INTRODUCTION

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Retroviral nucleocapsid (NC) proteins are typically small basic proteins containing one or two conserved 14-residue peptide segments (-C-[X]₂-C-[X]₄-H-[X]₄-C-) that bind zinc ions, forming structures referred to as zinc fingers (ZFs). NC proteins with native ZFs are required for viral replication and are apparently utilized at many stages of the replication cycle. The biological roles of retroviral NC proteins and their ZFs have been reviewed recently (18, 19) and include functions during viral assembly, maturation, and infection. Regarding particle morphology, the basic residues of NC are required for consistent and proper particle size (21), and the ZFs, when mutated or deleted, result in virions that do not mature properly (7). In fact, a number of the functions of NC are attributed to the ZFs.

All human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) NC proteins contain two ZFs. It is currently believed that each ZF is required for viral replication; however, they are not necessarily equally required for each and every specific function during the replication cycle. In HIV-1, the proximal Zn²⁺ finger (ZF1) functions in genomic RNA incorporation, viral core morphogenesis, and proviral DNA synthesis (44, 45), while the intact distal Zn²⁺ finger (ZF2) is required for Gag polyprotein stability during proteolytic processing (35). It is clear in HIV-1 that one finger cannot replace the function of another (25), so there is some degree of independence in ZF activity, but there is also an overlap of many of the NC functions attributed to both ZFs (23, 35, 44, 45). However, these functions may not be consistent among all NC proteins that contain two ZFs.

The functions of HIV-1 and SIV NC ZFs are of particular interest because of their vital roles in viral replication and their potential as targets for viral inactivation (mutational as well as chemical targets). At present, little work has been accomplished to address ZF function in SIV (23). Because these viruses are presently used in many of the in vivo models of HIV-1 disease and vaccination (3, 29, 30, 39, 48) and the retroviral NC protein is presently a recognized target for therapeutic (6) and vaccine (2, 43) development, it is imperative to understand the viral physiology dictated by this protein in the SIV system. To address this, we have mutated the specific Zn²⁺-binding Cys residues to Ser of each or both fingers of SIV(Mne) NC to eliminate ion binding (8), yet maintain as much of the protein conformation as possible. We have also deleted the first 4 amino acids of each finger or the entire finger-linker sequence to assess finger function differences between the Zn²⁺-coordinated conformation and primary amino acid sequence conformation. In this report, we have focused only on SIV(Mne) NC function relative to genomic RNA incorporation, Gag polyprotein processing, particle formation, and maturation. Here we provide a detailed analysis of the subtle differences in function attributed to, but not unique to, the individual ZFs of SIV(Mne) NC.

	MATERIALS AND METHODS

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Mutagenesis and plasmid construction. SIV(Mne) was isolated and cloned as described previously (4). The generation of the full-length proviral clone and deletion mutants of SIV(Mne) NC has been described previously (23). The subclone pPP1 (23) of SIV(Mne) was used as a template for the generation of point mutants of NC ZFs. The Cys coding sequences in the NC gene were changed to Ser by site-directed mutagenesis (QuickChange site-directed mutagenesis kit; Stratagene, La Jolla, Calif.) with primers (obtained from Life Technologies, Rockville, Md.) designed specifically for each ZF as follows (the mutagenic nucleotides are underlined): ZF1 CS, plus strand 5'-TAA GAG TTG GAA TAG TGG GAA AGA GGG ACA CTC TGC AAG GCA AAG CAG [5' end corresponding to nucleotide [nt] position 1705 of the SIV(Mne) sequence; GenBank accession no. M32741], producing pPP1-4.1r; and ZF2 CS, plus strand 5'-GGC AGC TGG AAA AGT GGA CAA ATG GGC CAT GTT ATG GCC AAA AGC C [5' end corresponding to nt position 1769 of the SIV(Mne) sequence], producing pPP1-6.15. CysSer changes in both ZFs were introduced by mutagenesis of pPP1-4.1r with the ZF2 CS mutational oligonucleotide, producing pPP1-4.1.5. All mutations were confirmed by nucleotide sequencing and are noted in the 52-amino-acid SIV(Mne) NC protein sequence (31) depicted in Fig. 1.

View larger version (33K): [in this window] [in a new window]   FIG. 1.   Protein sequence of WT and mutant SIV(Mne) NC proteins. Zinc-coordinating residues are indicated with residue numbers. Dotted lines indicate Zn2+ coordination. A dashed line indicates deletions.

