hnRNP E1 and E2 have distinct roles in modulating HIV-1 gene expression
© Woolaway et al; licensee BioMed Central Ltd. 2007
Received: 05 December 2006
Accepted: 23 April 2007
Published: 23 April 2007
Pre-mRNA processing, including 5' end capping, splicing, and 3' end cleavage/polyadenylation, are events coordinated by transcription that can influence the subsequent export and translation of mRNAs. Coordination of RNA processing is crucial in retroviruses such as HIV-1, where inefficient splicing and the export of intron-containing RNAs are required for expression of the full complement of viral proteins. RNA processing can be affected by both viral and cellular proteins, and in this study we demonstrate that a member of the hnRNP E family of proteins can modulate HIV-1 RNA metabolism and expression. We show that hnRNP E1/E2 are able to interact with the ESS3a element of the bipartite ESS in tat/rev exon 3 of HIV-1 and that modulation of hnRNP E1 expression alters HIV-1 structural protein synthesis. Overexpression of hnRNP E1 leads to a reduction in Rev, achieved in part through a decrease in rev mRNA levels. However, the reduction in Rev levels cannot fully account for the effect of hnRNP E1, suggesting that hmRNP E1 might also act to suppress viral RNA translation. Deletion mutagenesis determined that the C-terminal end of hnRNP E1 was required for the reduction in Rev expression and that replacing this portion of hnRNP E1 with that of hnRNP E2, despite the high degree of conservation, could not rescue the loss of function.
Prior to their export to the cytoplasm, eukaryotic pre-mRNAs undergo a number of processing events that include capping, splicing and 3' end processing (cleavage of the nascent transcript and polyadenylation). These processing events occur as the transcript is being synthesized, and each one is able to influence the efficiency and specificity of the others [1–5]. RNA processing is also required for efficient export of the mRNA to the cytoplasm and its translation [6–10]. Retroviruses such as HIV-1 however, export unspliced and incompletely spliced viral RNAs normally retained in the nucleus. By encoding proteins from these intron containing RNAs, retroviruses increase their coding potential . Indeed, suboptimal splicing of the primary HIV transcript generates over 30 mRNAs, many of which (e.g. Gag, Gagpol, Vpr, Vif, Vpu and Env) contain introns. HIV is able to overcome the requirement for complete splicing of a transcript prior to its export to the cytoplasm by the action of the virally encoded protein Rev. Rev functions by forming multimers that interact directly with a cis-acting Rev response element (RRE). This complex is exported via an interaction with host cellular Crm1/Exportin 1 through a pathway normally used by snRNA [12, 13].
Numerous host cellular factors can influence the processing and transport of HIV-1 viral RNAs, including Sam68 [14–17], hStaufen [18, 19], eIF5A [20, 21] and hRIP . hnRNPs are another class of proteins that associate with nascent transcripts to influence many stages of RNA metabolism [23–25], including that of HIV-1 [26–31]. The hnRNP E proteins (or α-complex proteins (αCPs) or poly(C)-binding proteins (PCBPs)) were first functionally characterized as components of a complex that stabilizes human α-globin [32–34]. Subsequent studies demonstrated the existence of five major isoforms, hnRNP E1 to hnRNP E4 and hnRNP E2-KL (a splice variant of hnRNP E2 differing from the original by a 31 amino acid deletion in the region between KH2 and KH3), encoded by four genetic loci [35, 36]. Since their initial characterization, hnRNP E proteins have been implicated in a wide array of processes including mRNA stabilization, translational enhancement and translational silencing [32–34]. Studies have also indicated a role for the hnRNP E proteins in the post-transcriptional regulation of a number of viruses. Examples include the stabilization and translational enhancement of poliovirus RNA, and translational silencing of human papillomavirus L2 mRNA [37–39].
