1. HIV-1 Reverse Transcription is Completed in the Nucleus and is Inhibited by DBR1 shRNA
Alvaro E. Galvis 1,2,3,4,5, Hugh E. Fisher 2,3,4,5, Hung Fan1,3,4 and David Camerini2,3,4,5*
1Department of Molecular Biology & Biochemistry, 2Division of Infectious Disease, 3Cancer Research Institute, 4Center for Virus Research and 5Institute for Immunology, University of California, Irvine CA
Corresponding author: David Camerini, Division of Infectious Disease, University of California
Irvine, CA ; Phone: (949) ,
Abstract
Background: During HIV-1 cDNA synthesis nascent retroviral minus-strand cDNA molecules translocate from the 5’ end of the RNA genome to near the 3’ end. The mechanism by which this occurs and the subcellular compartment where this event takes place remain poorly characterized. By analogy to the yeast retroelement Ty1, we hypothesize that the HIV-1 genome forms an RNA lariat intermediate that facilitates the minus-strand template switch during cDNA synthesis. To test this hypothesis, the cellular RNA lariat debranching enzyme (DBR1) was downregulated using short hairpin RNAs (shRNA) and HIV cDNA synthesis was monitored temporally and spatially within infected cells.
Methods: DBR1 shRNAs were introduced into GHOST-R5X4 cells and followed 48 hours later by infection with HIV-1 or an HIV-1 derived vector. DNA and RNA were isolated from whole cells as well as from cytoplasmic and nuclear fractions at 0, 1, 2, 4, 6, 12 and 24 hours post-infection (p.i.). HIV-1 reverse transcription was monitored by quantitative PCR (QPCR) for early, intermediate and late products of reverse transcription. Student’s t-test was utilized to analyze the data for statistical significance.]
Results: Inhibition of DBR1 caused a significant reduction in the detection of intermediate-length and full-length cDNA at all measured time points (p<0.01) but had only a minor effect on formation of the initial cDNA product from the primer binding site to the 5’ end of the genome (strong-stop cDNA,). We further observed that strong-stop signal rapidly accumulated in the cytoplasm at the beginning of infection (greater than 70%, 2 hours p.i., p= 2.7 x 10-7) but shifted to the nuclear fraction at later time points beginning 4 hours p.i. (50%, p= 4 x 10-5). Moreover, the shift of strong stop cDNA to the nucleus occurred even when intermediate and full-length cDNA synthesis was inhibited by DBR1 knockdown. Finally, regardless of DBR1 inhibition the vast majority of intermediate-length and full-length HIV-1 cDNA product was found in the nuclear fraction at all detectable time points (p<0.01).
Conclusion:. These data indicate the cellular RNA lariat debranching enzyme is involved in an early step in HIV-1 cDNA synthesis, and are consistent with the hypothesis that HIV-1 utilizes an RNA lariat intermediate during minus-strand cDNA synthesis to facilitate template switching. Moreover, our data show that HIV-1 cDNA synthesis is initiated in the cytoplasm and completed in the nucleus.
Results
DBR1 shRNA suppressed HIV-1 reverse transcription after strong stop cDNA synthesis.
Strong-stop cDNA synthesis was not affected by DBR1 shRNA, D4, until four hours post-infection and was inhibited only by one-third at later times compared to the triple mismatched control shRNA, M4 (Fig 1B). In contrast, the levels of intermediate and late products of reverse transcription were substantially decreased in the presence of DBR1 shRNA at all detectable time-points post-infection (p<0.001; Fig. 1C, 1D). Furthermore, as time after infection progressed, the difference between the amounts of intermediate and full-length HIV-1 vector cDNA copies in pHyper-D4 versus pHyper-M4 treated cells increased with a maximal fold difference of 8.5 (p = 3.1 x 10-6) and 7.6 (p = 5.9 x 10-10) respectively twenty-four hours post-infection. In the DBR1 shRNA, D4, treated cells, therefore, cDNA synthesis was inhibited after minus-strand transfer compared to mismatch shRNA treated cells and the difference between the two groups widened over time
Summary of Results
Inhibition of DBR1 had a small but significant effect on HIV-1 strong-stop DNA detection because it detects all HIV cDNA.
HIV-1 strong-stop DNA rapidly accumulated in the cytoplasm at the beginning of infection but was associated with the nucleus at later time points.
Inhibition of DBR1 led to a decrease in detection of intermediate and full length HIV-1 cDNA products.
Regardless of DBR1 inhibition the majority of envelope and full-length HIV-1 cDNA was found in the nuclear fraction.
Analysis of the viral RNA genome demonstrated that it rapidly associated within the nucleus and that DBR1 inhibition protected the 3’ region from being degraded.
We obtained similar results with a clinical HIV-1 isolate.
Discussion
Early studies of HIV-1 reverse transcription demonstrated via Southern blot analysis that linear viral DNA could be detected in the cytoplasm but the vast majority was found in the nuclear fractions (Kim et al 1989, Barbosa et al 1994). Furthermore, functional pre-integration complexes can be isolated from both the cytoplasmic and nuclear fractions (Engelman et al 2009). Based on these reports the current model of HIV-1 reverse transcription states that this process occurs predominantly in the cytoplasm. However, utilizing QPCR in our study we saw that in GHOST cells greater than 95% of full length genomes were found in the nuclear fraction.
