Cellular APO3G is present in HMM ribonucleoprotein complexes. Analysis of the RNAs in these complexes revealed the presence of Alu RNAs and small Y RNAs, two of the most prominent non-autonomous mobile genetic elements in human cells 2342. We wanted to confirm and extend these observations by further investigating the association of APO3G with other small cellular RNAs such as 7SL RNA, Y RNAs, and U RNAs. Messenger RNAs encoding β-actin, GAPDH, or α-tubulin were included as additional controls for the specificity of APO3G-RNA interactions. HeLa cells were transfected with pcDNA-Apo3G-MycHis DNA. Cells were harvested 24 h after transfection, washed with PBS and divided into two fractions: 30% of the transfected cells were used to isolate total cellular RNA as described in Methods; the remaining 70% of the cells were lysed in Triton X-100 lysis buffer. A sample of the lysate (10%) was used as total protein control for the subsequent immunoblot analysis (Fig. 1A, Total). Equal fractions of the remaining lysate (45% of total lysate each) were either immunoprecipitated with a myc-specific polyclonal antibody (Fig. 1A/B, α-myc) or were exposed to Protein-A beads without antibody (Fig. 1A/B, mock). Half of the immunoprecipitated samples were used for immunoblotting to identify APO3G protein (Fig. 1A). Immunoblot analysis revealed the presence of APO3G in the cell extract (Fig. 1A, Total) and the APO3G immune complex (Fig. 1A, α-myc). As expected, APO3G was absent in the mock immunoprecipitated sample (Fig. 1A, mock). The second half of the precipitated samples was used for RNA extraction. RT-PCR was performed on total RNA and RNA from the immune complexes as described in Methods using a series of primer sets as listed in table 1. All RT-PCR reactions were done simultaneously. RT-PCR of total RNA identified all RNAs in the total cellular extract (Fig. 1B, Total). None of the RNAs was amplified from the mock precipitated sample demonstrating the lack of non-specific binding of these RNAs to Protein A beads (Fig. 1B, mock). In contrast, several of the RNAs, including 7SL, β-actin, and GAPDH, as well as hY3 and U6 RNA were recovered from APO3G immune complexes (Fig. 1B, α-myc). Alpha-tubulin mRNA, as well as hY1, hY4, and U4 RNAs were amplified only inefficiently from the APO3G immune complexes suggesting weak interaction of these RNAs with APO3G (Fig. 1B, α-myc). In contrast, hY5 cytoplasmic RNAs and U1 and U2 small nuclear RNAs did not appear to associate with APO3G immune complexes (Fig. 1B, α-myc).

To rule out non-specific binding of RNAs to the myc-specific antibody in figure 1B, plasmids encoding epitope tagged or untagged APO3G were separately transfected into HeLa cells. Cell extracts were subjected to immunoblot analysis and RT-PCR as described for figure 1B. Myc-tagged and untagged APO3G were efficiently expressed in the transfected cells (Fig. 1C, top panel, lanes 1–2). As expected, untagged APO3G was not immunoprecipitated by the myc-specific antibody (Fig. 1C, top panel, lane 4) while epitope-tagged APO3G-MycHis was identified in the immune complexes (Fig. 1C, top panel, lane 3). A shorter form of APO3G-MycHis co-migrating with the untagged form of APO3G in figure 1C presumably represents C-terminally truncated protein missing part or all of the epitope tag as it was not recognized by epitope-tag-specific antibodies (data not shown). To test non-specific binding of RNA to the myc-specific antibody, we performed RT-PCR as described for figure 1B using 7SL-specific primers. As expected, 7SL RNA was identified in immune complexes of myc-tagged APO3G (Fig. 1C, lower panel, lane 3). However, 7SL RNA was not amplified by RT-PCR from samples containing untagged APO3G (Fig. 1C, lower panel, lane 4). These results demonstrate that the presence of 7SL RNA in immune complexes of myc-tagged APO3G was due to the presence of APO3G and not caused by non-specific binding of the RNA to the myc antibody. Finally, the RT-PCR reaction was sensitive to treatment with RNase A as exemplified by the lack of 7SL RNA amplification in RNase-treated samples (Fig. 1D).