The hnRNP E proteins belong to the triple KH domain containing protein family that includes hnRNP K . All of the isoforms of hnRNP E contain three repeats of the type 1, 70 amino acid KH domain, designated KH1, KH2 and KH3. Each KH domain is able to interact independently with a target RNA sequence. Therefore, these proteins have the potential for complex and highly specific RNA interactions . The hnRNP E proteins are expressed in many human tissues and have been shown to bind poly C regions [35, 41, 42], although both hnRNP E1 and hnRNP E2 have been shown to interact with mRNAs of low C content . The most highly expressed and well characterized of the hnRNP E proteins are the hnRNP E1 and E2 isoforms, which are 89% similar at the amino acid level . While multiple splice isoforms of hnRNP E2 are known to exist, hnRNP E1 is encoded by an intronless gene believed to be the product of a retrotransposition event of a fully processed minor isoform of hnRNP E2 . It has been suggested that the conservation of hnRNP E1 indicates it serves a non-redundant function although, it is presently unclear what this role might be.
In this study, we began to look for endogenous trans-acting factors that acted on HIV-1 ESS3. Two of the factors identified, were hnRNP E1 and hnRNP E2 and in this paper, we investigate the effects of overexpression and depletion of hnRNP E1 and E2 on HIV-1 gene expression. We demonstrate that overexpression of hnRNP E1 but not hnRNP E2 can inhibit expression of the Rev-dependent RNAs encoding gp120 and p24. Our data is consistent with hnRNP E1 acting to decrease viral mRNA translation. Depletion of either hnRNP E1 or E2 resulted in increased production of HIV-1 structural proteins. Domain analysis of the protein indicates that only the two carboxy-terminal KH domains of hnRNP E1 are required for its inhibitory effect. The differential capacity of hnRNP E1 and E2 to modulate HIV-1 RNA processing and utilization suggest that, despite their high degree of similarity, these two proteins have distinct, non-redundant roles in modulating HIV-1 gene expression.
hnRNP E proteins are enriched in ESS3a containing columns
One of the sequences generated by mass spectrometry matched a region found in both hnRNP E1 and hnRNP E2 (Figure 1C). We obtained an antibody to hnRNP E2 (kind gift from Raul Andino) and western blotting analysis confirmed that hnRNP E2 was selectively retained on the wt ESS3a column (Figure 1B), at this stage we did not have an antibody to hnRNP E1. Work by several groups has demonstrated a role for members of the hnRNP E family in the post-transcriptional regulation of a number of viruses [37–39]. Therefore, further study of the effects of this family of trans-acting factors on HIV expression appeared justified.
hnRNP E1 But Not hnRNP E2 overexpression suppresses HIV-1 gene expression
Vectors expressing myc epitope tagged forms of hnRNP E1 and hnRNP E2 (mycE1, mycE2) were generated and confirmed by sequencing. These were co-transfected into 293 cells alongside the replication incompetent proviral HxBru R-/RI- construct and CMVPLAP plasmid expressing secreted alkaline phosphatase (SEAP). 48 hrs post transfection, cells were harvested and lysates analyzed by western blot. To compare the level of expression of mycE1 and mycE2, myc blots were performed. Equal loading was confirmed by blotting for tubulin. To determine whether any effects on virus expression were as a result of a general inhibition of translation, expression of secreted alkaline phosphatase (SEAP) was also measured.
hnRNP E1/E2 overexpression has limited effects on HIV-1 RNA levels and splicing
hnRNP E1 or E2 knockdown increase HIV-1 gene expression
hnRNP E1 overexpression reduces rev synthesis
Two pgTat derivatives, pgTatΔESE (lacking the ESE but retaining the ESS) and pgTatΔESEΔESS SL1 (where the ESE and ESS have been replaced with a sequence of similar size from the EDA exon of the human fibronectin gene; Figure 5A) were also used to assess the potential role of ESE and ESS3a in mediating any effects of mycE1/E2 on HIV RNA processing . In all cases, mycE1 overexpression reduced gp160/120 expression while mycE2 overexpression had no effect. Consistent with previous results , a low level of gp160/120 expression was observed from pgTatΔESE which was further reduced upon expression of mycE1. The results demonstrate that the inhibitory effect of mycE1 does not require either ESE3 or the ESS3a, as reduced gp160/120 expression was observed for all constructs tested (Figure 5B).
