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Journal of Virology, August 2004, p. 8486-8495, Vol. 78, No. 16
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.16.8486-8495.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Basic Residues of the Retroviral Nucleocapsid Play Different Roles in Gag-Gag and Gag-{Psi} RNA Interactions

Eun-Gyung Lee and Maxine L. Linial*

Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109

Received 7 January 2004/ Accepted 5 April 2004


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ABSTRACT
 
The Orthoretrovirus Gag interaction (I) domain maps to the nucleocapsid (NC) domain in the Gag polyprotein. We used the yeast two-hybrid system to analyze the role of Alpharetrovirus NC in Gag-Gag interactions and also examined the efficiency of viral assembly and release in vivo. We could delete either or both of the two Cys-His (CH) boxes without abrogating Gag-Gag interactions. We found that as few as eight clustered basic residues, attached to the C terminus of the spacer peptide separating the capsid (CA) and NC domains in the absence of NC, was sufficient for Gag-Gag interactions. Our results support the idea that a sufficient number of basic residues, rather than the CH boxes, play the important role in Gag multimerization. We also examined the requirement for basic residues in Gag for packaging of specific packaging signal ({Psi})-containing RNA. Using a yeast three-hybrid RNA-protein interaction assay, second-site suppressors of a packaging-defective Gag mutant were isolated, which restored {Psi} RNA binding. These suppressors mapped to the p10 or CA domains in Gag and resulted in either introduction of a positively charged residue or elimination of a negatively charged one. These results imply that the structural interactions of NC with other domains of Gag are necessary for {Psi} RNA binding. Taken together, our results show that while Gag assembly only requires a certain number of positively charged amino acids, Gag binding to genomic RNA for packaging requires more complex interactions inherent in the protein tertiary structure.


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INTRODUCTION
 
The Alpharetrovirus Gag protein, exemplified by Rous sarcoma virus (RSV), is synthesized as a precursor protein consisting of matrix (MA); two small peptides, p2 and p10; capsid (CA), nucleocapsid (NC), and protease (PR). Cleavage of the precursor by PR occurs after, or concomitant with, viral budding from the plasma membrane. Three Gag domains (M [membrane association], I [interaction], and L [late]) are necessary for viral assembly (reviewed in references 18 and 40). The interaction, or I, domain is required for Gag multimerization and virus particle assembly. Deletion of the NC domain has been shown to result in drastic reduction of viral assembly and release, suggesting that I domain function resides in NC. In Orthoretrovirinae, NC has two structural characteristics: one or two copies of the conserved sequence Cys-X2-Cys-X4-His-X4-Cys (Cys-His [CH] box), which coordinates Zn2+ ion binding, and clustered basic residues (BRs) flanking the CH box or boxes. NC plays important roles in essential steps of virus replication, including reverse transcription, integration, RNA packaging, viral assembly, and infectivity (reviewed in reference 15). Some of these functions have been shown to require distinct structural motifs. A previous and extensive yeast two-hybrid study (43) found that the minimal domain required for Gag-Gag interactions mapped to human immunodeficiency virus (HIV) NC but did not characterize the specific residues required within the minimal domain. Gag multimerization also requires nucleic acid, so the dual roles of NC in assembly and RNA packaging could be closely related (42).

Retroviral packaging of RNA is dependent only upon the Gag polyprotein. Since less than 1% of total cellular RNA in the cytoplasm is virus specific, mechanisms are required by which cis-acting sequences in genomic RNA (termed {Psi}) are selectively recognized and specifically bound to domains in Gag and packaged into virions. As in the case of Gag-Gag interactions, a number of studies have shown that the NC domain is involved in specific packaging of the genomic RNA (25, 37, 45). Previously, we showed that the stretches of positively charged residues surrounding the CH boxes in RSV NC appear to be determinants for selective recognition and specific binding to {Psi}-containing RNAs (23). In vitro binding studies have shown that NC can be either a rather nonspecific nucleic acid binding protein, which binds to either RNA or DNA with a preference for single strands, or a specific binding protein under some conditions (7, 14, 22, 27). It is most likely that selection of viral genomic RNA by NC occurs through the Gag precursor protein. At present, there are no three-dimensional structures of any retroviral Gag polyprotein alone or complexed with {Psi} RNA available, although nuclear magnetic resonance structures of HIV-1 NC have been successfully obtained complexed with two small RNA stem-loops which are part of {Psi}, but which are not autonomous packaging signals (3, 17).

In many retroviruses, such as murine leukemia virus, HIV-1, and RSV, packaging sequences are located in the 5'-untranslated leader sequences of the genome. For RSV, a 160-nucleotide contiguous packaging sequence (called M{Psi}) was identified at the 5' end of the genome between the primer binding site and the Gag start codon, which confers efficient packaging of heterologous RNAs into virions that are tethered to M{Psi} RNA (5).

