addition, an inverse correlation between miR-363 and IFIT5 mRNA level was found in human PCa specimens. Conclusion: We unveil IFIT5 complex as a new post-transcriptional regulatory mechanism specific for miR-363 turnover at the precursor stage, which determines the stability and unique functional role of miR-363 distinct from other oncogenic miRNA members in the miR-106a-363 cluster. Overall, this study provides an insight of miRNA biogenesis machinery in cancer metastasis and new strategies of therapeutic intervention of PCa. Citation Format: U-Ging Lo, Rey-Chen Pong, Diane Yang, Jiancheng Zhou, Leah Gandee, Shu-Fen Tseng, Jer-Tsong Hsieh. Identification of a new mechanism of microRNA turnover from miR-106a-363 cluster leading to epithelial-to-mesenchymal transition in prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2873. doi:10.1158/1538-7445.AM2015-2873

Evidence that avian reovirus σNS is an RNA chaperone: implications for genome segment assortment.

Reoviruses are important human, animal and plant pathogens having 10–12 segments of double-stranded genomic RNA. The mechanisms controlling the assortment and packaging of genomic segments in these viruses, remain poorly understood. RNA–protein and RNA–RNA interactions between viral genomic segment precursors have been implicated in the process. While non-structural viral RNA-binding proteins, such as avian reovirus σNS, are essential for virus replication, the mechanism by which they assist packaging is unclear. Here we demonstrate that σNS assembles into stable elongated hexamers in vitro, which bind single-stranded nucleic acids with high affinity, but little sequence specificity. Using ensemble and single molecule fluorescence spectroscopy, we show that σNS also binds to a partially double-stranded RNA, resulting in gradual helix unwinding. The hexamer can bind multiple RNA molecules and exhibits strand-annealing activity, thus mediating conversion of metastable, intramolecular stem-loops into more stable heteroduplexes. We demonstrate that the ARV σNS acts as an RNA chaperone facilitating specific RNA–RNA interactions between genomic precursors during segment assortment and packaging.

An unbiased proteomics approach to identify human cytomegalovirus RNA-associated proteins

Post-transcriptional events regulate herpesvirus gene expression, yet few herpesvirus RNA-binding proteins have been identified. We used an unbiased approach coupling oligo(dT) affinity capture with proteomics to identify viral RNA-associated proteins during infection. Using this approach, we identified and confirmed changes in the abundance or activity of two host RNA-associated proteins, DHX9 and DDX3, in cells infected with human cytomegalovirus (HCMV). We also identified and confirmed previously unreported activities for the HCMV US22 and pp71 proteins as RNA-associated viral proteins and confirmed that a known viral RNA-binding protein, pTRS1, associates with RNA in infected cells. Further, we found that HCMV pp71 co-sedimented with polysomes, associated with host and viral RNAs, and stimulated the overall rate of protein synthesis. These results demonstrate that oligo(dT) affinity capture coupled with proteomics provides a rapid and straightforward means to identify RNA-associated viral proteins during infection that may participate in the post-transcriptional control of gene expression.

Virus-mediated compartmentalization of the host translational machinery.

ABSTRACTViruses require the host translational apparatus to synthesize viral proteins. Host stress response mechanisms that suppress translation, therefore, represent a significant obstacle that viruses must overcome. Here, we report a strategy whereby the mammalian orthoreoviruses compartmentalize the translational machinery within virus-induced inclusions known as viral factories (VF). VF are the sites of reovirus replication and assembly but were thought not to contain ribosomes. It was assumed viral mRNAs exited the VF to undergo translation by the cellular machinery, and proteins reentered the factory to participate in assembly. Here, we used ribopuromycylation to visualize active translation in infected cells. These studies revealed that active translation occurs within VF and that ribosomal subunits and proteins required for translation initiation, elongation, termination, and recycling localize to the factory. Interestingly, we observed components of the 43S preinitiation complex (PIC) concentrating primarily at factory margins, suggesting a spatial and/or dynamic organization of translation within the VF. Similarly, the viral single-stranded RNA binding protein σNS localized to the factory margins and had a tubulovesicular staining pattern that extended a short distance from the margins of the factories and colocalized with endoplasmic reticulum (ER) markers. Consistent with these colocalization studies, σNS was found to associate with both eukaryotic translation initiation factor 3 subunit A (eIF3A) and the ribosomal subunit pS6R. Together, these findings indicate that σNS functions to recruit 43S PIC machinery to the primary site of viral translation within the viral factory. Pathogen-mediated compartmentalization of the translational apparatus provides a novel mechanism by which viruses might avoid host translational suppression.