To probe the basis for the observed inhibition of gp160/120 expression, we first looked at whether hnRNP E1 was altering viral RNA processing, in particular preventing the formation of unspliced/cleaved env RNA, the preferred substrate for Rev export [17, 46]. However, only a two-fold decrease in the level of unspliced, polyadenylated env RNA was seen upon overexpression of mycE1 (data not shown). This observation is consistent with the reduction in proviral RNA seen in Figure 3A, but it is unable to fully account for the loss of viral protein expression. Alternatively, mycE1 may act indirectly by affecting Rev, as export of env RNA is dependent upon Rev. Indeed, co-expression of mycE1 decreased the number of Rev positive cells as assayed by immunofluorescence (data not shown). To test this further, we transfected cells with the Rev-dependent reporter, GagRRE, and assessed expression of both Gag (α p24) and Rev by Western blot in the presence or absence of the myc-tagged hnRNP E proteins. As shown in Figures 5C and 5D, cotransfection of mycE1 with the GagRRE reporter construct reduced both p24 production and Rev expression. In contrast, mycE2 had no effect. No significant change in cotransfected SEAP expression was seen, suggesting that the inhibition of Rev expression by mycE1 is not a generalized effect (Figure 5E). Thus, hnRNP E1 and E2 isoforms appear to differ in their ability to inhibit HIV-1 gene expression both from the provirus and from HIV-1 expression constructs. Also, the response to mycE1 appears to be independent of ESS3 within the HIV-1 env reporter. However, the sequence of ESS3 is also present in Rev, possibly explaining why hnRNP E1 regulates its expression.
Deletion of the C terminal KH domain of hnRNP E1 abrogates its ability to inhibit Rev expression
Given the effect of the N- and C-terminal domain mutants on hnRNP E1 subcellular distribution, we were interested to determine if these changes in protein localization correlated with a change in the ability of mycE1 to suppress viral protein expression. 293T cells were transfected with the env expression vector (pgTat), as well as plasmids expressing Rev and mycE1 (or domain mutants thereof) or mycE2. As shown in Figure 6C, mycE1ΔN mutant retains the ability to inhibit gp160/120 and Rev expression (Figure 6C). In contrast, deletion of the C-terminal KH domain of hnRNP E1 completely abrogates the ability of the protein to inhibit gp160/120 and Rev expression. Interestingly, the domain swap mutant mycE1N/2C had reduced activity relative to mycE1 indicating that the C-terminus of hnRNP E1 performs a unique function. As before, no effect on Rev or gp160/120 expression was observed upon co-expression with mycE2. None of the constructs induced any significant alteration in expression of cotransfected secreted alkaline phosphatase (SEAP, Fig. 6D). Thus, the C-terminal KH domain of hnRNP E1 is required both for the localization of the protein to the nucleus and, in part, for the inhibition of viral protein (gp160/120, Rev) expression.
Effects of hnRNP E1 and its domain mutants on HIV-1 RNA abundance, processing and subcellular distribution
To investigate the basis for the effect of mycE1 on Rev expression, its effect on rev RNA abundance was also examined. Total RNA was isolated from 293T cells transfected with pgTat, and plasmids expressing Rev, and mycE1, mutants thereof, or mycE2, and RPAs performed using a rev specific probe. Co-transfection of a VA RNA expression vector served as an internal control. mycE1, mycE1ΔN, and mycE1N/2C resulted in only a modest (~2 fold) decrease in the level of rev RNA (Figure 7C, D). No significant changes in rev RNA levels were detected upon cotransfection with mycE1ΔC, or mycE2. Reduction in RNA levels is observed even for those constructs lacking an intron (SV Rev), consistent with an alteration in stability of the affected RNAs.
In this study, we have demonstrated that hnRNP E1 is a trans-acting factor able to modulate HIV-1 gene expression. This fact, coupled with the identification of hnRNP E1 in the virion particle, suggests that this factor plays an important role in HIV-1 expression and assembly . Although hnRNP E1 were initially examined due to an interaction with ESS3 in vitro, subsequent work determined that it does not function through altering the splicing of HIV-1 RNA to any significant extent (Fig. 3B, C). Indeed, hnRNP E1 overexpression led to dramatic suppression of reporter constructs (GagRRE, pgTatΔESEΔESS/SL1) that lacked ESS3 (Fig. 5). However, since all viral RNAs include the terminal exon containing ESS3, the effect of hnRNP E1 can be explained by its interaction with the mRNAs encoding the HIV-1 regulatory proteins Tat and Rev. Although the ESS3 lacks a polycytosine tract similar to the consensus hnRNP E1 binding site, other studies have demonstrated binding to sequences that do not fit the consensus . In addition, recent work has shown that hnRNP E1 can be recruited to an RNA through protein-protein interaction (in particular via hnRNP A2) to modulate translation of a mRNA . Given the previously determined role of hnRNP A1/A2 in mediating the activity of ESS3 [50, 51], recruitment of hnRNP E1/E2 to this sequence may be indirect. However, the loss of hnRNP E1/E2 binding to mutants of ESS3 that retain hnRNP A1 binding (Fig. 1) and the independence of the response to the presence of the ESS3 suggests that hnRNP E1 may be interacting with sequences elsewhere in the viral RNA. Previous work on hnRNP E1 and E2 has identified roles for these factors in the translational control of both viral and cellular mRNAs [33, 37–39, 52–54] as well as in the mediation of mRNA stabilization [55–57]. These effects are the result of either steric protection of the mRNA 3'UTR from endonucleolytic attack  or protection of the poly (A) tail from degradation by interacting with the poly(A)-binding protein (PABP; [59–61]). Little is known as to whether the different hnRNP E proteins have unique functions or if they show redundancy in their actions. In this study, we establish that hnRNP E1 and hnRNP E2 elicit different responses as only overexpression of hnRNP E1 inhibited the expression of several HIV-1 genes. This difference was observed in the context of both the HIV provirus and subviral expression plasmids. The failure to detect significant alterations in cotransfected SEAP expression suggests that the response to hnRNP E1 is somewhat restricted. Consistent with the overexpression assays, depletion of hnRNP E1 or E2 by siRNA resulted in increased expression of HIV-1 Gag and Env. These findings underline the differential roles of these factors in modulating HIV-1 gene expression.
The basis for the observed effect of hnRNP E1 remains only partially defined. Overexpression of hnRNP E1 resulted in a decrease in the levels of Rev protein. The loss of Rev is explained in part by effects at the RNA level. Forms of hnRNP E1 (mycE1 and mycE1ΔN) that yielded reduced Rev protein expression elicited a 2 fold reduction in rev RNA abundance. A similar change in rev RNA but not protein levels was also observed upon co-expression of mycE1N/2C. However, the fold change in rev RNA levels is not consistent with the extent of reduction in Rev protein (decreased 5 fold, Fig, 5D). Therefore, it is likely that the loss of Rev expression is also due to reduced translation of its RNA, a phenomenon consistent with the known role of hnRNP E1 in translational silencing of RNAs in other systems. A similar decrease in abundance is also seen for the spliced form (S/C) of pgTat (Fig. 7) and proviral RNAs (Fig. 3) although, in the context of pgTat, all constructs except hnRNP E2 reduced S/C RNA abundance by ~2 fold. However, despite reduced Rev expression, viral RNAs encoding structural proteins still accumulate in the cytoplasm (Fig. 8). Therefore, the loss of HIV Gag and Env expression upon hnRNP E1 overexpression is not due to a block in export of these RNAs but rather an inhibition of their translation.
Deletion analysis identified domains of hnRNP E1 essential for the modulation of HIV-1 expression and revealed a correlation with the extent of nuclear accumulation of the protein. Analysis of the subcellular localization of our hnRNP E1 domain mutants is consistent with earlier studies  that identified a nuclear localization signal (NLS) between KH2 and KH3 of hnRNP E1. Deletion of the C-terminal 148 amino acids from hnRNP E1 (mycE1ΔC) resulted in the redistribution of the protein from the nucleus to the cytoplasm. Removal of the C-terminal KH domain also resulted in loss of inhibition of HIV-1 gene expression. However, our work failed to show localization of full length hnRNP E1 to nuclear speckles. Swapping the C-terminal domain (KH3) of hnRNP E1 and E2 (mycE1N/2C) only partially restored the nuclear accumulation of the mutant, the distribution of the fusion protein being comparable to that observed for mycE2. The failure of mycE1N/2C to inhibit Rev and p24 expression suggests that the C-terminal domains of hnRNP E1 and E2 are not functionally interchangeable and implicate that sequence differences in this region partially account for the different activities of these two proteins.
In summary, despite high levels of sequence identity between the hnRNP E1 and E2 isoforms, the emerging model is that these proteins are capable of fulfilling distinct cellular functions. These differences are reflected in the failure of domain swaps to maintain the activity of the proteins. The isoform specific effects for these highly similar proteins is surprising. Work by other groups has also suggested differential roles for hnRNP E1 and hnRNP E2. This includes the finding that hnRNP E1 has a greater affinity for AUF/hnRNP D than hnRNP E2 , as well as differential regulation of hnRNP E1 and E2 in response to hypoxic stressing of cortical neurons . This observation supports the notion that despite a high degree of similarity at the amino acid level, these proteins possess non-redundant functions. To our knowledge, the present work is among the few examples of a system that allows for examination of the unique effects of the hnRNP E isoforms. Further analysis using this system may allow us to elucidate the amino acid differences between hnRNP E1 and E2 that account for their differential effect on HIV-1 RNA metabolism and refine the mechanism by which hnRNP E1 modulates HIV-1 expression.
Materials and methods
The following plasmids have been previously described: SVH6Rev, pgTat, pgTatΔESE, pgTatΔESEΔESS SL1, pgTatΔESE S5-2, Gag RRE, pSPVA, HxBruR-/RI- [26, 47]. pTRAP plasmid was generously provided by Dr. H. Krause, University of Toronto. CMVmycE1 was constructed by amplifying HeLa cDNA with primers hnRNP E1-F-Hind (5' CCC AAG CTT ATG GAT GCC GGT GTG ACT GAA 3') and hnRNPE1-R-Pst (5' AAA ACT GCA GCT AGC TGC ACC CCA TGC CCT T 3'). The resulting amplicon was digested with HindIII/PstI and cloned into the corresponding sites of the CMVmyc3xterm vector (a N-terminal myc-tagged CMV immediate early promoter, with a 3xterm cassette and SV40 polyadenylation signal in a Bluescript (Stratagene) backbone). CMVmycE2 was constructed by amplifying HeLa cDNA with primers hnRNPE2-F-Hind (5' CCC AAG CTT ATG GAC ACC GGT GTG ATT GAA 3') and hnRNPE2-R-Bam (5' CGC GGA TCC CTA GCT GCT CCC CAT GCC ACC 3'). The resulting amplicon was HindIII/BamHI digested and cloned into the corresponding sites of the CMVmyc3Xterm vector. CMVmycE1ΔN was constructed by amplifying CMVmycE1 with primers E1Δ1FHind (5' CCC AAG CTT ATG ACC AAC AGT ACC GCG GCC 3') and hnRNPE1-R-Pst. Amplicons were HindIII/PstI digested and cloned into the corresponding sites of CMVmyc3xterm. CMVmycE1ΔC was constructed by digesting CMVmycE1 with XbaI and religating the backbone. CMVmycE1N/2C was constructed by ligating the 500 bp fragment generated upon XbaI digest of CMVmycE2 with the CMVmycE1 XbaI digested backbone. pTRAP ESS and S5-2 were constructed by PCR amplification of pgTatΔESE and pgTatΔESE S5-2 with primers ESS-F (5' CCC AAG CTT GGG ATC CCC GAA GAA ATA GTG G 3') and ESS-R (5' CCG CTC GAG GCC AAG GTC TGA AGG TCA CTC GA 3'). Amplicons were digested with HindIII and XhoI and cloned into the corresponding sites of pTRAP. All constructs were confirmed by sequencing.
Transfections and SEAP assays
HeLa, 293 and 293T cells were maintained in Iscove's modified Dulbecco's media (IMDM) supplemented with 10% fetal bovine serum (FBS), 50 μg/ml gentamycin sulfate and 2.5 μg/ml amphotericin B. For transient expression studies, vectors were introduced by calcium phosphate transfection . Transfections using HIV-1 provirus, were performed using 1:2:0.5 ratio (provirus : CMVmyc expression construct : CMVPLAP). Transfections using HIV-1 expression constructs were performed at 1:4:0.4 ratio (HIV-1 expression construct (pgTat and derivatives thereof or GagRRE) : CMVmyc expression construct : Rev expressing construct), depending on the experiment either 0.4 μg VA or 0.5 μg CMVPLAP was also included.
Cell lysates were prepared by harvesting cells 48 hrs post transfection in either RIPA buffer (50 mM Tris-HCl pH 7.5, 1% NP40, 0.05% SDS, 0.5% Sodium Deoxycholate, 1 mM EDTA, 150 mM NaCl) followed by shaking for 20 mins at room temperature and pelleting by centrifugation or 9 M urea, 5 mM Tris pH8 followed by boiling for 10 mins and pelleting by centrifugation. Total RNA was prepared by lysing cells in 4 M GT solution as described in . The RNA was treated for 30 mins at 30°C with TURBO DNase as outlined by the manufacturer (Ambion), then extracted for a second time in 4 M GT solution as before.
Secreted alkaline phosphatase (SEAP) levels were assayed by diluting an aliquot of cell media in water and adding 10 mM diethanolamine (pH 9.5, 0.5 mM MgCl2) containing 4-nitrophenyl phosphate (1 mg/ml). SEAP levels were measured at 405 nm using a Titertek multiscan® Plus ELISA plate reader.
Proteins were fractionated on sodium dodecyl sulfate-7% (gp160/120) or 10% polyacrylamide gels (Rev, p24, hnRNP E1, hnRNP E2, myc and tubulin) and transferred to PVDF membrane (PALL Corporation). Blots were probed with antibody to p24 (hybridoma line 183-H12-5C), gp160/120 (kindly provided by H. Schaal, Heinrich-Heine-University of Duesseldorf, Germany), hnRNP E1 (Santa Cruz Biotechnology Inc), hnRNP E2 (Santa Cruz Biotechnology Inc), myc (hybridoma line 9E10), Rev (rabbit polyclonal raised against recombinant rev) and tubulin (Sigma) and detected using HRP-conjugated anti-mouse (p24, gp120, myc and tubulin; Jackson ImmunoResearch Laboratories Inc), anti-rabbit (Rev; Jackson ImmunoResearch Laboratories Inc) or anti-goat (hnRNP E1 and hnRNP E2; Santa Cruz Biotechnology Inc) antibody and the Western Lightning kit (Perkin-Elmer). To quantitate changes in Rev protein expression, developed films were scanned and analyzed using Imagequant software.
293 cells were washed with media minus serum/antibiotics and transfected with 10 nM final concentration of 27mer siRNAs (IDT) against hnRNP E1 (E1 (3): 5'CUU GAA UCG AGU AGG CAU CUA GAG3'; E1 (16): 5' GUA CUG UUG GUC AUG GAG CUG UUG AUA 3'), hnRNP E2 (5' AGA CUG UUG CAU UGC CAA CUG GUG CAG 3') or a scrambled negative control (5' CUU CCU CUC UUU CUC UCC CUU GGA 3'). 10 μM siRNA was combined with OPTI-MEM (Gibco) and transfected with Oligofectamine™ Reagent (Invitrogen) as per manufacturer's instructions. The siRNA/Oligofectamine solutions were added to cell media minus serum and antibiotics to a final volume of 1 ml. 5 hrs later, the transfection media was removed and replaced with growth media. The following day, cells were transfected with HxBruR-/RI- provirus and CMVPLAP at a ratio of 1:0.5 using Fugene (Roche) as described in the manufacturers instructions. Two days post transfection the cells were harvested and lysed in 9 M urea, 5 mM Tris pH8.
HeLa cells were plated on glass coverslips, transfected, and processed as previously described . Briefly, cells were processed 48 hrs post transfection by washing with 1× PBS and fixing in 4% paraformaldehyde, 1× PBS for 30 min at room temperature. Cells were washed twice with 1× PBS, 10 mM glycine and permeabilized with 1% Triton X-100 in 1× PBS for 5 min. Cells were washed twice with 1× PBS, 10 mM glycine and blocked in 3% BSA overnight at 4°C. Coverslips were inverted onto primary antibody and incubated for 1 hr at room temperature. After washing twice in 1× PBS, 10 mM glycine, coverslips were inverted over fluorescently (FITC and Texas Red; Jackson ImmunoResearch Laboratories Inc) labeled secondary antibody and incubated for 1 hr. Samples were washed with 1× PBS, 10 mM glycine, and stained for DAPI by washing in 1× PBS, DAPI (50 ng/ml) for 10 min. Coverslips were mounted in 90% glycerol and analyzed using a Leica DMR epifluorescent microscope.
RNase protection assays (RPAs) were performed as described previously using 10 μg of total RNA . Alternatively, nuclear and cytoplasmic fractions were prepared as previously described  prior to RNA extraction. Three RPA probes were used. The rev probe was a BamH1/XhoI fragment of HXB-2 cloned into Bluescript. The env and VA probes are described elsewhere (env probe: ; VA probe: ). Position of bands was determined following exposure to phosphor screens and scanning using a Phosphor Imager.
For Northern blots, RNA (20 μg) was run on a 1% formaldehyde agarose gel in 1× gel running buffer (0.1 M MOPS pH7, 40 mM NaAc, 5 mM EDTA pH8). The RNA was transferred to nitrocellulose membrane (HYBOND N+, Amersham) overnight in 5 × SSC, 10 mM NaOH and immobilized by UV crosslinking. Membranes were prehybridized at 50°C for 2 hrs in 20 ml Church buffer (0.5 M Phosphate buffer, 7% SDS, 1 mM EDTA) and hybridized with probe overnight at 50°C. Probe to HIV LTR was designed to the LTR region found in all 3 classes of HIV-1 RNA and generated by PCR using the primers (F 5'CTA ATT CAC TCC CAA CGA AGA 3'; R 5' TGC TAG AGA TTT TCC ACA CTG 3'). GAPDH probe was generated by end labeling the primer (5' AAA GGT GGA GGA GTG GGT GTC GCT GTT GAA 3') with 32P γATP. Membranes were washed in 6 × SSC for 5 mins, 6 × SSC, 0.1%SDS for 15 mins and 2 × SSC, 0.1%SDS for 15 mins all at 45°C, followed by a brief wash in 2 × SSC at room temperature before exposure to a phosphor screen. Membranes were stripped by washing in boiling 0.1% SDS for 5 mins followed by rinsing in 2 × SSC at room temperature.
