Impairment of HIV-1 cDNA Synthesis by DBR1 Knockdown

Impairment of HIV-1 cDNA Synthesis by DBR1 Knockdown Alvaro E. Galvis, a,b,c,d,e Hugh E. Fisher, b,c,d,e Takayuki Nitta, a,c,d Hung Fan, a,c,d David Camerini a,b,c,d,e Department of Molecular Biology & Biochemistry, a Division of Infectious Disease, b Cancer Research Institute, c Center for Virus Research, d and Institute for Immunology, e University of California, Irvine, California, USA ABSTRACT 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 infec- tions 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 fac- tors and occur in different intracellular locations. IMPORTANCE This study shows that HIV-1 reverse transcription starts in the cytoplasm but is completed in or on the surface of the nucleus. Moreover, we show that nuclear reverse transcription is dependent on the activity of the human RNA lariat debranchng enzyme (DBR1), while cytoplasmic reverse transcription is not. These findings may provide new avenues for inhibiting HIV-1 replication and therefore may lead to new medicines for treating HIV-1-infected individuals. H uman immunodeficiency virus type 1 (HIV-1) is the caus- ative agent of AIDS. Like all retroviruses, HIV-1 must con- vert its RNA genome into DNA and then integrate its linear, dou- ble-stranded DNA into the cellular genome to program transcription of new viral RNA. The HIV-1 RNA- or DNA-depen- dent DNA polymerase reverse transcriptase (RT) synthesizes dou- ble-stranded viral DNA using the single-stranded RNA genome as the template (1). Reverse transcription is initiated from a tRNA primer bound at the primer binding site located 183 nucleotides from the 5= end of the HIV-1 RNA genome (nucleotides 183 to 201 [1, 2]). Since the RNA genome is positive sense, the first prod- uct of reverse transcription is minus-sense cDNA. Initially, the cellular tRNA Lys3 primes minus-strand strong-stop DNA synthe- sis, whereby the 5= end of the viral positive-sense RNA genome is copied into minus-strand cDNA while the RNA template is de- graded by the RNase H activity of RT (1, 2). After minus-strand strong-stop DNA synthesis, transfer of this nascent cDNA strand from the 5= end of the genome to the 3= end is required to continue synthesis of complete minus-strand cDNA (3–5) The precise mechanism of this strand transfer, however, has not been eluci- dated. Retrotransposons are mobile genetic elements that resemble retroviruses and contain long terminal repeats (LTRs). They rep- licate and transpose via RNA intermediates. The Ty1 retroelement is among the best characterized of the retrotransposons of the jvi.asm.org Journal of Virology yeast Saccharomyces cerevisiae (6). Using a genetic screen aimed at identifying cellular factors involved in Ty1 transposition, Chap- man and Boeke found that debranching enzyme 1 (DBR1) plays a role in Ty1 transposition (7). DBR1 is a nuclear 2=-5= phosphodi- esterase that cleaves branch-point bonds in excised intron RNA lariats after mRNA splicing, facilitating turnover and recycling of lariat ribonucleotides. Yeast DBR1 mutant strains produce ma- ture mRNAs but accumulate intron lariats, and they are defective in both Ty1 cDNA formation and transposition (6, 8, 9). Cheng and Menees (8) provided evidence that during cDNA synthesis the Ty1 RNA genome contains a 2=-5= branch characteristic of an RNA lariat, although these data remain controversial (10). The location of this branch connecting the 5= end of the genome to the 3= nucleotide of the U3 region suggested that it may play a role during Ty1 cDNA synthesis by facilitating the transfer of nascent minus-strand cDNA from the 5= end of the Ty1 RNA template to Received 14 March 2014 Accepted 18 March 2014 Published ahead of print 26 March 2014 Editor: K. L. Beemon Address correspondence to David Camerini, david.camerini@uci.edu. Copyright © 2014, American Society for Microbiology. All Rights Reserved. doi:10.1128/JVI.00704-14 p. 7054 –7069 June 2014 Volume 88 Number 12