Previous studies on murine and avian retroviruses found that these viruses encapsidate a variety of host RNAs 2425282930313343. More recent studies have similarly identified cellular RNAs in HIV-1 particles 2832. The experiments described above are both consistent with our previous finding that APO3G has RNA binding properties in vitro 21 and other studies demonstrating association of APO3G with cellular RNAs as well as HIV-1 RNA 1923. Furthermore, we and others previously reported that viral genomic RNA enhances the encapsidation of APO3G into HIV-1 virions 1618. Contrasting these findings, other reports concluded that Gag is sufficient for the encapsidation of APO3G into VLP 91112131416. Interestingly, the APO3G-Gag interaction was found to be either RNA independent 13 or to be sensitive to RNase-treatment 14 and several studies concluded that nonspecific RNA was critical for APO3G packaging 911. Thus, the parameters determining APO3G packaging into HIV-1 virions remained unclear and warranted further investigation.

In our next experiment, we compared the packaging of APO3G and cellular RNAs into HIV-1 virions or VLP in an attempt to identify a possible correlation between APO3G packaging and encapsidation of cellular RNAs. Four types of particles were analyzed as shown in Fig. 2. All particles lacked a functional vif gene to prevent degradation of APO3G, which would make interpretation of our results more difficult. NL4-3ΔVif served as a positive control; C-HelpΔVif is a helper virus construct lacking both LTRs and carrying deletions in env and in the 5' untranslated region 44. C-HelpΔVif particles do not package detectable quantities of genomic RNA and we previously found that packaging of APO3G into C-HelpΔVif virions was impaired 18. The mS.1ΔVif construct carries mutations in stem-loop 1 of the 5' untranslated region of the viral RNA 18 mS.1ΔVif particles contain viral genomic RNA but are impaired in APO3G packaging due to the mutations in the stem loop 1 motif 18. Finally, DB653ΔVif was included to control for the requirement of NC in RNA and APO3G packaging. DB653ΔVif was derived from DB653 1845 and carries SSHS/SSHS mutations in the NC zinc finger motifs. The genomic RNA content of DB653 particles was reported to be less than 10% of wild type virus 45.

Particles were produced by transfecting HeLa cells with appropriate plasmid DNAs in the presence of APO3G. Viruses were purified and concentrated as described in Methods. Aliquots were used for immunoblot analysis to determine viral protein content and to verify APO3G packaging (Fig. 3A). Other aliquots of the concentrated viruses were used to extract particle-associated RNA, which was then used for RT-PCR analysis (Fig. 3C). Consistent with our previous report, immunoblot analysis showed that NL4-3ΔVif packaged significantly higher amounts of APO3G than C-HelpΔVif and mS.1ΔVif particles (Fig. 3A). Packaging of APO3G was quantified by densitometric scanning of the APO3G bands. Results were corrected for fluctuations in capsid (CA) levels and are presented as percentage of APO3G packaged into NL4-3ΔVif particles, which was defined as 100% (Fig. 3B). Consistent with our previous data 18, packaging of APO3G into C-HelpΔVif and mS.1ΔVif particles was reduced to about 25–30% of wild type levels.

Equal numbers of particles, as judged by reverse transcriptase activity, were used for extraction of RNA, which was then used for RT-PCR using a series of primers as shown in figure 3C and detailed in table 1. All RT-PCR reactions shown in figure 3C were done simultaneously. Amplification by an HIV-1 specific primer confirmed the presence of genomic RNA in NL4-3ΔVif and mS.1ΔVif particles and verified the lack of detectable amounts of genomic RNA in C-HelpΔVif preparations (Fig. 3C, HIV-1). In contrast, amplification of 7SL RNA as well as β-actin, GAPDH, and α-tubulin mRNAs yielded comparable amounts of PCR products indicative of the presence of similar levels of these cellular RNAs in all three particle preparations. These results suggest that packaging of these RNAs was independent of the presence or absence of viral genomic RNA (Fig. 3C). Similarly, U1, U2, U4, and U6 small nuclear RNAs were amplified with similar efficiency from all three particle preparations. while human Y5 RNA was virtually absent from the particles. On the other hand, hY1, hY3, and hY4 RNAs appeared to be packaged more efficiently into C-HelpΔVif particles than into NL4-3ΔVif and mS.1ΔVif virions. The less efficient packaging of hY1, hY3, and hY4 RNAs into NL4-3ΔVif and mS.1ΔVif particles is unrelated to APO3G encapsidation as APO3G levels in mS.1ΔVif particles were as low as in C-HelpΔVif (Fig. 3A &3B). Importantly, there was no obvious correlation between APO3G packaging and encapsidation of any of the tested cellular RNAs.