For RT-PCR, cDNA was generated by incubation of 3 μg total RNA with random hexamer and M-MLV RT as outlined by the manufacturer (Invitrogen). Separate RT-PCRs were performed for the 2 kb and 4 kb classes of HIV-1 RNA. For the 2 kb class of RNA, 1 μl of cDNA was incubated with 10 μM Forward primer Odp045 (5' CTG AGC CTG GGA GCT CTC TGG C 3') and 10 μM reverse primer Odp032 (5'CCG CAG ATC GTC CCA GAT AAG 3') and Taq. Reactions were heated at 94°C for 2 mins, then cycled at 94°C for 1 min, 57°C for 1 min, 68°C for 1 min (repeated 34 times) and 68°C for 5 mins. A 1/10 dilution of the PCR product was prepared and 3 μl used as the template for a second PCR containing 10 μM forward and reverse primers with Taq as before however this time 0.5 μl α32P dCTP was included. The second PCR was cycled at 94°C for 2 mins, then at 94°C for 1.5 mins, 57°C for 1 min, 68°C for 1 min (repeated 3 times) and 68°C for 5 mins.
To analyze the 4 kb class of HIV RNA, 3 μl of cDNA was incubated with the forward primer Odp045 and reverse primer Odp084 (5' TCA TTG CCA CTG TCT TCT GCT CT 3') and Taq and cycled as indicated above. For the second PCR, 3 μl of undiluted RT-PCR reaction was used as the template and cycled with primers Odp045 and Odp084, as described for the 2 kb class of RNAs.
RNA affinity chromatography and mass spectrometry
pTRAP ESS and pTRAP S5-2 were linearized with XhoI and in vitro transcribed with T7 RNA polymerase. Streptavidin beads (62.5 μl) were pre-bound to 5–10 μg of RNA for 30 mins at 4°C. Pre-cleared HeLa nuclear extract (120 μl, 10 mg/ml) containing 0.5 M ATP, 40 mM creatine phosphate, 4 mM MgCl2 and 4 mM DTT was subsequently added to the Streptavidin beads and incubated for 1 hr at 4°C. Beads were washed three times in 1× TBP (60 mM HEPES, 10 mM NaCl and 0.1% Triton × pH 7.4). Elutions were performed with 250 μl Biotin elution buffer (5 mM Biotin in 1× TBP) and collected proteins precipitated with acetone.
Column fractions were separated on SDS-PAGE gels and detected by silver staining or Coomassie blue. Bands enriched in the wildtype ESS3a sample were excised, and destained in 50% 100 mM NH4HCO3/50% acetonitrile. Gel slices were washed with 100 mM NH4HCO3, 1 mM DTT, then 100 mM NH4HCO3, 1 mM CaCl2 and finally dehydrated in acetonotrile. After drying to remove residual acetonitrile, gel was immersed in 100 mM NH4HCO3, 12.5 ng/μl trypsin for 45 min. on ice. An equal volume of 100 mM NH4HCO3, 1 mM CaCl2 was subsequently added and digest continued overnight at 30°C. Following digestion, peptides were separated and eluted with an organic gradient using a reverse phase C18 microcapillary column and analyzed in real time with a linear ion trap tandem mass spectrometer (LTQ, ThermoFinnigan) with a custom-built ion source. Precursor peptide ions were automatically selected for data-dependentfragmentation using dynamic exclusion. The resulting tandem mass spectra were searched against the full set of predicted ORFs downloaded from SGD using a distributed version of Sequest. Confidence scores were assigned and high-confidence protein identifications (>95% likelihood) were filtered using the Statquest probability algorithm .
The authors would like to thank H. Schaal and R. Andino for providing antibodies. KA was supported by a CIHR MD/Ph.D. Studentship and AC is an OHTN Scholar. Research was supported by a grant from OHTN.
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