Distinct Export Pathway Utilized by the Hepatitis B Virus Posttranscriptional Regulatory Element

Zang, Wei-Qing; Yen, T. S. Benedict

doi:10.1006/viro.1999.9777

Cited by 34 publications

(33 citation statements)

References 47 publications

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“…LMB was used as a tool with which to determine that SNV RU5-containing RNAs achieve nuclear export independently of the leucine-rich NES/CRM1-independent nuclear export pathway utilized by Rev/RRE. Recently, similar CRM1-independent RNA export of intron-containing RNA was reported for the Rous sarcoma virus direct repeat (DR) element, the MPMV constitutive transport element, and the hepatitis B virus posttranscriptional regulatory element (PRE) (33,34,45). SNV RU5 is distinct in affecting both intron-containing and intron-lacking reporter RNAs.…”

Section: Discussionmentioning

confidence: 61%

Nuclear Interactions Are Necessary for Translational Enhancement by Spleen Necrosis Virus RU5

Dangel¹,

Hull

Roberts

et al. 2002

J Virol

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The 5 long terminal repeat of spleen necrosis virus (SNV) facilitates Rev/Rev-responsive element (RRE)-independent expression of intron-containing human immunodeficiency virus type 1 (HIV-1) gag. The SNV RU5 region, which corresponds to the 165-nucleotide 5 RNA terminus, functions in a position-and orientationdependent manner to enhance polysome association of intron-containing HIV-1 gag RNA and also nonviral luc RNA. Evidence is mounting that association with nuclear factors during intron removal licenses mRNAs for nuclear export, efficient translation, and nonsense-mediated decay. This project addressed the relationship between the nuclear export pathway of SNV RU5-reporter RNA and translational enhancement. Results of RNA transfection experiments suggest that cytoplasmic proteins are insufficient for SNV RU5 translational enhancement of gag or luc RNA. Reporter gene assays, leptomycin B (LMB) sensitivity experiments, and RNase protection assays indicate that RU5 gag RNA accesses a nuclear export pathway that is distinct from the LMB-inhibited leucine-rich nuclear export sequence-dependent CRM1 pathway, which is used by the HIV-1 RRE. As a unique tool with which to investigate the relationship between different RNA trafficking routes and translational enhancement, SNV RU5 and Rev/RRE were combined on a single gag RNA. We observed a less-than-synergistic effect on cytoplasmic mRNA utilization. Instead, Rev/RRE diverts RU5 gag RNA to the CRM1-dependent, LMB-inhibited pathway and abrogates translational enhancement by SNV RU5. Our study is the first to show that a nuclear factor(s) directs SNV RU5-containing RNAs to a distinct export pathway that is not inhibited by LMB and programs the intron-containing transcript for translational enhancement.Retroviruses contain structured RNA elements that interact with viral and cellular proteins and modulate nuclear export and efficient translation of intron-containing transcripts (reviewed in references 3, 5, and 11). The long terminal repeat (LTR) of spleen necrosis virus (SNV) facilitates Rev/Rev-responsive element (RRE)-independent expression of unspliced human immunodeficiency virus type 1 (HIV-1) gag reporter RNA (7). Quantitative RNA and protein analysis identified a minor 2-fold increase in cytoplasmic accumulation of gag RNA but a greater-than-100-fold increase in Gag protein production in response to the SNV LTR. The 165-nucleotide 5Ј RNA terminus encoded by the RU5 region of the LTR functions in a position-and orientation-dependent manner to direct polysome association and detectable Gag protein synthesis. SNV RU5 functions in a cap-dependent manner (M. Butsch and K. Boris-Lawrie, unpublished data) and is not an internal ribosome entry site (IRES) (35). The SNV U5 region also augments translation of nonviral luciferase (luc) RNA by increasing polysome loading (35). Recently, the R region of human foamy virus (HFV) and RU5 of Mason-Pfizer monkey virus (MPMV) were also shown to program unspliced gag RNA templates for Gag protein synthesis (37; S. Hull and K. BorisL...

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Section: Discussionmentioning

confidence: 61%

Nuclear Interactions Are Necessary for Translational Enhancement by Spleen Necrosis Virus RU5

Dangel¹,

Hull

Roberts

et al. 2002

J Virol

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“…Due to the presence of HPRE␤, the construct pDM138HPRE␤-Bul(AS) exhibited a low level of activity (20% of the positive control) which was Rev-independent. This result was expected for the following reasons: (i) HPRE does not need Rev to function (48,64), and (ii) HPRE subelements function cooperatively; therefore, the presence of only one subelement results in minimal activity (7). When Rev was cotransfected with pDM138HPRE␤-Bul, the level of activation was 86% of that of the positive control with Rev.…”

Section: Resultsmentioning

confidence: 97%

“…Although the function of the PREs is unclear, their ability to work in a Rev-dependent assay and their requirement for RNA cytoplasmic accumulation have been suggestive of a role in export. Earlier experiments demonstrated that HPRE function is LMB resistant, indicating that, similar to CTE, it does not use CRM1 as an export receptor (48,64). Mapping studies have demonstrated that HPRE and WPRE contain two homologous cis-acting sequences, or subelements, designated PRE␣ and PRE␤ (7,8).…”

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confidence: 99%

CRM1-Dependent Function of a cis-Acting RNA Export Element

Popa

Harris

Donello

et al. 2002

Molecular and Cellular Biology

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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.The generation of mature cytoplasmic mRNAs requires numerous processing steps, namely, transcription, capping, splicing, polyadenylation, and transport to the cytoplasm. This process is tightly regulated, since aberrant transcripts are degraded in the nucleus and only properly processed mRNAs are exported to cytoplasm (43). Thus, nuclear export ensures that only completely processed mRNAs can be translated into protein.Much of our present understanding of nuclear export has come from the study of how viruses exploit host cell RNA processing and export pathways. The first viral export system studies were those of complex retroviruses, exemplified by human immunodeficiency virus type 1 (HIV-1). HIV-1 replication requires unspliced and partially spliced RNAs to be exported from the nucleus by the virally encoded Rev protein (9,13,44). Rev contains an RNA binding domain, which specifically binds to the Rev response element (RRE), located within the second intron of HIV-1 pre-mRNA, and a nuclear export signal (NES) that interacts with CRM1, a member of the importin ␤ family of transport receptors (6,15,17,44,58).The interaction between Rev and CRM1 is dependent upon CRM1 association with the GTP-bound form of the GTPase Ran protein (RanGTP). Once assembled, the RRE/Rev-CRM1-RanGTP ribonucleoprotein complex interacts with NPs, which trigger its nuclear export. CRM1 has been proposed to mediate this interaction by directly contacting selected nucleoporins (NPs), including CAN/Nup214. Binding of CRM1 to CAN has been mapped to the NP domain located within the extreme carboxy terminus of CAN (16). Overexpression of the isolated NP domain of CAN, termed ⌬CAN, is able to inhibit Rev-mediated export by competing with the NPs for binding to CRM1 (2). Rev-mediated export is also inhibited by the antibiotic leptomycin B (LMB), w...

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“…To do this, 10 nmol/L LMB was added to the culture medium 18 h after transfection, and cells were further incubated for 6 h. Total protein extracts were then prepared and used to determine the concentration of CAT by ELISA. As negative control pDM138-PRE(+) transfected HuH-7 cells were used since it was previously reported that export of HBV PRE(+) is not sensitive to LMB [23,34] . Figure 7 displays the obtained results.…”

Section: Analysis Of the Nee Function In The Context Of The Fulllengtmentioning

confidence: 99%

“…In the case of human immunodeficiency virus-1 (HIV-1), the association of CRM1 with intron-containing mRNAs is mediated by the virus protein Rev which recognizes a specific sequence named rev-responsive element (RRE) [22] . Additionally, the HBV posttranscriptional regulatory element (PRE), which was reported to play a crucial role in export of virus mRNAs to the cytoplasm, seems to use a distinct, not yet identified nuclear export pathway [23] .…”

Section: Introductionmentioning

confidence: 99%

Searching for nuclear export elements in hepatitis D virus RNA

Freitas¹

2013

WJV

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AIM:To search for the presence of cis elements in hepatitis D virus (HDV) genomic and antigenomic RNA capable of promoting nuclear export. METHODS:We made use of a well characterized chloramphenicol acetyl-transferase reporter system based on plasmid pDM138. Twenty cDNA fragments corresponding to different HDV genomic and antigenomic RNA sequences were inserted in plasmid pDM138, and used in transfection experiments in Huh7 cells. The relative amounts of HDV RNA in nuclear and cytoplasmic fractions were then determined by realtime polymerase chain reaction and Northern blotting. The secondary structure of the RNA sequences that displayed nuclear export ability was further predicted using a web interface. Finally, the sensitivity to leptomycin B was assessed in order to investigate possible cellular pathways involved in HDV RNA nuclear export. RESULTS:Analysis of genomic RNA sequences did not allow identifying an unequivocal nuclear export element. However, two regions were found to promote the export of reporter mRNAs with efficiency higher than the negative controls albeit lower than the positive control. These regions correspond to nucleotides 266-489 and 584-920, respectively. In addition, when analyzing antigenomic RNA sequences a nuclear export element was found in positions 214-417. Export mediated by the nuclear export element of HDV antigenomic RNA is sensitive to leptomycin B suggesting a possible role of CRM1 in this transport pathway. CONCLUSION:A cis-acting nuclear export element is present in nucleotides 214-417 of HDV antigenomic RNA.© 2013 Baishideng. All rights reserved.Key words: Hepatitis D virus; Genomic RNA; Antigenomic RNA; Nuclear export; Nuclear export element Core tip: Hepatitis D virus (HDV) replicates in the nucleus and export of HDV RNPs to the cytoplasm is thought to be mediated by cis-elements present in virus RNA. We used a chloramphenicol acetyl-transferase reporter system in an attempt to identify the RNA sequences that mediate export to the cytoplasm. Several cDNA constructs coding for different HDV RNA (genomic and antigenomic) sequences were tested. Our results show that a cis-acting nuclear export element is present in positions 214-417 of antigenomic RNA. Two regions in genomic RNA were found to promote nuclear export with efficiency higher than the negative control although lower that the positive control.Freitas N, Cunha C. Searching for nuclear export elements in hepatitis D virus RNA. World J Virol 2013; 2(3): 123-135 Available from:

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Distinct Export Pathway Utilized by the Hepatitis B Virus Posttranscriptional Regulatory Element

Cited by 34 publications

References 47 publications

Nuclear Interactions Are Necessary for Translational Enhancement by Spleen Necrosis Virus RU5

Nuclear Interactions Are Necessary for Translational Enhancement by Spleen Necrosis Virus RU5

CRM1-Dependent Function of a cis-Acting RNA Export Element

Searching for nuclear export elements in hepatitis D virus RNA

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