The increased packaging of hY RNAs into particles lacking genomic RNA could indicate a competitive mechanism in which viral genomic RNA competes for a common packaging domain. Since viral genomic RNA is packaged through an interaction with the NC zinc finger domain, we investigated the impact of zinc finger mutations on the packaging of hY RNAs. In addition, we assessed the impact of zinc finger mutations on the packaging of genomic RNA and 7SL RNA as well as APO3G (Fig. 4). NL4-3ΔVif and DB653ΔVif particles were produced from transfected HeLa cells as described for figure 3. Cell lysates and concentrated cell-free virions were subjected to immunoblot analysis to verify comparable amounts of viral Gag proteins and to assess the encapsidation of APO3G into NC zinc finger mutant particles. Consistent with previous reports 91112131416 we found that mutation of the NC zinc finger domain abolished packaging of APO3G into virus-like particles (Fig. 4A, DB653ΔVif).

For RT-PCR analysis, C-HelpΔVif RNA from figure 3C was included for comparison. As before particles were normalized for equal reverse transcriptase activity. RT-PCR analysis using HIV-1-specific primers confirmed the absence of viral genomic RNA in C-HelpΔVif and the DB653ΔVif zinc finger mutant (Fig. 4B). As before, hY5 RNA was virtually absent from all particle preparations including the zinc finger mutant. Interestingly, packaging of 7SL RNA was not affected by mutation of the NC zinc fingers suggesting that 7SL RNA is packaged in an NC-independent manner. In contrast, packaging of hY1, hY3, and hY4 RNAs was critically dependent on intact NC zinc finger domains (Fig. 4B). Thus, packaging of hY RNAs is indeed NC-dependent and the absence of hY RNAs from NL4-3ΔVif particles is best explained by competitive binding of viral genomic RNA and hY RNA to NC.

7SL RNA (also referred to as SRP RNA) is a component of the signal recognition particle (SRP), which is critical for the targeting of nascent secretory and membrane proteins to the endoplasmic reticulum membrane (for review see 46). SRP54 is one of six protein subunits that constitute mammalian SRPs and is responsible for high affinity assembly of 7SL RNA into the SRP complex (reviewed in 47). Given the fact that 7SL RNA was efficiently packaged into HIV-1 virions, we wanted to test whether intracellular high affinity 7SL RNA-SRP54 interactions would result in the recruitment of SRP54 rather than APO3G into HIV-1 virions.

First, we verified the association of 7SL RNA with SRP54 in normal HeLa cells. For that purpose, HeLa cell lysates were adsorbed to SRP54 reactive autoantibodies and immunoprecipitation of SRP54 was confirmed by immunoblotting using an SRP54-specific antibody (Fig. 5A, top panel, SRP). The specificity of the reaction was verified by the absence of SRP54 protein in mock-immunoprecipitated samples (Fig. 5A, mock) and by the absence of α-tubulin in SRP54-specific and mock precipitates (Fig. 5A, middle panel). Total RNA extracted from the immunoprecipitates revealed the presence of 7SL RNA in SRP54-specific but not in mock immunoprecipitated samples (Fig. 5A, lower panel).

Next, the packaging of SRP54 protein into HIV-1 virions was tested. Virus particles were produced as described for figure 3 except that APO3G was omitted in these samples. Cell lysates and concentrated virus preparations were used for immunoblotting and for RT-PCR analysis as described for figure 3. The results are shown in figure 5B. All cell lysates contained equal amounts of SRP54 and viral capsid proteins as well as 7SL RNA (Fig. 5B, cell). Furthermore, all samples produced comparable amounts of cell-free virions as judged from the immunoblot (Fig. 5B, CA) and packaged comparable amounts of 7SL RNA (Fig. 5B, 7SL). Of note, SRP54 was virtually absent from the virus preparations (Fig. 5B, SRP54), thus confirming and extending a recent study that also did not find SRP54 protein in HIV-1 virions 28. These results demonstrate that intracellular RNA-protein interactions are not a predictor for subsequent targeting of the proteins into viral particles.

The vif-defective molecular clone pNL4-3ΔVif 50 was used for the production of vif-defective HIV-1 virus stocks. Plasmid pC-HelpΔVif was used for the production of vif-defective Ψ^- virus-like particles (VLP). These particles contain undetectable levels of viral genomic RNA 18. Plasmid pNL4-3mS.1ΔVif carries mutations in stem-loop 1 of the 5'-untranslated region 51 and was constructed by subcloning the mutated stem-loop 1 region into the vif-defective pNL4-3 vector 18. NL4-3mS.1ΔVif particles are Ψ⁺ but do not support the encapsidation of APO3G 18. A vif-defective variant of DB653 45 was constructed by transferring the Gag region of DB653 into pNL4-3Vif(-) using standard cloning techniques. The structures of these constructs are schematically shown in figure 2. Construction of pcDNA-Apo3G-MycHis for the expression of C-terminally epitope-tagged wild type human APO3G proteins was described previously 7 and untagged version, pcDNA-Apo3G, was construction by introducing a stop coding at the end of the APO3G gene 52.

HeLa cells, which do not express endogenous APO3G, were propagated in Dulbecco's modified Eagles medium (DMEM) containing 10% fetal bovine serum (FBS). For transfection, HeLa cells were grown in 25 cm² flasks to about 80% confluency. Cells were transfected using LipofectAMINE PLUS™ (Invitrogen Corp, Carlsbad CA) following the manufacturer's recommendations. A total of 5 μg of plasmid DNA per 25 cm² flask (5 × 10⁶ cells) was generally used. Cells were harvested 24 h post transfection.

Virus stocks were prepared by transfecting HeLa cells with pNL4-3ΔVif, pC-HelpΔVif, or pNL4-3mS.1ΔVif DNAs in the presence or absence of APO3G expression vector as indicated in the text. Virus-containing supernatants were harvested 24 h after transfection. Cellular debris was removed by centrifugation (5 min, 1500 rpm) and clarified supernatants were filtered (0.45 μm) to remove residual cellular contaminants. For immunoblot analysis of viral proteins and RNA extraction, virus-containing supernatants (7 ml) were concentrated by ultracentrifugation through 2 ml of 20% sucrose in PBS as described previously 7.

APO3G was identified using a polyclonal rabbit serum against a synthetic peptide comprising the 17 C-terminal residues of APO3G. Serum from an HIV-positive patient (APS) was used to detect HIV-1-specific capsid (CA) proteins. Tubulin was identified using an α-tubulin-specific monoclonal antibody (Sigma-Aldrich, Inc., St. Louis MO). SRP54 protein was detected with a SRP54-specific monoclonal antibody (BD Biosciences, San Jose, CA). Immunoprecipitation of APO3G was done using a polyclonal antibody raised against the myc tag (Sigma-Aldrich, Inc., St. Louis, MO). A human SRP54-reactive autoimmune serum was used for immunoprecipitation of SRP54 protein (kind gift of Frederick W. Miller, NIEHS, NIH, Bethesda, MD, USA).