Transfections. 293T cells were transfected as previously described (26) with equimolar amounts of proviral plasmids by calcium phosphate precipitation (5'3', Inc., Boulder, Colo.). Supernatants were harvested and clarified as previously described (24).

Electron microscopy and particle analysis. Supernatants from transfected 293T cells were clarified by centrifugation, and virus was pelleted at 100,000 × g through a 20% (wt/vol) sucrose cushion in phosphate-buffered saline (PBS). Virus pellets were fixed in 1.25% (vol/vol) glutaraldehyde in PBS. Electron microscopy was performed with a Hitachi H-7000 microscope at 75 kV as previously described (23). Six photographs were taken of each virus pellet, with an average number of 119 virions per photo. Virus particles (magnification of ×90,000) were digitized with a ScanAce III scanner (Pacific Image Electronics Co., Ltd., Torrance, Calif.) at a 600-dpi resolution and processed with Adobe Photoshop (Adobe Systems, Inc., Mountain View, Calif.).

SIV p28^CA antigen capture assay. Virus was produced and pelleted as described above and then resuspended in PBS. Serial dilutions of sample aliquots and standards were prepared and assayed as previously described for aldrithiol-2-treated HIV p24^CA (43), except that the capture antibody (AIDS Vaccine Program, Biological Products Laboratory [AVP-BPL] inventory no. 0089A-9B5-8BE5-6AC10) and detector antibody (AVP-BPL inventory no. DJ-40115, bleed 10263) were specific for SIV(Mne). Assay standards were based on a wild-type (WT) SIV(Mne), clone E11S stock, produced from infected HuT78 cells that had been accurately quantitated by reversed-phase high-performance liquid chromatography (RP-HPLC) and amino acid composition.

Viral protein western analysis. Virus was produced and pelleted as described above. Samples containing equivalent reverse transcriptase (RT) activity (3.8 × 10⁵ cpm by exogenous template RT activity assay [27]) were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using 4 to 20% Tris-glycine Novex gels (Invitrogen Corp.), immunoblotted as described previously (25), and then incubated with mouse monoclonal anti-p28^CA antibodies (AVP-BPL inventory no. 96-0020 89A P9B5 H8B1) and visualized with the Amersham ECL (enhanced chemiluminescence) system (Arlington Heights, Ill.). Membranes were stripped according to manufacturer's recommendations (Amersham ECL system) and then incubated with rabbit polyclonal anti-gp120 and anti-p8^NC antibodies (AVP-BPL no. DJ-36603 003545 and DJ-37545 005970, respectively) concomitantly and visualized with ECL as well. Lyophilized fractions from RP-HPLC were fractionated by SDS-PAGE and immunoblotted as described above. Membranes were sequentially incubated no more than three times with rabbit polyclonal anti-p28^CA (no. B2119-5), anti-p8^NC (no. 62837), anti-p6^Gag (no. 63157), and anti-p2^Gag (no. 63158) antibodies supplied by Raoul Benveniste, Basic Research Laboratory, National Cancer Institute at Frederick, Frederick, Md. Each hybridization was followed by ECL visualization. Blots were completely stripped of all antisera before being reprobed with the next antibody.

RP-HPLC separation of SIV(Mne) WT and ZF mutant proteins. Pelleted virus from equivalent supernatant volumes of transfected 293T cultures was first resuspended in 50 µl of PBS and then reduced in 10 mM dithiothreitol (Calbiochem, La Jolla, Calif.) in the presence of 50 µl of 8 M sequenal-grade aqueous concentrate of high-purity guanidine-HCl (Pierce, Rockford, Ill.) and 10% (vol/vol) acetonitrile (EM Science, Gibbstown, N.J.) for 5 min at 37°C and loaded onto a Poros R2/H narrow-bore column (2.1 by 100 mm) (PerSeptive Biosystems, Inc., Framingham, Mass.). RP-HPLC was performed with a Shimadzu system as described previously (36). Fractions were eluted at a flow rate of 0.3 ml/min with a variable gradient of acetonitrile in 0.1% (vol/vol) Spectro-grade trifluoroacetic acid (Applied Biosystems, Warrington, United Kingdom) as follows: from 10 to 36.5% (vol/vol) acetonitrile for 12 min, 36.5 to 37% for 4 min, 37 to 41% for 7 min, 41 to 70% for 12 min, and 70% for 5 min. Peaks were detected by UV absorption at 206, 280, and 260 nm and analyzed by sequencing with an automated Applied Biosystems, Inc., Procise Protein Sequencer, SDS-PAGE, and immunoblot detection with the ECL system (Amersham Life Science). Fractions were collected every 30 s. Selected fractions were also analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry as described previously (36).

