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Using the Fermentas First Strand cDNA Synthesis Kit.

RNA/Reverse Transcription (RT) & cDNA Synthesis Protocols Reverse Transcription (RT) protocols.

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(RT) provided with the 1st-strand cDNA synthesis kit is Agilent's.

Results of infection of GHOST-R5X4 cells in which DBR1 was knocked down are also shown in , , and . As described above, the initial cytoplasmic accumulation of strong-stop DNA was largely unaffected by DBR1 shRNA expression. In contrast, DBR1 shRNA treatment strongly inhibited the formation of products after the minus-strand template switch; this effect was observed in the nuclear fractions. Consistent with the results shown in and our previous work (), nuclear env region cDNA synthesis was inhibited 8.1-fold and nuclear full-length cDNA synthesis was inhibited 8.2-fold by DBR1 shRNA at 24 h postinfection. Moreover, these results are also consistent with the hypothesis that DBR1, which is a nuclear factor, may facilitate HIV-1 cDNA synthesis during or after minus-strand transfer.

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To further investigate the relationship of the nuclear transport of the RTC and the completion of reverse transcription, we employed a HeLa-derived cell line that expresses a truncated dominant negative form of the cleavage and polyadenylation factor 6 (CPSF6) known as CPSF6-358 (). Lee et al. have shown that expression of CPSF6-358 blocks HIV-1 infection at the level of nuclear transport (). We transfected HeLa cells expressing CPSF6-358 with D4 or the M4 control for 48 h and then infected the cells with VSV-G-pseudotyped HIV-1 vector HR-E and harvested the cells at 2 to 24 h postinfection. Cells were fractionated into total, cytoplasmic, and nuclear fractions, and synthesis of HIV-1 cDNA was measured by qPCR, as described above. As previously reported (), the expression of CPSF6-358 caused the accumulation of strong-stop cDNA in the cytoplasm, which did not occur in infected control HeLa cells ( and ). At later times, there was low-level transfer of strong-stop cDNA to the nucleus, which may have resulted from the fact that these cells were dividing. In CPSF6-358 cells transfected with the D4 plasmid for DBR1 knockdown, there was little effect on the initial amount of total strong-stop cDNA, which was largely retained in the cytoplasm (). As described above, the total amounts of strong-stop cDNA at later times were reduced due to the lack of generation of the second LTR during late reverse transcription in the nucleus.

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We have previously reported that short hairpin RNA (shRNA)-mediated knockdown of human DBR1 in HIV-1-infected cells inhibits the detection of late reverse transcription products and HIV capsid protein p24 at 24 h postinfection but has little effect on minus-strand strong-stop DNA synthesis. Moreover, this effect can be reversed if cells are cotransfected with a DBR1 expression plasmid (). These results are consistent with a mechanism in which the 5′ and 3′ ends of the genomic RNA are brought together during reverse transcription into an RNA lariat-like structure in the absence of sequence homology; if a lariat-like intermediate is formed, resolution of the structure by DBR1 would be important for the completion of cDNA synthesis, since lariat branch points cause RT pausing or termination (). Alternatively, DBR1 might play a different role in HIV-1 reverse transcription independent of lariat resolution.

Previous studies showed that short hairpin RNA (shRNA) knockdown of the RNA lariat debranching enzyme (DBR1) led to a decrease in the production of HIV-1 cDNA. To further characterize this effect, DBR1 shRNA was introduced into GHOST-R5X4 cells, followed by infection at a multiplicity near unity with HIV-1 or an HIV-1-derived vector. DNA and RNA were isolated from whole cells and from cytoplasmic and nuclear fractions at different times postinfection. Inhibition of DBR1 had little or no effect on the formation of minus-strand strong-stop cDNA but caused a significant reduction in the formation of intermediate and full-length cDNA. Moreover, minus-strand strong-stop DNA rapidly accumulated in the cytoplasm in the first 2 h of infection but shifted to the nuclear fraction by 6 h postinfection. Regardless of DBR1 inhibition, greater than 95% of intermediate-length and full-length HIV-1 cDNA was found in the nuclear fraction at all time points. Thus, under these experimental conditions, HIV-1 cDNA synthesis was initiated in the cytoplasm and completed in the nucleus or perinuclear region of the infected cell. When nuclear import of the HIV-1 reverse transcription complex was blocked by expressing a truncated form of the mRNA cleavage and polyadenylation factor CPSF6, the completion of HIV-1 vector cDNA synthesis was detected in the cytoplasm, where it was not inhibited by DBR1 knockdown. Refinement of the cell fractionation procedure indicated that the completion of reverse transcription occurred both within nuclei and in the perinuclear region. Taken together the results indicate that in infections at a multiplicity near 1, HIV-1 reverse transcription is completed in the nucleus or perinuclear region of the infected cell, where it is dependent on DBR1. When nuclear transport is inhibited, reverse transcription is completed in the cytoplasm in a DBR1-independent manner. Thus, there are at least two mechanisms of HIV-1 reverse transcription that require different factors and occur in different intracellular locations.