Host and viral RNA-binding proteins involved in membrane targeting, replication and intercellular movement of plant RNA virus genomes.

Many plant viruses have positive-strand RNA [(+)RNA] as their genome. Therefore, it is not surprising that RNA-binding proteins (RBPs) play important roles during (+)RNA virus infection in host plants. Increasing evidence demonstrates that viral and host RBPs play critical roles in multiple steps of the viral life cycle, including translation and replication of viral genomic RNAs, and their intra- and intercellular movement. Although studies focusing on the RNA-binding activities of viral and host proteins, and their associations with membrane targeting, and intercellular movement of viral genomes have been limited to a few viruses, these studies have provided important insights into the molecular mechanisms underlying the replication and movement of viral genomic RNAs. In this review, we briefly overview the currently defined roles of viral and host RBPs whose RNA-binding activity have been confirmed experimentally in association with their membrane targeting, and intercellular movement of plant RNA virus genomes.

Middle East Respiratory Syndrome Coronavirus 4a Protein Is a Double-Stranded RNA-Binding Protein That Suppresses PACT-Induced Activation of RIG-I and MDA5 in the Innate Antiviral Response

Middle East respiratory syndrome coronavirus (MERS-CoV) is an emerging pathogen that causes severe disease in human. MERS-CoV is closely related to bat coronaviruses HKU4 and HKU5. Evasion of the innate antiviral response might contribute significantly to MERS-CoV pathogenesis, but the mechanism is poorly understood. In this study, we characterized MERS-CoV 4a protein as a novel immunosuppressive factor that antagonizes type I interferon production. MERS-CoV 4a protein contains a double-stranded RNA-binding domain capable of interacting with poly(I · C). Expression of MERS-CoV 4a protein suppressed the interferon production induced by poly(I · C) or Sendai virus. RNA binding of MERS-CoV 4a protein was required for IFN antagonism, a property shared by 4a protein of bat coronavirus HKU5 but not by the counterpart in bat coronavirus HKU4. MERS-CoV 4a protein interacted with PACT in an RNA-dependent manner but not with RIG-I or MDA5. It inhibited PACT-induced activation of RIG-I and MDA5 but did not affect the activity of downstream effectors such as RIG-I, MDA5, MAVS, TBK1, and IRF3. Taken together, our findings suggest a new mechanism through which MERS-CoV employs a viral double-stranded RNA-binding protein to circumvent the innate antiviral response by perturbing the function of cellular double-stranded RNA-binding protein PACT. PACT targeting might be a common strategy used by different viruses, including Ebola virus and herpes simplex virus 1, to counteract innate immunity. Middle East respiratory syndrome coronavirus (MERS-CoV) is an emerging and highly lethal human pathogen. Why MERS-CoV causes severe disease in human is unclear, and one possibility is that MERS-CoV is particularly efficient in counteracting host immunity, including the sensing of virus invasion. It will therefore be critical to clarify how MERS-CoV cripples the host proteins that sense viruses and to compare MERS-CoV with its ancestral viruses in bats in the counteraction of virus sensing. This work not only provides a new understanding of the abilities of MERS-CoV and closely related bat viruses to subvert virus sensing but also might prove useful in revealing new strategies for the development of vaccines and antivirals.

Chloroplast Phosphoglycerate Kinase Is Involved in the Targeting of Bamboo mosaic virus to Chloroplasts in Nicotiana benthamiana Plants