Viral RNA analysis. Viral RNA was prepared as previously described (23). Samples were normalized for equivalent RT activity (4.2 × 10⁶ cpm) determined from pelleted virus samples. RNA was electrophoresed through a formaldehyde-agarose gel, transferred to a nitrocellulose membrane, and treated as described previously (27). WT RNA was additionally analyzed at dilutions of 1:4, 1:16, 1:64, and 1:128. The full-length WT SIV(Mne) proviral plasmid pRB85 (23) was cut with XhoI restriction enzyme, random-prime ³²P labeled (DECAprimeII kit; Ambion, Inc., Austin, Tex.), and used as a probe for full-length genomic RNA. RNA blots were hybridized overnight at 42°C in a mixture of 50% (vol/vol) formamide, 120 mM Na₂HPO₄ (pH 7.2), 250 mM NaCl, 7% (wt/vol) SDS, and the entire Sephadex G-50 (Amersham Pharmacia Biotech, Inc., Piscataway, N.J.) spin column-purified labeled probe. Blots were washed as previously described (23). Phosphorimager analysis was performed with bands of full-length genomic RNA with a Bio-Rad Molecular Imager FX phosphorimager with a K-Imaging Screen (Hercules, Calif.).

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Virion morphology. To characterize the effect of the removal of zinc-binding residues of NC on particle formation and maturation, mutations were introduced into the SIV(Mne) viral coding sequence. Specifically, the 52-amino-acid NC domain of Gag was altered to produce the mutants listed in Fig. 1. Virus particles were produced from 293T cells by transfection of DNA plasmids containing the complete coding sequence of WT or the various NC mutants of SIV(Mne). Virions were collected by centrifugation and examined by electron microscopy with positive staining. Electron micrographs were digitized so that individual virions could be sorted according to specific morphological criteria. Virus particles were isolated from two representative photographs of each mutant and categorized as (i) mature morphology if a condensed core and thin capsid membrane were observed; (ii) immature morphology if a noncondensed core and thick capsid membrane were observed, or (iii) abnormally large or small morphology if particles deviated from the average 100-nm diameter of WT particles by greater than 10% (Fig. 2). Particles were excluded if they contained both characteristics and/or cores were not visible because of the plane of thin sectioning (Table 1). Those particles of condensed core morphology were subcategorized as (i) cone, or WT core of high electron density; (ii) rod core of low electron density; or (iii) multiple cores, all of which were rod morphologies.

View larger version (82K): [in this window] [in a new window]   FIG. 2.   Electron microscopic classification of virion morphology. Three typical examples of each type of morphology (except irregular large) are shown. The upper two virions in each category are typical longitudinal cross-sections, and the lower virion in each category is a typical latitudinal cross-section. Virus was pelleted from clarified supernatants of 293T cells 72 h after transfection with mutant or WT plasmid DNA. Images are digital and were scanned from electron micrographs at a magnification of ×90,000.

Virus particle production. To determine the ratio of particle-associated Gag-Pol polyprotein to Gag produced by the NC mutant and WT transfections, virus was quantitated by RT activity in an exogenous template assay (to estimate Gag-Pol levels) and Gag expression through a p28^CA antigen capture assay. Table 2 shows that the Gag-Pol/Gag ratios are relatively constant among the mutant and WT viruses, except for the ZF1+2 mutant. RT activity was measured in viral pellets as well as polyethylene glycol (PEG)-precipitated culture supernatants to observe any differences in particle-associated and supernatant RT. These results are also summarized in Table 2. All mutants demonstrate similar ratios of particle-associated to supernatant RT levels.