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In this study, we confirmed the findings of our earlier work showing that DBR1 shRNA-mediated inhibition of HIV-1 replication occurs after minus-strand strong-stop cDNA synthesis but prior to reverse transcription of the env gene or the completion of cDNA synthesis (). We extended the previous studies significantly by using qPCR and cellular fractionation to study the intracellular location of different steps in reverse transcription and the role of DBR1. We found that while minus-strand strong-stop cDNA formation initially takes place in the cytoplasm, minus-strand transfer and the completion of reverse transcription take place in the nucleus or in the perinuclear region under the conditions used at an MOI near 1. Furthermore, when we inhibited viral nuclear import by CPSF6-358 expression in dividing HeLa cells, the completion of late steps in reverse transcription in the cytoplasm or perinuclear regions was enhanced. Moreover, nuclear completion of full-length cDNA synthesis was dependent on DBR1, while cytoplasmic completion of cDNA synthesis in CPSF6-358-expressing cells was not; perinuclear completion of cDNA synthesis appeared to be sensitive to DBR1 knockdown in control HeLa cells but less so in CPSF6-358-expressing cells. Our results suggest that there are two mechanisms for the completion of HIV-1 cDNA synthesis. In the predominant mechanism under our experimental conditions, the completion of reverse transcription occurs in the nucleus or in the perinuclear region and requires DBR1, perhaps to resolve an RNA structure such as a lariat to allow the completion of reverse transcription. In the second mechanism, which is enhanced in CPSF6-358-expressing cells, the completion of reverse transcription occurs in the cytoplasm and is DBR1 independent; whether the configuration of the template RNA genome is the same for these two mechanisms remains to be determined.

When infected HeLa cells expressing CPSF6-358 (and transfected with the control M4 plasmid) were analyzed by the double-detergent fractionation procedure, the intermediate and late reverse transcription products that accumulated in the C+P fraction represented higher proportions of the total cDNA (compare and with and ). This indicated that some of the RTCs in CPSF6-358-expressing cells were likely accumulating at the perinuclear region and completing reverse transcription there. When CPSF6-358-expressing cells were also treated with the D4 DBR1 shRNA, there was a ca. 50% reduction in the accumulation of intermediate and full-length cDNA in the nucleus, while accumulation in the cytoplasm (C+P) was less affected (compare and with and ). This might suggest that, unlike the situation in control HeLa cells, some of the perinuclear RTCs accumulating in CPSF6-358-expressing cells are completing reverse transcription without the involvement of DBR1.

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Protocol for First-strand cDNA Synthesis - protocol …

Reverse transcriptase is an enzyme capable of synthesizing a OneScript® cDNA Synthesis Kit (25 rxns) | Applied Biological 2) A higher random primer ratio could result in a shorter cDNA being synthesized.

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In the present study, we confirmed our previous results that DBR1 inhibition decreases the amount of detectable intermediate and late products of reverse transcription in cells infected by an HIV-1-based vector or a clinical HIV-1 isolate but that it has minimal effect on the formation of minus-strand strong-stop DNA. We extended these studies by performing cell fraction experiments and detected the great majority of intermediate and late reverse transcription products in the nuclear or perinuclear region, indicating that under the conditions employed at a multiplicity of infection (MOI) of nearly 1, reverse transcription is initiated in the cytoplasm but completed in the nuclear fraction. When HIV-1 nuclear entry was blocked in cells expressing a truncated form of cleavage and polyadenylation factor 6 (CPSF6) known as CPSF6-358, intermediate and full-length cDNA synthesis was detected in the cytoplasm; cytoplasmic cDNA synthesis was not dependent on DBR1. Refinements of the cell fractionation procedure indicated that the completion of cDNA synthesis can occur either in the nucleus or in the perinuclear region. Taken together, the results indicate that strand transfer and the completion of HIV-1 cDNA synthesis predominantly take place in the nucleus or perinuclear region but that it can also take place in the cytoplasm. Nuclear completion of cDNA synthesis is dependent on DBR1, while the completion of cDNA synthesis in the cytoplasm is not. These results provide evidence of two alternate mechanisms of HIV-1 reverse transcription.

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RNA purification, cDNA synthesis and preparation for Illumina microread sequencing in cDNA Synthesis - MIT OpenCourseWare First strand cDNA Synthesis using the NEB Protoscript kit.

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