In this report, we have examined the importance of NC basic amino acids for both Gag-Gag and Gag-{Psi} RNA binding. We used a yeast two-hybrid system to specifically analyze the role of basic residues of NC in the context of a Gag molecule (containing all of the domains except PR) in Gag-Gag interactions and examined the effects of mutation of important residues on viral assembly and release in vivo. We also employed a yeast three-hybrid system (44) to examine {Psi} RNA-Gag protein interactions required for specific genomic RNA binding. Previously, we could demonstrate that for RSV, there is a strong correlation between the yeast three-hybrid binding and in vivo packaging assays (23-25). With the advantage of having a defined small RSV packaging sequence and this rapid binding assay, we took a genetic approach to examine the structural interactions of NC with other domains in Gag important for {Psi} RNA binding and packaging. Using a Gag mutant, NC-SKL, which does not bind to {Psi} RNA or package genomes (25), we selected second-site suppressors in the Gag polyprotein which restored {Psi} RNA binding to the NC-SKL mutant and examined the effects on the {Psi} RNA packaging and viral replication. Our results show that the interactions of NC with other Gag molecules and with RNA have greatly different requirements for basic amino acids, with NC-RNA interactions being more stringent.


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MATERIALS AND METHODS
 
Site-directed mutagenesis of RSV Gag. All NC mutations were made in the context of Gag (RSV-PrC; GenBank accession no. J02342) lacking the PR domain (Gag{Delta}PR). Both deletion and substitution mutations in NC were engineered by two rounds of PCR. The first round of DNA amplification was performed with a primer set of outer oligonucleotides which annealed to the 5' or 3' end of a gag gene containing two engineered unique restriction sites at each end and inner mutagenic oligonucleotides that are complementary to the NC sequences with introduced mutations. The amplified DNA fragments were gel extracted and used for the second round of PCR with outer primers. Each PCR-amplified gag mutated sequence was digested with the flanking restriction enzymes and ligated to DNA restriction fragments of the appropriate vectors. For each mutant construct, sequencing was performed to confirm designed mutational changes in NC sequences.

Yeast two-hybrid binding assay. All gag mutations were fused with both an activation domain (AD) and a DNA binding domain (DBD) of the yeast two-hybrid plasmids. The AD plasmid, pACTII (26), carrying a leu gene, has the AD domain of a transcriptional activator Gal4. The DBD plasmid pSH2-1 (2) contains the DBD of lexA with a selectable his gene. The yeast strain CTY 10-5d (28), which expresses either LacZ or His downstream of the activation site of a lexA gene, was used to obtain double transformants expressing the Gal4AD-Gag hybrid plasmid and the LexADBD-Gag hybrid plasmid. Transformation was performed in accordance with the manufacturer's instructions (Frozen-EZ yeast transformation II; Zymo Research). Plates lacking histidine and leucine were used to select transformants carrying both the AD and DBD hybrid plasmids. A filter ß-galactosidase (ß-Gal) assay was used to qualitatively measure the enzyme activity of cotransformants after directly transferring colonies to filters. Cells were permeabilized by three cycles of freeze-thaw treatment of filters in a pool of liquid nitrogen, and the filters were placed on another filter that was presoaked in Z-buffer-X-Gal solution (110 mM Na2HPO4, 46 mM NaH2PO4, 10 mM KCl, 1 mM MgSO4, 330 µg of 5-bromo-4-chloro-3-indolyl-ß-D-galactoside [X-Gal] per ml, 38.5 mM ß-mercaptoethanol). Filters were incubated at 30°C, and the appearance of blue ß-Gal-positive colonies on the filters was monitored. A positive result indicates that colonies turned dark blue within 1 h. Negative colonies did not turn blue in 3 h. At least four colonies were examined for each interaction in each experiment. Similar qualitative assays have been used to monitor HIV Gag-Gag interactions (43).

Yeast protein detection using Western blot analysis. Double yeast cell transformants were made carrying the Gal4AD-Gag hybrid plasmid and the LexADBD-Gag hybrid plasmid. Such cells were grown in selective medium lacking histidine and leucine. Cells in the log phase (optical density at 600 nm [OD600] of 0.6 to 0.8) were harvested, and cell pellets were resuspended in 1x sample buffer (80 mM Tris [pH 6.8], 2% sodium dodecyl sulfate [SDS], 10% glycerol, 10 mM EDTA [pH 8.0], 0.00013% bromophenol blue, 2-mercaptoethanol, 2 mM phenylmethylsulfonyl fluoride [PMSF], 1-µg/ml leupeptin, 1-µg/ml pepstatin A). Cells were disrupted with glass beads, and cell lysates were loaded onto SDS-10% polyacrylamide gels. Proteins were transferred to Immobilon-P membranes (Millipore) and reacted with polyclonal rabbit serum against whole RSV virions (anti-RSV-PrB antibody) (4). ECL enhanced chemiluminescence reagents (Amersham) were used for signal detection with X-ray film.

Cell cultures and transfections. Cells of the quail cell line QT35 (33) or the chicken cell line DF-1 (19) were grown in GM+D+CK (Ham's F-10 medium containing 10% tryptose phosphate broth [Difco], 5% calf serum, 1% heat-inactivated chicken serum, and 1% dimethyl sulfoxide). Polyfect reagent (QIAGEN) was used for DNA transfection, in accordance with the manufacturer's protocol. Stably transfected mass cultures of G418-resistant cells, at either 0.15-mg/ml G418 for QT35 cells or 0.3 mg/ml for DF-1 cells, were obtained after 2 weeks of selection.