HeLa cells transfected with APO3G were used to detect cellular APO3G expression and untransfected HeLa cells were used for the detection of endogenous SRP54 protein by immunoblotting with appropriate antibodies. For immunoblot analysis of cellular proteins, whole cell lysates were prepared as follows. Cells were washed once with PBS, suspended in 450 μl/10⁷ cells with X-100 lysis buffer (50 mM Tris-HCL pH7.5, 150 mM NaCl, 0.5% Triton X-100). For Western blot analysis 50 μl aliquot was taken and mixed with equal volume of sample buffer (4% sodium dodecyl sulfate [SDS], 25 mM Tris-HCL, pH 6.8, 10% 2-mercaptoethanol, 10% glycerol, and 0.002% bromphenol blue). Proteins were solubilized by boiling for 5 min at 95°C with occasional vortexing of the samples to shear chromosomal DNA. Residual insoluble material was removed by centrifugation (2 min, 15,000 rpm in an Eppendorf Minifuge). For immunoblot analysis of virus-associated proteins, concentrated viral pellets were suspended in a 1:1 mixture of PBS and sample buffer and boiled. Cell lysates and viral extracts were subjected to SDS-polyacrylamide gel electrophoresis; proteins were transferred to polyvinylidene difluoride membranes and reacted with appropriate antibodies as described in the text. Membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies (Amersham Bioscience, Piscataway, NJ) and visualized by enhanced chemiluminescence (Amersham Bioscience).

HeLa cells were transfected with pcDNA-APO3G-MycHis. Cells were harvested at 24 h post transfection cell lysates were prepared as follows: Cells were divided into two unequal fractions (30% and 70%). The larger fraction was used for immunoprecipitation studies and the smaller fraction was used for RNA extraction (see below). For immunoprecipitation, cells were washed once with PBS and lysed in 450 μl of lysis buffer (50 mM Tris, pH7.5, 150 mM, NaCl 0.5% and Triton X-100). The cell extracts were clarified by centrifugation (13,000 × g, 3 min) and the supernatant was incubated on a rotating wheel for 1 h at 4°C with protein A-Sepharose beads (Sigma-Aldrich, Inc., St. Louis MO) coupled with (IP) or without (Ctrl) anti-myc rabbit polyclonal antibody (Sigma-Aldrich, Inc., St. Louis MO). Immunocomplexes were washed three times with wash buffer (50 mM Tris, 300 mM NaCl, and 0.1% Triton X-100 (pH 7.4). Bound proteins were eluted form beads by heating in sample buffer for 5 min at 95°C and analyzed by immunoblotting using antibodies as indicated in the text. For immunoprecipitation of APO3G-RNA complexes, cell extracts were subjected to immunoprecipitation by antibody covered beads or control beads as described above and washed three times with RNA-protein binding buffer (20 mM HEPES, 25 mM KCl, 7 mM 2-Mercaptoethanol, 5% Glycerol and 0.1% NP-40). Bound RNA was then extracted as described below.

Total cellular RNA was extracted from untransfected and transfected HeLa cells using the RNeasy RNA extraction kit (QIAGEN, Valencia, CA) following the manufacturer's instructions. To isolate RNA from immunocomplexes, beads were washed three times with RNA-protein binding buffer (20 mM HEPES, 25 mM KCl, 7 mM 2-Mercaptoethanol, 5% Glycerol and 0.1% NP-40). RNA was then extracted using RNeasy RNA extraction kit. For isolation of SRP54-associated RNA, SRP54 was precipitated with SRP54-reactive human autoantibodies derived from a patient with polymyositis (53; gift of Frederick W Miller, NIEHS, NIH, Bethesda, MD, USA). RNA was then extracted from the immunocomplexes as before

RNA extracted from cells, viruses, or immunocomplexes was treated with RNase-free DNase I (10 units, 30 min, 37°C) prior to the RT-PCR reaction. RNA concentrations were determined by spectrophotometry. RT-PCR was performed using equal amounts of RNA and the one-step RT-PCR kit (QIAGEN, Valencia, CA) according to the manufacturer's instruction. Primers for the amplification of specific RNAs are listed in table 1. RNA was first reverse transcribed at 50°C for 30 minutes followed by 30 PCR cycles (denaturation at 94°C; 15 sec; annealing at 55°C, 30 sec; and extension at 72°C, 1 min) and one 10-minute extension cycle at 72°C. RT-PCR products were mixed with DNA loading buffer (EDTA 20 mM, TAE 5×, Glycerol 50% and 0.002% Bromphenol Blue dye), electrophoresed in 1% agarose gels, and visualized by staining with ethidium bromide. A DNA size marker was run in parallel.