The Bamboo mosaic virus (BaMV) is a positive-sense, single-stranded RNA virus. Previously, we identified that the chloroplast phosphoglycerate kinase (chl-PGK) from Nicotiana benthamiana is one of the viral RNA binding proteins involved in the BaMV infection cycle. Because chl-PGK is transported to the chloroplast, we hypothesized that chl-PGK might be involved in viral RNA localization in the chloroplasts. To test this hypothesis, we constructed two green fluorescent protein (GFP)-fused mislocalized PGK mutants, the transit peptide deletion mutant (NO TRANSIT PEPTIDE [NOTP]-PGK-GFP) and the nucleus location mutant (nuclear location signal [NLS]-PGK-GFP). Using confocal microscopy, we demonstrated that NOTP-PGK-GFP and NLS-PGK-GFP are localized in the cytoplasm and nucleus, respectively, in N. benthamiana plants. When NOTP-PGK-GFP and NLS-PGK-GFP are transiently expressed, we observed a reduction in BaMV coat protein accumulation to 47% and 27% that of the wild-type PGK-GFP, respectively. To localize viral RNA in infected cells, we employed the interaction of NLS-GFP-MS2 (phage MS2 coat protein) with the modified BaMV RNA containing the MS2 coat protein binding sequence. Using confocal microscopy, we observed that BaMV viral RNA localizes to chloroplasts. Furthermore, elongation factor1a fused with the transit peptide derived from chl-PGK or with a Rubisco small subunit can partially restore BaMV accumulation in NbPGK1-knockdown plants by helping BaMV target chloroplasts.

Suppression of PACT-Induced Type I Interferon Production by Herpes Simplex Virus 1 Us11 Protein

Herpes simplex virus 1 (HSV-1) Us11 protein is a double-stranded RNA-binding protein that suppresses type I interferon production through the inhibition of the cytoplasmic RNA sensor RIG-I. Whether additional cellular mediators are involved in this suppression remains to be determined. In this study, we report on the requirement of cellular double-stranded RNA-binding protein PACT for Us11-mediated perturbation of type I interferon production. Us11 associates with PACT tightly to prevent it from binding with and activating RIG-I. The Us11-deficient HSV-1 was indistinguishable from the Us11-proficient virus in the suppression of interferon production when PACT was compromised. More importantly, HSV-1-induced activation of interferon production was abrogated in PACT knockout murine embryonic fibroblasts. Our findings suggest a new mechanism for viral evasion of innate immunity through which a viral double-stranded RNA-binding protein interacts with PACT to circumvent type I interferon production. This mechanism might also be used by other PACT-binding viral interferon-antagonizing proteins such as Ebola virus VP35 and influenza A virus NS1.

Viral interference with host mRNA surveillance, the nonsense-mediated mRNA decay (NMD) pathway, through a new function of HTLV-1 Rex: implications for retroviral replication

Nonsense-mediated mRNA decay (NMD) is an essential and conserved cellular mRNA quality control mechanism. RNA signals to express viral genes from overlapping open reading frames potentially initiate NMD, nevertheless it is not clear whether viral RNAs are sensitive to NMD or if viruses have evolved mechanisms to evade NMD. Here we demonstrate that the genomic and full-length mRNAs of Human-T-cell Leukemia Virus type-I (HTLV-1), a retrovirus responsible for Adult T-cell Leukemia (ATL), are sensitive to NMD. They exhibit accelerated turnover in NMD-activated cells, while siRNA-mediated knockdown of NMD-master-regulator, UPF1, promotes enhanced stability of them. These effects on RNA stability were recapitulated by a reporter construct encoding the HTLV-1 translational frameshift signal of gag-pol. In agreement with the RNA stability, viral protein expression from the integrated provirus was inversely correlated with cellular NMD activity. We further demonstrated that the viral RNA-binding protein, Rex, approves the stability of viral RNA by inhibiting NMD. Significantly, Rex establishes a general block to NMD, as both NMD-responsive reporter transcripts and natural host-encoded NMD substrates were stabilized in the presence of Rex. Thus, we suggest that Rex not only stabilizes viral transcripts, but also perturbs cellular mRNA metabolism and host cell homeostasis via inhibition of NMD.