Our results do not necessarily contradict previous reports. Rather based on those studies and our data we propose that while reverse transcription initiates in the cytoplasm, the vast majority occurs after import of the reverse transcription complex to the nucleus where it interacts with DRB1 to facilitate minus-strand transfer.
Acknowledgements
Medical Scientist Training Program. University of California, Irvine.
Center for Virus Research. University of California, Irvine.
California HIV-AIDS Research Grants Program Office of the University of California. Grant number ID07-I-124
Results
DBR1 inhibition protects the 3’ region of the viral RNA genome from being degraded during cDNA synthesis.
To more precisely assess the effects of DBR1 inhibition during reverse transcription we followed the degradation of the HIV-1 vector RNA genome in cytoplasmic and nuclear fractions of infected cells We found that the 5’ region of the vector genome was rapidly degraded, consistent with early reverse transcription of minus-strand strong-stop DNA and degradation of the template. As expected, DBR1 shRNA had no effect at any time on the degradation of the 5’ region of the vector genome since knockdown of DBR1 did not inhibit minus-strand strong-stop DNA synthesis (Figs. 3A, 3B). In contrast, knockdown of DBR1 prevented degradation of the 3’ region of the HIV-1 vector (Figs. 3C, 3D), consistent with its inhibition of late reverse transcription products. As a result, there was an increasing difference between the level of HIV vector RNA 3’ region detected in the DBR1 shRNA and mismatch treated groups over time post-infection with a maximum fold difference of 6.1 (p = 1.96 x 10-6) twelve hours post-infection (Figure 3C, 3D)Furthermore, the 3’ region of the HIV vector RNA genome was found almost exclusively in the nuclear fraction by six hours post-infection. Taken together these data are consistent with the view that HIV-1 initiates reverse transition in the cytoplasm but must be transported to the nucleus to recruit DBR1 and complete cDNA synthesis
Figure 1
Figure 3
Background
Human immunodeficiency virus type 1 (HIV-1) is the causative agent of the acquired immunodeficiency syndrome (AIDS). Like all retroviruses, HIV-1 must convert its RNA genome into DNA and then integrate its linear, double-stranded DNA into the cellular genome to produce new viral RNA. The HIV-1 RNA or DNA dependent DNA polymerase, reverse transcriptase (RT), synthesizes the conversion of the single-stranded RNA genome to DNA. Reverse transcription is initiated from a tRNA primer bound at the primer binding site (PBS) that is located 183 nucleotides from the 5’ end of the HIV-1 RNA genome and spans 18 nucleotides (nucleotides ). Since the RNA genome is positive sense, the first product of reverse transcription is minus sense cDNA. Early in infection, the cellular tRNALys3 primes minus-strand strong-stop DNA synthesis, whereby the 5’ end of the viral positive-sense RNA genome is copied into minus-strand cDNA while the RNA template is degraded by the RNase H activity of RT. After minus-strand strong-stop DNA synthesis, a strand transfer of this nascent cDNA from the 5’ end of the genome to the 3’ end, is required to continue synthesis of complete minus-strand cDNA. The precise mechanism of this strand transfer, however, has not been identified.
In the present study, we demonstrated that DBR1 inhibition early in reverse transcription decreases the amount of detectable intermediate and late products of reverse transcription but had minimal effect on detectable minus-strand strong-stop DNA. By cell fractionation, we determined that minus-strand strong-stop DNA signal rapidly accumulated in the cytoplasm but migrated to the nucleus within six hours. Moreover, intermediate and late products of reverse transcription were detected only in the nucleus regardless of DBR1 inhibition. Our results imply that reverse transcription begins in the cytoplasm and is paused at the completion of minus-strand strong-stop DNA synthesis. Subsequently, the reverse transcription complex migrates into the nucleus where DBR1 facilitates the continuation of minus-strand cDNA synthesis and ultimately the formation of full-length cDNA.
Results
Analysis of cytoplasmic and nuclear fractions demonstrated that reverse transcription was initiated in the cytoplasm and completed in the nucleus.
Detection of HIV-1 vector cDNA in subcellular fractions demonstrated that at one and two hours post-infection, strong-stop cDNA was predominantly found in the cytoplasm in both control shRNA, M4, treated cells (Fig 2A) and DBR1 shRNA, D4, treated cells (Fig 2B). By four hours post-infection, however, almost 50% of the strong-stop cDNA was associated with the nuclear fraction and at later times it was increasingly nuclear localized in both DBR1 shRNA treated and control cells. As above, the accumulation of HIV-1 vector strong-stop DNA was only slightly inhibited by DBR1 shRNA expression. In contrast, DBR1 shRNA treatment strongly inhibited the formation of post minus-strand template switch products but did not affect their localization. Both intermediate and full-length products of reverse transcription, were found almost exclusively in the nuclear fraction at all times regardless of DBR1 shRNA treatment (Figs. 2C-2F). Consistent with figure 1 above), env region cDNA synthesis was inhibited 8.1 fold and full-length cDNA was inhibited 8.2 fold by DBR1 shRNA at twenty-four hours post-infection. The cell fractionation data strongly suggest that transport of the reverse transcription complex (RTC) to the nucleus facilitates steps in reverse transcription that occur after strong-stop cDNA synthesis. This is consistent with the hypothesis that a nuclear factor such as DBR1 is required for the completion of HIV-1 cDNA synthesis.
Figure 2
Methods: