Mutation of the major 5′ splice site renders a CMV‐driven HIV‐1 proviral clone Tat‐dependent: connections between transcription and splicing

Bohne, Jens; Kräusslich, Hans‐Georg

doi:10.1016/s0014-5793(04)00277-7

Cited by 30 publications

(29 citation statements)

References 16 publications

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“…A variant of pNL4-3 containing mutations in the tat and rev genes (pNL4-3 tr− ) was used for insertion of the BGI sequence into the 5′ UTR (Figure 7A), and Tat and Rev expression vectors were cotransfected as indicated. For insertion into the 3′ region, a pNL4-3 derivative containing a CMV promoter driving HIV-1 transcription [pNLC4-3; (44)] and with mutations in the tat and rev genes was used (Figure 7A). Transfection of pNLC4-3 was previously shown to cause production of infectious virus with a similar efficiency as pNL4-3 (44).…”

Section: Resultsmentioning

confidence: 99%

“…For insertion into the 3′ region, a pNL4-3 derivative containing a CMV promoter driving HIV-1 transcription [pNLC4-3; (44)] and with mutations in the tat and rev genes was used (Figure 7A). Transfection of pNLC4-3 was previously shown to cause production of infectious virus with a similar efficiency as pNL4-3 (44). Figure 7B indicates that similar amounts of Gag-derived products were observed when transfection of pNLC4-3 was compared with transfection of pNLC4-3 tr− and pNL4-3BGB tr− in the presence of Tat and Rev (Figure 7B, upper panel, compare lane 2 with lanes 4 and 6).…”

Section: Resultsmentioning

confidence: 99%

“…Plasmid pNLenvM3 was generated by overlap PCR and contains three point mutations in the 5′ss #4 (Figure 5; details of the cloning procedure available upon request). The proviral plasmid pNLC4-3 has been described previously (44). The BGI sequence was cloned into the 3′region of a tat/rev negative derivative of pNLC4-3 by exchanging the BamHI with XhoI fragment (nucleotide 8465–8887) for that from pNLenvBGIs and pNLenvBGIas, respectively.…”

Section: Methodsmentioning

confidence: 99%

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Splicing of human immunodeficiency virus RNA is position-dependent suggesting sequential removal of introns from the 5' end

Bohne¹

2005

Nucleic Acids Research

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Transcription of the HIV-1 genome yields a single primary transcript, which is alternatively spliced to >30 mRNAs. Productive infection depends on inefficient and regulated splicing and appears to proceed in a tight 5′ to 3′ order. To analyse whether sequential splicing is mediated by the quality of splice sites or by the position of an intron, we inserted the efficient β-globin intron (BGI) into the 3′ region or 5′UTR of a subgenomic expression vector or an infectious proviral plasmid. RNA analysis revealed splicing of the 3′ BGI only if all upstream introns were removed, while splicing of the same intron in the 5′UTR was efficient and independent of further splicing. Furthermore, mutation of the upstream splice signal in the subgenomic vector did not eliminate the inhibition of 3′ splicing, although the BGI sequence was the only intron in this case. These results suggest that downstream splicing of HIV-1 RNAs is completely dependent on prior splicing of all upstream intron(s). This hypothesis was supported by the mutation of the major 5′ splice site in the HIV-1 genome, which completely abolished all splicing. It appears likely that the tight order of splicing is important for HIV-1 replication, which requires the stable production of intron containing RNAs, while splicing of 3′ introns on incompletely spliced RNAs would be likely to render them subject to nonsense-mediated decay.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Splicing of human immunodeficiency virus RNA is position-dependent suggesting sequential removal of introns from the 5' end

Bohne¹

2005

Nucleic Acids Research

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“…Plasmids-All HIV plasmids were based either on the proviral construct pNLC4-3 (20), which encodes the complete HIV-1 NL4-3 genome (21) under the control of the cytomegalovirus promoter, or on the subviral plasmid pCHIV (22), which expresses all NL4-3 proteins except Nef but cannot replicate because of the lack of long terminal repeat regions. Derivatives carrying fluorescent proteins fused to the C terminus of the MA domain of the Gag open reading frame (pCHIVeCFP and pCHIVeYFP) have been characterized previously (22,23).…”

Section: Methodsmentioning

confidence: 99%

HIV-1 Gag Processing Intermediates Trans-dominantly Interfere with HIV-1 Infectivity

Müller

Anders

Akiyama

et al. 2009

Journal of Biological Chemistry

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105

140

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Protease inhibitors (PI) act by blocking human immunodeficiency virus (HIV) polyprotein processing, but there is no direct quantitative correlation between the degree of impairment of Gag processing and virion infectivity at low PI concentrations.To analyze the consequences of partial processing, virus particles were produced in the presence of limiting PI concentrations or by co-transfection of wild-type proviral plasmids with constructs carrying mutations in one or more cleavage sites. Low PI concentrations caused subtle changes in polyprotein processing associated with a pronounced reduction of particle infectivity. Dissection of individual stages of viral entry indicated a block in accumulation of reverse transcriptase products, whereas virus entry, enzymatic reverse transcriptase activity, and replication steps following reverse transcription were not affected. Co-expression of low amounts of partially processed forms of Gag together with wild-type HIV generally exerted a trans-dominant effect, which was most prominent for a construct carrying mutations at both cleavage sites flanking the CA domain. Interestingly, co-expression of low amounts of Gag mutated at the CA-SP1 cleavage site also affected processing activity at this site in the wild-type virus. The results indicate that low amounts (<5%) of Gag processing intermediates can display a trans-dominant effect on HIV particle maturation, with the maturation cleavage between CA and SP1 being of particular importance. These effects are likely to be important for the strong activity of PI at concentrations achieved in vivo and also bear relevance for the mechanism of action of the antiviral drug bevirimat.

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“…Transfections of HEK 293T cells with plasmid DNA and the preparation of total or fractionated RNA were performed as described previously (13). The nuclear and cytoplasmic fractions were subjected to RNA extraction with the RNeasy kit (Qiagen) according to the manufacturer's protocol.…”

Section: Methodsmentioning

confidence: 99%

Foamy Virus Nuclear RNA Export Is Distinct from That of Other Retroviruses

Bodem

Schied

Rinaldi

et al. 2011

J Virol

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Most retroviruses express all of their genes from a single primary transcript. In order to allow expression of more than one gene from this RNA, differential splicing is extensively used. Cellular quality control mechanisms retain and degrade unspliced or partially spliced RNAs in the nucleus. Two pathways have been described that explain how retroviruses circumvent this nuclear export inhibition. One involves a constitutive transport element in the viral RNA that interacts with the cellular mRNA transporter proteins NXF1 and NXT1 to facilitate nuclear export. The other pathway relies on the recognition of a viral RNA element by a virus-encoded protein that interacts with the karyopherin CRM1. In this report, we analyze the protein factors required for the nuclear export of unspliced foamy virus (FV) mRNA. We show that this export is CRM1 dependent. In contrast to other complex retroviruses, FVs do not encode an export-mediating protein. Crosslinking experiments indicated that the cellular protein HuR binds to the FV RNA. Inhibition studies showed that both ANP32A and ANP32B, which are known to bridge HuR and CRM1, are essential for FV RNA export. By using this export pathway, FVs solve a central problem of viral replication.The nuclear export of RNA molecules in eukaryotic cells is a tightly regulated process (18,59,63,70,71). Nuclear exit is usually allowed only for fully spliced cellular mRNAs, while intron-containing mRNAs are retained in the nucleus and subsequently degraded (17,18,59,63,70). This defines a specific problem in the replication of retroviruses (RVs), since they must export not only fully spliced but also unspliced or partially spliced mRNAs into the cytoplasm. For the export of the two latter RNA species, retroviruses have found ways to escape both the splicing machinery and the degradation of incompletely spliced mRNAs by making use of either of two strategies for nuclear export of mRNAs with intact splice donor (SD) and acceptor (SA) pairs.In complex retroviruses, such as lentiviruses, some betaretroviruses, and all deltaretroviruses, virus-encoded regulatory proteins (Rev, Rem, and Rex, respectively) bind to the unspliced or incompletely spliced viral mRNA on one hand and contact the karyopherin CRM1 on the other (1,29,33,48,49). Subsequently, this complex shuttles to the cytoplasm, where it delivers the RNA cargo in a regulated fashion that involves Ran in GTP-bound form. Normally CRM1 is used for nuclear export of ribosomal subunits, 5S rRNAs, cellular proteins containing a nuclear export signal (NES), and snRNAs (18,27,53,63). This pathway can also be hitchhiked by endogenous human retroviruses (12,47,74). The presence of regulatory proteins acting at the posttranscriptional level enables complex retroviruses to use a biphasic mode of gene expression ("early" versus "late" phase), resulting in a gain of complexity better known from DNA viruses (16).Alternatively, more simple retroviruses, such as the betaretrovirus Mason-Pfizer monkey virus (MPMV), can (42) contain a cis-acting constitu...

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Mutation of the major 5′ splice site renders a CMV‐driven HIV‐1 proviral clone Tat‐dependent: connections between transcription and splicing

Cited by 30 publications

References 16 publications

Splicing of human immunodeficiency virus RNA is position-dependent suggesting sequential removal of introns from the 5' end

Splicing of human immunodeficiency virus RNA is position-dependent suggesting sequential removal of introns from the 5' end

HIV-1 Gag Processing Intermediates Trans-dominantly Interfere with HIV-1 Infectivity

Foamy Virus Nuclear RNA Export Is Distinct from That of Other Retroviruses

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