Viral Double-Strand RNA-Binding Proteins Can Enhance Innate Immune Signaling by Toll-Like Receptor 3

Toll-like Receptor 3 (TLR3) detects double-stranded (ds) RNAs to activate innate immune responses. While poly(I:C) is an excellent agonist for TLR3 in several cell lines and in human peripheral blood mononuclear cells, viral dsRNAs tend to be poor agonists, leading to the hypothesis that additional factor(s) are likely required to allow TLR3 to respond to viral dsRNAs. TLR3 signaling was examined in a lung epithelial cell line by quantifying cytokine production and in human embryonic kidney cells by quantifying luciferase reporter levels. Recombinant 1b hepatitis C virus polymerase was found to enhance TLR3 signaling in the lung epithelial BEAS-2B cells when added to the media along with either poly(I:C) or viral dsRNAs. The polymerase from the genotype 2a JFH-1 HCV was a poor enhancer of TLR3 signaling until it was mutated to favor a conformation that could bind better to a partially duplexed RNA. The 1b polymerase also co-localizes with TLR3 in endosomes. RNA-binding capsid proteins (CPs) from two positive-strand RNA viruses and the hepadenavirus hepatitis B virus (HBV) were also potent enhancers of TLR3 signaling by poly(I:C) or viral dsRNAs. A truncated version of the HBV CP that lacked an arginine-rich RNA-binding domain was unable to enhance TLR3 signaling. These results demonstrate that several viral RNA-binding proteins can enhance the dsRNA-dependent innate immune response initiated by TLR3.

Role of the RNA recognition motif of the E1B 55 kDa protein in the adenovirus type 5 infectious cycle

Although the adenovirus type 5 (Ad5) E1B 55 kDa protein can bind to RNA in vitro, no UV-light-induced crosslinking of this E1B protein to RNA could be detected in infected cells, under conditions in which RNA binding by a known viral RNA-binding protein (the L4 100 kDa protein) was observed readily. Substitution mutations, including substitutions reported to inhibit RNA binding in vitro, did not impair synthesis of viral early or late proteins or alter significantly the efficiency of viral replication in transformed or normal human cells. However, substitutions of conserved residues in the C-terminal segment of an RNA recognition motif specifically inhibited degradation of Mre11. We conclude that, if the E1B 55 kDa protein binds to RNA in infected cells in the same manner as in in vitro assays, this activity is not required for such well established functions as induction of selective export of viral late mRNAs.

Multifaceted Evasion of the Interferon Response by Cytomegalovirus

Human cytomegalovirus (HCMV), which infects the majority of the population worldwide, causes few, if any, symptoms in otherwise healthy people but is responsible for considerable morbidity and mortality in immunocompromised patients and in congenitally infected newborns. The evolutionary success of HCMV depends in part on its ability to evade host defense systems. Here we review recent progress in elucidating the remarkable assortment of mechanisms employed by HCMV and the related beta-herpesviruses, murine cytomegaloviruses (MCMV) and rhesus cytomegaloviruses (RhCMV), for counteracting the host interferon (IFN) response. Very early after infection, cellular membrane sensors such as the lymphotoxin beta receptor initiate the production of antiviral cytokines including type I IFNs. However, virion factors, such as pp65 (ppUL83) and viral proteins made soon after infection including the immediate early gene 2 protein (pUL122), repress this response by interfering with steps in the activation of IFN regulatory factor 3 and NF-kappaB. CMVs then exert a multi-pronged attack on downstream IFN signaling. HCMV infection results in decreased accumulation and phosphorylation of the IFN signaling kinases Jak1 and Stat2, and the MCMV protein pM27 mediates Stat2 down-regulation, blocking both type I and type II IFN signaling. The HCMV immediate early gene 1 protein (pUL123) interacts with Stat2 and inhibits transcriptional activation of IFN-regulated genes. Infection also causes reduction in the abundance of p48/IRF9, a component of the ISGF3 transcription factor complex. Furthermore, CMVs have multiple genes involved in blocking the function of IFN-induced effectors. For example, viral double-stranded RNA-binding proteins are required to prevent the shutoff of protein synthesis by protein kinase R, further demonstrating the vital importance of evading the IFN response at multiple levels during infection.

Multi-scale modeling of HIV-1 proteins

The efficiency of protease inhibiting drugs is hampered by the rapid emergence of protease variants. Understanding this phenomenon requires the characterization of the salient steps of HIV-1 protease’s catalytic cycle. We summarize our investigations on the reactive geometry of the protease-substrate complex based on first principles, QM/MM and classical atomistic molecular dynamics simulations. Previous and novel analysis indicates that the reactive geometry is assisted by a mechanical coupling between the local structural fluctuations at the active site and large scale-motion of the entire protein. Additional coarse-grained modeling further allows uncovering unexpected analogies of concerted large-scale movements across members of the aspartyl-protease family. Taken together, these results may help understand some aspects of the resistance against drugs targeting HIV-1 protease. We further present computational studies on HIV-1 transactivator of transcription (Tot) viral RNA binding protein. Interfering with Tat/TAR interactions is a promising strategy for anti-AIDS intervention. We have identified conserved structural and energetic features among different protein isolates and predicted the structural determinants of Tat in complex with one of the host cell cognate proteins, p/CAF. These findings may help the design of ligands interfering with Tat function.