Metabolic radiolabeling of transfected cells and purification of virus particles. Stably transfected QT35 cells were labeled with [35S]methionine for 5 h and chased for 18 to 24 h, as previously described (24). The supernatants were collected and cell debris was cleared by low-speed centrifugation and filtration through a 0.45-µm-pore-diameter syringe filter. Virus particles were pelleted at 24,000 rpm for 2 h with an L7 ultracentrifuge (Beckman) through 20% sucrose cushions. The viral pellets were resuspended in standard buffer (100 mM NaCl, 10 mM Tris-HCl [pH 7.4], 1 mM EDTA). The labeled cells were washed twice with cold phosphate-buffered saline, scraped from plates, and lysed with lysis buffer (Direct Protect kit; Amersham, Inc.). Lysates and pelleted virus particles were used for both radioimmunoprecipitation assay (RIPA) and RNase protection assay (RPA) analyses, as described below.

RIPA. [35S]methionine-labeled cellular lysates or concentrated viral particles were reacted with anti-RSV PrB antibody, in antibody buffer (20 mM Tris-HCl [pH 7.4], 50 mM NaCl, 0.5% NP-40, 0.5% deoxycholic acid [DOC], 0.5% SDS, 0.5% aprotinin, 1 mM EDTA [pH 8.0]) with 35 µl of protein A-Sepharose beads (Pharmacia LKB Biotechnology, Inc.), for 90 min at room temperature. The antigen-antibody complexes were washed twice in RIPA buffer (10 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1% NP-40, 1% DOC, 0.1% SDS, 0.5% aprotinin), once in high-salt buffer (10 mM Tris-HCl [pH 7.4], 2 M NaCl, 1% NP-40, 0.5% DOC), and once more in RIPA buffer. The bound proteins were eluted in SDS sample buffer and loaded onto SDS-10% polyacrylamide gels. The gels were directly scanned with the Molecular Dynamics PhosphorImager. Radioactive bands were quantitated with ImageQuant software (Molecular Dynamics).

Random mutagenesis of Gag and yeast three-hybrid screening. Gag sequences of the nSKL{Delta}PR mutant were randomly mutagenized by transforming the pACTII-nSKL{Delta}PR protein hybrid plasmid (see Fig. 4B) into an E. coli mutator strain (Epicurian Coli XL 1-Red; Stratagene). This strain is deficient in three of the primary DNA repair pathways: mutS (error-prone mismatch repair), mutD (deficient in 3'-to-5' exonuclease of DNA polymerase III), and mutT (unable to hydrolyze 8-oxo-dGTP). A library of mutant DNAs was extracted from pools of transformant colonies, digested with SfiI and EcoRI, which cleave 5' and 3' ends of the Gag sequences, respectively, and ligated with the SfiI-EcoRI fragments of the pACTII{Delta}PR plasmid (25). In order to allow for screening of a large number of mutants, first, a yeast strain, L40-coat (39), was stably transformed with the pIIIA/ms2-M{Psi} RNA hybrid vector (25), in accordance with the manufacturer's instructions (Frozen-EZ yeast transformation II; Zymo Research), and then the mutant library DNA plasmids were introduced into these M{Psi}+ yeast cells. The transformant colonies were directly transferred onto filters, and enzymatic activity was assayed by a filter ß-Gal assay with X-Gal as a substrate, as previously described in the section describing the yeast two-hybrid binding assay. Filters were incubated at 30°C, and the appearance of blue ß-Gal-positive colonies on the filters was monitored.



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  		FIG. 4. The yeast three-hybrid system. (A) Diagram of the vector used and the detection strategy. Reconstitution of the two separable domains of a transcriptional activator is derived by the interactions between RNA (RSV M{Psi} RNA in our system) and protein (Gag{Delta}PR in our system), which are hybridized with a known RNA (MS2 phage RNA) and the AD, respectively. MS2 phage RNA has been shown to bind to the MS2 phage coat protein that is hybridized with the DBD (DB). Bringing together the DBD and AD leads to the expression of a lacZ reporter gene. The assay was performed as described in reference 25. (B) Experimental design for in vivo screening of second-site suppressors using the yeast three-hybrid system. Pools of randomly mutated Gag sequences of the NC-nSKL mutant generated by the Escherichia coli mutator strain XL-1 Red were swapped for the WT Gag sequence of the pACTII{Delta}PR protein hybrid plasmid. The ligated mutant DNAs were cotransformed into a yeast three-hybrid strain along with the M{Psi}-expressing RNA hybrid plasmid and plated onto selection plates lacking Ura and Leu. Filter ß-Gal assays were done with filters directly lifted from the plates, and ß-Gal-positive clones were selected and sequenced to locate second-site mutations in the Gag sequences. (C) Amino acid changes found in in vivo-selected suppressors. MHR, major homology region in the CA domain; SP, spacer peptide between the CA and NC domains. One-letter abbreviations for amino acid sequence were used. Two primary RNA binding (–) mutants are indicated in NC (BR2alaP and nSKL; thick solid lines). The name of each suppressor is represented as Supp with numbering starting from the amino terminus of each domain in Gag and listed below the secondary mutation sites (Supp 50, 94, and 127; solid lines). (D) Additional mutant sites created by site directed mutagenesis in Gag (dotted lines). MA99 indicates a mutational change at glycine 99 (numbering starting from the N terminus of MA) to arginine. Two additional suppressors (Supp 51 and Supp 128) in either p10 or CA are shown below the secondary mutation sites, with numbering starting from the amino terminus of each domain in Gag.

Yeast three-hybrid liquid ß-Gal assay. Liquid cultures were used to quantitatively measure enzymatic activity by using chlorophenol red-ß-D-galactopyranoside (CPRG) as a substrate (20). The color changes of the substrate at 37°C incubation were measured by OD574. Units were calculated as (1,000 x OD574)/[(volume in ml x time in min) x OD600]. For each transformant, at least three independent colonies were assayed, and their values were averaged.

RPA. The cell lysates and viral pellets were prepared as described above. The amounts of cellular or viral RNAs were measured with either the antisense neo probe or the antisense gapdh probe. 32P-labeled probe RNA was in vitro transcribed with T7 RNA polymerase. The labeled RNA was purified with phenol-chloroform and passed through a G-25 column (Amersham Pharmacia) to remove unincorporated 32P. The probes were ethanol precipitated and used for hybridization with cellular and viral RNAs. RPAs were performed by the method specified for the Direct Protect kit (Ambion, Inc.). Reaction mixtures were run on 5% polyacrylamide gel, and the dried gels were directly scanned with a Molecular Dynamics PhosphorImager. Radioactively labeled protected bands were quantitated with ImageQuant software.

Calculation of packaging efficiencies. The packaging efficiencies for the heterologous RNAs were determined by calculating the amount of neo RNA in virions (measured by RPA) normalized to the level of neo RNA in the cells (normalized to a constitutive cellular mRNA, gapdh, both measured by RPA of the cell lysate). This calculated level of RNA was further normalized to the number of virions, as determined by RIPA.

RT assay. Reverse transcriptase (RT) activity was determined by the incorporation of [32P]TTP by using a poly(A):(dT)12 template, as previously described (32). A 10-µl volume of the concentrated viral particles was added to 50 µl of reaction cocktail, consisting of 45 mM Tris (pH 8.0), 50 mM NaCl, 5 mM dithiothreitol, 2.5 mM MgCl2, 10 µg of poly(A):(dT)12 per ml, 0.1% NP-40, and 10 µl of [32P]TTP per ml, and the reaction mixtures were incubated at 37°C for 1 h. Then 5-µl volumes of reaction were transferred to DE81 filters (Whatman), and the filters were washed in 2x SSC (0.3 M sodium chloride, 30 mM sodium citrate) in 95% ethanol and dried. The filters were resuspended in 2 ml of scintillation counter fluid, and radioactivity was counted in a Beckman scintillation counter.


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RESULTS
 
Basic residues, but not Cys-His boxes, are required for Gag-Gag interactions. In order to examine what constitutes the interaction (I) domain for RSV assembly, we made substitutions or deletions in NC in the context of Gag sequences lacking the PR domain (Gag{Delta}PR) (Fig. 1A). The yeast two-hybrid binding assay was used to measure the interactions between mutant Gag proteins that were hybridized with either the DBD or the AD of the yeast two-hybrid plasmids, as described in Materials and Methods. This binding brings together the DBD and AD to reconstitute expression of a reporter gene, lacZ. The enzymatic activity of ß-Gal was directly measured by a filter ß-Gal assay. Interactions between mutant Gag proteins in both vectors or between mutant and wild-type (WT) Gag proteins were examined. All Gag mutants, including those with deletion of either or both of the two Cys-His (CH) boxes, substitution mutations of Zn-coordinating residues in the CH boxes, and alanine substitutions for basic residues flanking CH boxes, interacted with either the WT or the cognate mutant Gag (Fig. 1A). These data show that both of the two CH boxes can be deleted without abrogating Gag-Gag interactions. Thus, it is likely that in NC, components other than the two CH boxes play important roles in Gag multimerization or that there are redundant I domains in NC or in other regions of Gag.



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  		FIG. 1. Gag constructs used in the yeast two-hybrid binding assays. Substitution and deletion mutations in NC (A) and carboxy-terminal Gag truncation mutations (B) were made in the context of Gag{Delta}PR (the dotted box indicates the region of Gag that has been deleted). Carboxy-terminal Gag truncation mutations were made to completely delete the C terminus of NC, including the second Cys-His box and 3' sequences along with the PR domain. The amino acid sequence of NC is shown by one-letter abbreviations with numbering starting from the amino terminus of NC. The positively charged residues are indicated in boldface type, and the two Cys-His motifs are boxed in gray. (A and B). Interactions of the indicated Gag mutants with either WT or cognate mutant Gag were examined with yeast two-hybrid binding assays. Gag constructs were fused to either the AD of a transcriptional activator Gal4 or the DBD of LexA to reconstitute expression of a reporter gene, lacZ. ß-Gal activities were qualitatively measured with the filter ß-Gal assay, as described in Materials and Methods. For each construct, at least four transformant colonies were assayed. +, colonies turned dark blue in a 1-h incubation at 30°C; –, no color changes detected after at least 3 h of incubation. The number of positively charged amino acids in NC of the constructs is shown in the last column of panel B. Wild-type NC contains 16 positively charged residues.

Since all of Gag mutants tested in the context of a full-length NC were competent for Gag-Gag interactions, we next made carboxyl-terminal truncations in NC (Fig. 1B). Deletion of the distal portion of NC, including the second CH box and the 3' sequences, did not prevent Gag-Gag interactions (mutant CT in Fig. 1B). However, this deletion, in conjunction with alanine substitutions of six basic residues (BRs) between the two CH boxes, prevented interactions (mutant BR2alaACT). The BR2alaACT mutant showed about the same level of DBD- and AD-fusion protein expression as WT or CT (Fig. 2). Upstream of the first CH box, substitutions for two of the four BRs did not affect the Gag binding (mutant BR1A2CT), whereas all four changes abolished binding (mutant BR1A4CT). The AD-BR1A4CT fusion protein was not expressed, leading to a failure to interact (Fig. 2). However, the DBD-BR1A4CT protein was expressed similar to WT and CT (Fig. 2) and still failed to interact with WT or CT. The C21S mutation, which is predicted to lead to a large conformational change in the Zn-finger binding motif of the CH box, also did not inhibit Gag binding in the context of C-terminal-truncated NC (mutant C21SCT in Fig. 1B). Mutants that retained eight BRs in NC did interact with Gag, but those with either six or four did not (Fig. 1B). In order to test whether clusters of basic residues are sufficient to drive Gag-Gag interactions, we engineered a minimal sequence containing 8 BRs plus 10 flanking amino acids in place of NC (mutant 8BR in Fig. 1B). We found that eight BRs attached to the C terminus of the spacer peptide separating the CA and NC domains in Gag were enough to drive Gag-Gag interactions. Thus, as few as eight BRs, rather than the CH boxes, may have the major role in Gag assembly.



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  		FIG. 2. Yeast protein detection using Western blotting. Cell lysates using double transformants expressing both the DBD- and AD-hybrid proteins were prepared as described in Materials and Methods. Proteins were separated on the SDS-10% polyacrylamide gel, transferred to the membrane, and reacted with polyclonal anti-RSV antiserum. Two independent clones for each transformant were used for analysis, shown in the paired brackets. The horizontal lines indicate the AD and DBD fusion proteins from the full-length Gag protein lacking the PR domain (WT). The sizes of the fusion proteins vary in the different deletion mutants. The Gag constructs used are described in Fig. 1.

Since we previously found that deletion of either MA or CA alone did not affect efficient viral assembly and release in the context of a PR-defective virus (25), we examined whether other domains in Gag are necessary for Gag multimerization. We found that deletion of the p10 domain did not prevent Gag-Gag interactions if the Gag polyprotein contains a sufficient number of basic residues in place of NC (data not shown). We tried to engineer a minimal construct sufficient for Gag interactions, based on these results. We made a small construct containing clustered basic residues in place of NC, which were attached to the 20 amino acids comprising the terminus of CA and the spacer peptide separating CA and NC. These 20 amino acids correspond to a region just upstream of HIV-1 NC, which has been shown to be important for Gag multimerization and virus production (1, 29, 30). However, this minimal construct did not allow Gag-Gag interactions (data not shown), indicating that additional, possibly redundant, sequences in Gag are required for NC functioning as the interaction domains.

In vivo phenotypes of NC mutants. In order to examine whether putative Gag-Gag interactions measured by the yeast two-hybrid binding assays predict in vivo viral assembly, we cloned several C-terminal truncation mutations into the full-length RSV proviral vector, pASY165, which lacks PR (25). QT35 cells stably transfected with mutant DNAs were labeled with [35S]methionine for 5 h, and cells and virus particles were collected as described in Materials and Methods. Labeled viral proteins from cell lysates and pelleted virus particles were detected by RIPA using polyclonal antiserum against whole virions (Fig. 3). The C-terminal NC truncation mutant (CT in Fig. 1B) lacking the second CH box and downstream sequences produced about one-third as many virus particles as Gag{Delta}PR RSV (data not shown), indicating that loss of the second CH box and the C-terminal sequences of NC did not prevent viral assembly. All the C-terminal truncation mutants tested showed about equivalent amounts of proteins expressed in the cells (Fig. 3A). However, while the CT and eight-BR mutants showed significant virus particle production, both alanine substitution mutants (BR2alaACT and BR1A4CT) released very few virus particles into the media (Fig. 3B). Quantitation (Fig. 3C) showed that the eight-BR mutation resulted in about 40% of the level of CT particles in the supernatants, and these were of WT density (data not shown). In contrast, the small number of BR1A4CT particles had a very light density of 1.05 g/ml compared with 1.14 g/ml for WT particles (data not shown). Viral density has been used as a measure of proper viral assembly for several retroviruses (6, 8, 38, 41). One group (12), however, reported that the I domain was not the sole determinant of viral density, and there may be other Gag domains that contribute. Nonetheless, our result with BR1A4CT is consistent with a deficient Gag multimerization phenotype. The results imply that the mutants showing Gag-Gag interactions in yeast are competent to assemble and release virus particles of proper density. Thus the minimal NC construct with eight BRs can assemble in vivo, although at reduced levels.



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  		FIG. 3. Viral assembly and release analysis. [35S]methionine-labeled cells (A) and virus particles in the supernatants (B) were collected and immunoprecipitated with polyclonal anti-RSV antiserum. Complex proteins were run on the SDS-10% polyacrylamide gel, and the gel was directly scanned with the Molecular Dynamics PhosphorImager. Radioactive bands were quantitated with ImageQuant software. (C) Efficiency of viral assembly and release was calculated by normalizing the levels of viral Gag proteins with those of cellular Gag and is shown as the ratio relative to the CT parental construct. The results are averages from three independent assays.

Role of NC basic residues in the tertiary structure of Gag required for {Psi} RNA binding. We previously described an RSV Gag mutant, NC-SKL, which did not affect particle formation, but failed to package genomic RNA (25). We used this mutant to isolate second-site suppressors, which restore ability of Gag to bind to {Psi} RNA. For in vivo screening, we used random mutagenesis of the Gag sequences in the NC-SKL mutant and yeast three-hybrid screening. Briefly, the yeast three-hybrid binding assay measures the ability of Gag protein fused with the Gal4 AD to bind to M{Psi} RNA fused to the MS2 phage RNA (Fig. 4A). This binding brings together the DBD coupled to the MS2 phage coat protein with Gag-AD to reconstitute the expression of lacZ reporter gene. In order to prevent the NC-SKL mutation from reverting to the WT sequence (RKR), we first added four synonymous mutations to the NC sequences in NC-SKL, resulting in six nucleotide changes out of nine. As predicted, a newer version of NC-SKL (nSKL in Table 1) also had low ß-Gal activity in the three-hybrid RNA binding assay (Table 1). The nSKL mutation was then cloned into the gag gene lacking a PR domain (nSKL{Delta}PR in Fig. 4B), which we previously found to be the optimal construct for the yeast three-hybrid screening (25). The Gag sequences of the nSKL{Delta}PR mutant were randomly mutagenized, and ß-Gal-positive colonies were screened in the yeast three-hybrid system, as described in Materials and Methods. Of about 400 colonies screened in three separate experiments, three colonies were found to be darker blue than those transformed with the NC-nSKL mutant. The entire Gag regions of these three suppressor mutants were sequenced to locate the mutations. We expected that we would see mutations that increase the positive charges in NC. Unexpectedly, each Gag gene contained a single second-site mutation in either p10 (Supp 50) or the CA domain (Supp 94 and 127) (Fig. 4C). Supp 50 contained a mutation of glutamic acid 50 to glycine (numbering starting from the amino terminus of p10), and the other two had mutational changes at glycine 94 or 127 to aspartic acid or arginine, respectively (numbering starting from the N terminus of CA). Liquid cultures were used to quantitate ß-Gal activity by directly measuring the enzyme activity using CPRG as a substrate (20). Both the Supp 50 and 127 mutants had about eightfold higher ß-Gal activity than that of the nSKL mutant, while Supp 94 had only a threefold increase (Table 1). In order to test whether the secondary mutations alone led to the increase {Psi} RNA binding, we cloned each single second-site mutation into a WT Gag{Delta}PR plasmid and transformed them into yeast cells. The recloned second-site mutants (p10-50, CA-127, and CA-94) displayed about the same ß-Gal activity as WT Gag{Delta}PR (Table 1), indicating that the second-site mutations did not alter the ability for {Psi} RNA binding on their own.


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  		TABLE 1. Relative ß-Gal activities of Gag mutants

In order to examine whether the suppressor mutations could alter the deficient packaging phenotype of NC-nSKL, we cloned Supp 50 and 127 into an RSV packaging vector containing the neo gene (25). Because of the lower level of ß-Gal activity, we did not further characterize Supp 94. Mutant proviruses were transfected into QT35 quail cells, and stably transfected mass cultures of G418-resistant cells were used to measure the packaging efficiency in vivo. Cells containing the mutant proviruses were labeled with [35S]methionine for 5 h, the supernatants were collected, and virus particles were purified as described in Materials and Methods. To quantitate cellular and virion levels of Gag protein, RIPA was performed with anti-Gag serum. The results of a typical experiment are shown in Fig. 5A. The nSKL mutant was much more efficient in production of virus particles compared to the other mutants, which had equivalent amounts of cell-associated viral proteins, for unknown reasons.



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  		FIG. 5. Viral RNA packaging assays. (A) RIPA used to measure the Gag protein in cells transfected with Gag constructs and virus particles collected from the supernatants. All constructs are in the context of Gag{Delta}PR, except SKL, which has a point mutation at the active site in PR. (B) RPA to measure the amount of neo RNA in virions and the amount of neo RNA and cellular gapdh RNA in transfected cells. The RNase+ lane contains a mixture of the neo probe RNA and gapdh probe RNA treated with an RNase cocktail (RNase A and T1); the RNase– lane was not treated with RNase cocktail. (C) Relative packaging efficiency to Gag{Delta}PR in vivo. The packaging efficiency was calculated as described in Materials and Methods. Each experiment was done three times, and the averages with standard deviations (error bars) are shown. The fold increase in packaging efficiency compared to that of nSKL is shown above the bar.

As the pASY165 packaging vector contains neo in the place of the src gene, we used a neo probe for RPAs. Since the transfected cultures produced variable levels of genomic viral RNA in the cells, we also measured the levels of both cellular neo RNA and the constitutively expressed cellular gapdh RNA, which were used to normalize the level of viral RNAs (Fig. 5B). To calculate the packaging efficiency of viral RNA into particles, the normalized level of viral RNA was corrected by the number of particles, as measured by the level of 35S-labeled Gag protein in the pelletable particles normalized to the level of 35S-labeled cellular Gag (Fig. 5A). The packaging efficiency of each mutant relative to Gag{Delta}PR, taken from three independent assays, is shown in Fig. 5C. As predicted, the nSKL and SKL mutants are both deficient in {Psi} RNA packaging. Supp 50 and Supp 127 each increased the packaging efficiency by 4- to 10-fold compared with that of the nSKL mutant, and the levels reached about 15 to 35% that of Gag{Delta}PR, indicating that suppressors at least partially restored in vivo packaging of viral RNA.

We next examined whether the effect of these second-site suppressors is specific to the NC-nSKL mutation. We engineered a double mutant that combined the second-site mutation of Supp 50 with another NC mutant. We previously found that the BR2alaP mutant (Fig. 4C), which has four alanine substitutions for contiguous BRs distal to the first Cys-His box, completely abolished {Psi}-RNA binding and RNA packaging (23). A p10-50/BR2alaP double mutant showed a ca. 500-fold increase in ß-Gal activity compared to BR2alaP, and thus {Psi} RNA binding was restored to the WT level (Table 1). These results show that the p10-50 mutation can suppress the RNA binding defect in two mutants with different deficiencies of basic amino acids.

Since the second-site substitutions resulted in introduction of a positively charged residue or elimination of a negatively charged residue in either the p10 or CA domain, it is possible that in the native conformation of Gag, the NC domain is in proximity to the regions defined by the suppressor mutations and the loss of positive charge at the primary mutation site is directly compensated for by the second-site mutations. This idea was tested by generating additional mutations. Basic amino acids were introduced at positions adjacent to the selected suppressor mutations in p10 or CA or at a site in the MA domain (Fig. 4D) in the context of the nSKL mutant and tested in the three-hybrid assay. Supp 128 showed ß-Gal activity as high as that in the original suppressor (Supp 127), and the ß-Gal activity of Supp 51 was similar to that of WT Gag{Delta}PR. In contrast, the mutational change at the MA domain did not restore {Psi}-RNA binding (Table 1). These results show that addition of basic residues to either the CA and p10 domain can suppress loss of function in NC, but a charge change in MA cannot.

Suppressor mutations do not restore viral replication. Since the selected Gag suppressor mutations Supp 50 and Supp127 allowed a high level of RNA packaging, we engineered these mutations into pASY155, a replication-competent proviral construct containing the neo gene in place of src, with or without the nSKL mutation. Plasmids containing WT or mutant proviruses were transfected into DF-1 chicken cells. Stably transfected cell cultures of G418-resistant cells were established, and viruses were harvested from the supernatants. Virus particles were enumerated by measuring RT activities, and equal numbers of virus particles were used to infect new DF-1 cells. Culture supernatants were harvested, viral particles were pelleted through sucrose, and RT activities were measured (Fig. 6). We found that the second-site mutations alone did not prevent viral infection, although in the case of the p10 mutation, viral replication was reduced to about 50% of that of the WT. However, these mutations were not able to restore replication competence to the nSKL mutation. Thus, the changes in NC BRs distal to the second CH box might affect the function of other viral processes needed for replication.



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  		FIG. 6. Infectivity of suppressor mutations. The suppressor mutations alone (p10-50 and CA-127) or the double mutations with nSKL (Supp 50 and Supp 127) were introduced into the replication-competent proviral construct pASY155. Plasmids were transfected into DF-1 cells, and stably transfected pools were selected with G418. Amounts of supernatant normalized for RT were used to infect fresh cultures of DF-1 on 60-mm-diameter dishes. Cells were passaged two times to new 100-mm dishes, and viral supernatants were harvested. Viral particles were pelleted through 20% sucrose, and resuspended viral pellets were used for RT assays. The relative RT activity compared to that of the WT is shown as the average with standard deviation (error bars) of the values for two independent infection assays and duplicate RT reactions for each infection assay. The WT 32P value is about 12,000 cpm.


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DISCUSSION
 
In this study, we have examined the role of NC BRs in two of the most important functions of this protein, virus assembly and genomic RNA packaging. Although the most striking conserved features of NC are one or two Cys-His boxes, previous work has indicated that it is the basic residues that are indispensable for these interactions (8, 13, 16). We have used two different rapid genetic assays in yeast to explore the necessity for specific BRs and total charge on Gag-Gag and Gag-RNA binding. We can conclude that although the total number of BRs is the key factor for Gag-Gag interactions, specific BRs and their contacts in the tertiary protein structure appear essential for the interaction of Gag with its cognate genomic RNA packaging signal. We would predict that the regions of CA and p10 defined by the suppressor mutations are closely associated with NC in the Gag tertiary structure.

One of the important questions about retroviral assembly is the role of RNA in initiation of the Gag interactions. The weight of the current data suggests that nucleic acid is a required scaffold for initial Gag contacts required to initiate higher-order assembly (21, 47). These initial contacts can occur by using any nucleic acid—even short oligonucleotides—at least in vitro (42). The yeast two-hybrid system mirrors in vitro virus-like particle (VLP) assembly, as there is no {Psi}-containing RNA present in the yeast cells, and thus any Gag interactions that occur use only nonspecific nucleic acids. Paradoxically, in infected cells, virions specifically package genomic RNA and are likely to use their cognate genomes as the initial scaffold, an interaction that requires more specific Gag contacts. In the in vivo situation, it is highly likely that Gag-Gag and Gag-{Psi} RNA binding are intimately coupled, with basic amino acids in NC being instrumental for both interactions, and it is possible that the specific RNA interactions are essential. Whether the less-specific Gag-Gag contacts, which are sufficient in vitro and in yeast to allow Gag multimerization and particle formation, actually are important for binding to {Psi}-containing RNA in vivo is not known, since they would be masked by the less-specific interactions that are sufficient in these situations.

Previous studies have examined viral determinants of assembly and packaging in RSV, murine leukemia virus, and HIV. Our finding that substitutions for zinc-coordinating residues in the CH boxes did not affect Gag multimerization is consistent with many previous studies (35, 42). Yu et al. found that a minimal number of basic residues in NC are required to assemble VLPs in vitro, and we found that the whole NC domain could be replaced with a small stretch of BRs, which agrees with this conclusion, as well as additional studies (16, 46). In vitro binding assays using a series of HIV-1 Gag mutant proteins showed that deletion of NC abolished Gag binding when the amino-terminal basic residues in the MA domain were also removed (9), so the basic region of MA was proposed to substitute for the NC RNA binding motif to promote Gag-Gag interactions. Similarly, VLPs of a spherical morphology were assembled in vitro when the RSV NC domain was replaced with a dimer-forming leucine zipper domain (21), and similar results were found for HIV (47). Taken together, these findings show that the I domain of NC functions to either promote the formation of Gag dimers, which are intermediates in Gag polymerization, and/or nonspecifically bind to RNA, which serves as a scaffold for Gag multimerization (10, 11, 21, 31, 34, 42). Results from the present study and many other laboratories suggest that the function of NC can be substituted for with unrelated RNA binding motifs, but always in the context of additional domains in Gag. Gag multimerization seems to involve both initial indirect contacts via RNA binding and subsequent direct protein-protein interactions mediated by domains in Gag. Our finding that none of the NC mutations we made in full-length Gag affected Gag-Gag interactions, whereas in the context of C-terminal truncated NC alanine substitution mutations for basic residues flanking the first CH box did, indicates that the I domain function is redundant within NC. This agrees with work showing that multiple regions within the HIV-1 NC domain act concomitantly to promote the Gag-Gag interactions that drive formation of dense HIV particles (38).

Our results indicating that NC is a part of I domain are consistent with what has been shown from previous studies cited above. However, one group recently found that deletion of all but seven amino acids of NC in a full-length HIV-1 proviral clone still allowed production of virus particles, albeit at lower levels and with lower density than the WT (36). These authors also found that inactivation of protease activity suppressed the defect in the NC deletion mutant and allowed production of WT virion levels, suggesting that HIV-1 NC is not necessarily required for particle formation, in contrast to the plethora of studies that have assigned an obligate role for NC in viral assembly, including this work.

Our previous results (23) showed that loss of as few as two specific positively charged amino acids in NC leads to a pronounced defect in RNA binding and packaging. We now have found that several such mutants can be compensated for by addition of a positively charged residue at several sites in the Gag polyprotein. We would predict that the regions defined by the suppressor mutants at amino acid 50 in p10 and amino acid 127 in CA are situated near NC in the tertiary Gag structure and can compensate for removal of critical basic residues from NC in {Psi}-RNA binding.

Taken together, our results show that basic residues in NC play different roles in Gag-Gag and Gag-{Psi} RNA interactions. Gag-Gag interactions leading to particle assembly through nonspecific nucleic acid binding require a certain concentration of basic residues, but their location is not important. In contrast, for Gag-{Psi} RNA interactions while the number of basic residues is also important, so is their specific location. The fact that mutations in either p10 or CA can compensate for changes in NC charge suggests a complex interaction of domains required for specific recognition of RNA. Thus, it is likely that in retroviral assembly, Gag multimerization, mediated by RNA, can occur via rather nonspecific NC binding to RNA, but encapsidation of genomic RNA requires a highly structured RNA-protein interaction.


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ACKNOWLEDGMENTS
 
We thank Jaisri Lingappa and Michael Emerman for helpful comments on the manuscript.

This work was supported by Public Health Service grant CA18282 from the National Cancer Institute to M.L.L.


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FOOTNOTES
 
* Corresponding author. Mailing address: Division of Basic Sciences, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave. N., Seattle, WA 98109-1024. Phone: (206) 667-4442. Fax: (206) 667-5939. E-mail: mlinial{at}fhcrc.org. Back


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Journal of Virology, August 2004, p. 8486-8495, Vol. 78, No. 16
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.16.8486-8495.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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