Localization of the Carnation Italian ringspot virus replication protein p36 to the mitochondrial outer membrane is mediated by an internal targeting signal and the TOM complex.

BackgroundCarnation Italian ringspot virus (CIRV) is a positive-strand RNA virus that causes massive structural alterations of mitochondria in infected host cells, the most conspicuous being the formation of numerous internal vesicles/spherules that are derived from the mitochondrial outer membrane and serve as the sites for viral RNA replication. While the membrane-bound components of the CIRV replication complex, including a 36-kD RNA-binding protein (p36), are known to be essential for these changes in mitochondrial morphology and are relatively well characterized in terms of their roles in nascent viral RNA synthesis, how these proteins are specifically targeted and inserted into mitochondria is poorly defined.ResultsHere we report on the molecular signal responsible for sorting p36 to the mitochondrial outer membrane. Using a combination of gain-of-function assays with portions of p36 fused to reporter proteins and domain-swapping assays with p36 and another closely-related viral RNA-binding protein, p33, that sorts specifically to the peroxisomal boundary membrane, we show that the mitochondrial targeting information in p36 resides within its two transmembrane domains (TMDs) and intervening hydrophilic loop sequence. Comprehensive mutational analysis of these regions in p36 revealed that the primary targeting determinants are the moderate hydrophobicity of both TMDs and the positively-charged face of an amphipathic helix within the intervening loop sequence. We show also using bimolecular fluorescence complementation (BiFC) that p36 interacts with certain components of the translocase complex in the mitochondrial outer membrane (TOM), but not with the sorting and assembly machinery (SAM).ConclusionOur results provide insight to how viruses, such as CIRV, exploit specific host-cell protein sorting pathways to facilitate their replication. The characterization of the targeting and insertion of p36 into the mitochondrial outer membrane also sheds light on the mechanisms involved in sorting of host-cell membrane proteins to mitochondria, a process that has been largely unexplored in plants.

Genetic Adaptation to Untranslated Region-Mediated Enterovirus Growth Deficits by Mutations in the Nonstructural Proteins 3AB and 3CD

Both untranslated regions (UTRs) of plus-strand RNA virus genomes jointly control translation and replication of viral genomes. In the case of the Enterovirus genus of the Picornaviridae family, the 5'UTR consists of a cloverleaf-like terminus preceding the internal ribosomal entry site (IRES) and the 3' terminus is composed of a structured 3'UTR and poly(A). The IRES and poly(A) have been implicated in translation control, and all UTR structures, in addition to cis-acting genetic elements mapping to the open reading frame, have been assigned roles in RNA replication. Viral UTRs are recognized by viral and host cell RNA-binding proteins that may co-determine genome stability, translation, plus- and minus-strand RNA replication, and scaffolding of viral replication complexes within host cell substructures. In this report, we describe experiments with coxsackie B viruses with a cell type-specific propagation deficit in Sk-N-Mc neuroblastoma cells conferred by the combination of a heterologous IRES and altered 3'UTR. Serial passage of these constructs in Sk-N-Mc cells yielded genetic adaptation by mutations within the viral nonstructural proteins 3A and 3C. Our data implicate 3A and/or 3C or their precursors 3AB and/or 3CD in a functional complex with the IRES and 3'UTR that drives viral propagation. Adaptation to neuroblastoma cells suggests an involvement of cell type-specific host factors or the host cell cytoplasmic milieu in this phenomenon.

HIV-1 TAR RNA Subverts RNA Interference in Transfected Cells through Sequestration of TAR RNA-binding Protein, TRBP

TAR RNA-binding protein, TRBP, was recently discovered to be an essential partner for Dicer and a crucial component of the RNA-induced silencing complex (RISC), a critical element of the RNA interference (RNAi) of the cell apparatus. Human TRBP was originally characterized and cloned 15 years ago based on its high affinity for binding the HIV-1 encoded leader RNA, TAR. RNAi is used, in part, by cells to defend against infection by viruses. Here, we report that transfected TAR RNA can attenuate the RNAi machinery in human cells. Our data suggest that TAR RNA sequesters TRBP rendering it unavailable for downstream Dicer-RISC complexes. TAR-induced inhibition of Dicer-RISC activity in transfected cells was partially relieved by exogenous expression of TRBP.

Subcellular localization of the severe acute respiratory syndrome coronavirus nucleocapsid protein

The coronavirus nucleocapsid (N) protein is a viral RNA-binding protein with multiple functions in terms of virus replication and modulating cell signalling pathways. N protein is composed of three distinct regions containing RNA-binding motif(s), and appropriate signals for modulating cell signalling. The subcellular localization of severe acute respiratory syndrome coronavirus (SARS-CoV) N protein was studied. In infected cells, SARS-CoV N protein localized exclusively to the cytoplasm. In contrast to the avian coronavirus N protein, overexpressed SARS-CoV N protein remained principally localized to the cytoplasm, with very few cells exhibiting nucleolar localization. Bioinformatic analysis and deletion mutagenesis coupled to confocal microscopy and live-cell imaging, revealed that SARS-CoV N protein regions I and III contained nuclear localization signals and region II contained a nucleolar retention signal. However, cytoplasmic localization was directed by region III and was the dominant localization signal in the protein.

The transport of prolamine RNAs to prolamine protein bodies in living rice endosperm cells.

RNAs that code for the major rice storage proteins are localized to specific subdomains of the cortical endoplasmic reticulum (ER) in developing endosperm. Prolamine RNAs are localized to the ER and delimit the prolamine intracisternal inclusion granules (PB-ER), whereas glutelin RNAs are targeted to the cisternal ER. To study the transport of prolamine RNAs to the surface of the prolamine protein bodies in living endosperm cells, we adapted a two-gene system consisting of green fluorescent protein (GFP) fused to the viral RNA binding protein MS2 and a hybrid prolamine RNA containing tandem MS2 RNA binding sites. Using laser scanning confocal microscopy, we show that the GFP-labeled prolamine RNAs are transported as particles that move at an average speed of 0.3 to 0.4 microm/s. These prolamine RNA transport particles generally move unidirectionally in a stop-and-go manner, although nonlinear bidirectional, restricted, and nearly random movement patterns also were observed. Transport is dependent on intact microfilaments, because particle movement is inhibited rapidly by the actin filament-disrupting drugs cytochalasin D and latrunculin B. Direct evidence was obtained that these prolamine RNA-containing particles are transported to the prolamine protein bodies. The significance of these results with regard to protein synthesis in plants is discussed.

CRM1-dependent function of a cis-acting RNA export element.

Viruses often contain cis-acting RNA elements, which facilitate the posttranscriptional processing and export of their messages. These elements fall into two classes distinguished by the presence of either viral or cellular RNA binding proteins. To date, studies have indicated that the viral proteins utilize the CRM1-dependent export pathway, while the cellular factors generally function in a CRM1-independent manner. The cis-acting element found in the woodchuck hepatitis virus (WHV) (the WHV posttranscriptional regulatory element [WPRE]) has the ability to posttranscriptionally stimulate transgene expression and requires no viral proteins to function. Conventional wisdom suggests that the WPRE would function in a CRM1-independent manner. However, our studies on this element reveal that its efficient function is sensitive to the overexpression of the C terminus of CAN/Nup214 and treatment with the antimicrobial agent leptomycin B. Furthermore, the overexpression of CRM1 stimulates WPRE activity. These results suggest a direct role for CRM1 in the export function of the WPRE. This observation suggests that the WPRE is directing messages into a CRM1-dependent mRNA export pathway in somatic mammalian cells.

Characterization of Viral Double-Stranded RNA-Binding Proteins

Several viruses have been shown to code for proteins that specifically bind to double-stranded RNA or RNA with large amounts of secondary structure. These proteins have been implicated in providing interferon resistance to viruses and in inhibiting induction of apoptosis by viruses, and have been suggested to be involved in regulation of viral and cellular protein synthesis in infected cells. This article describes methods for detecting and analyzing viral double-stranded RNA-binding proteins.