Genomic RNA incorporation. To determine if the morphological differences observed could be attributed to variations of full-length genomic RNA incorporation, particle-associated RNA was isolated and analyzed by Northern blotting and quantitative RT-PCR (Fig. 3) (23). It appears that the alteration of NC ZF1 does not significantly affect genomic RNA incorporation, yet the alteration of NC ZF2 results in reduced incorporation of full-length genomic RNA. Similar to the ZF1+2 mutant (23), ZF1+2 CS incorporates <20% WT levels of full-length genomic RNA. These results suggest that there is no correlation between genomic RNA incorporation and viral core condensation. While it is apparent that loss of zinc coordination results in some reduction of genomic RNA encapsidationand ZF2 appears to function in this capacity more so than ZF1the observations with the ZF1+2 mutant suggest that it is the linker region and basic charge of NC that influence genomic RNA encapsidation the greatest.

View larger version (30K): [in this window] [in a new window]   FIG. 3.   Analysis of genomic RNA incorporation into WT and NC mutant SIV(Mne) virions. Virus was pelleted from clarified supernatants of transfected 293T cells, and viral RNA was isolated by phenol-chloroform extraction-ethanol precipitation. Samples, equalized for pelletable RT activity, were probed with the pRB85 vector containing full-length SIV(Mne) that had been linearized with XhoI and then labeled with 32P (23). The mock control lane contains an RNA preparation from carrier DNA-transfected 293T supernatants. Full-length genomic RNA (9.6 kbp) indicated on the left was quantitated by phosphorimaging (PI) and quantitative real-time RT-PCR (PCR). Positions of RNA markers are shown on the right. ND, not determined.

Polyprotein processing. To determine if improper or incomplete proteolytic cleavage of the Gag, Gag-Pol, and Env polyproteins could be associated with the observed morphological differences, WT and mutant viruses were examined by Western analysis and fractionation by RP-HPLC. Because this laboratory focuses on the development of novel vaccines against HIV and SIV, a plasmid vector (pS8) was designed for use as a naked DNA vaccine. pS8 consists of the full-length SIV(Mne) coding sequence, but the LTRs have been partially deleted (see Materials and Methods) for increased in vivo safety purposes. Briefly, the R and U5 regions in the 3' LTR have been replaced by an exogenous SV40 polyadenylation sequence to prevent first-strand transfer during viral DNA synthesis, yet provide the polyadenylation necessary for proper viral gene expression. The LTR deletions, in combination with the specific NC mutations, render these viruses replication incompetent (data not shown). Because of the changes in LTR structure in the pS8 expression vector, it was necessary to demonstrate that particle assembly and polyprotein processing were not affected. Viral proteins of the WT and an example of one of the NC mutants, ZF2, were compared in the context of the pS8 vector or a complete proviral vector (Fig. 4). By gp120^SU, p28^CA, and p8^NC Western analysis, no significant differences in polyprotein processing were observed if the LTR sequences were altered as described for the pS8 vector. While these LTR modifications decrease infectivity at least 3 logs in the clone containing WT NC (data not shown), Fig. 4 demonstrates that the processing differences observed were clearly the result of the mutation present in the NC domain and were not due to the LTR alterations.

View larger version (46K): [in this window] [in a new window]   FIG. 4.   Immunoblot comparison of viral protein processing with complete or deleted LTR ends. WT or ZF2 mutant SIV(Mne) virus was produced by transfection of 293T cells with plasmids containing complete LTRs (provirus [PV]) or deleted LTRs (S8) as described in Materials and Methods. Virus pelleted from clarified supernatants 72 h after transfection was resuspended in SDS-PAGE lysis buffer and then subjected to Western blot analysis. Protein samples were adjusted to equivalent RT activity before SDS-PAGE. Blots were initially incubated with mouse monoclonal p28CA antibody (A) and then stripped and reprobed concomitantly with rabbit polyclonal gp120 and p8NC antibodies (B). Molecular markers are indicated on the left, and immunodetected proteins are indicated on the right.

View larger version (58K): [in this window] [in a new window]   FIG. 5.   Immunoblot analysis of viral protein processing in WT and NC mutant SIV(Mne). Virus was pelleted as described in Materials and Methods and resuspended in SDS-PAGE lysis buffer. Samples of equivalent RT activity (3.8 × 105 cpm by exogenous template RT assay) were heat denatured, subjected to 4 to 20% SDS-PAGE, and transferred to Immobilon-P (Millipore Corp.). Membranes were probed with mouse monoclonal p28CA antibodies and visualized with the Amersham ECL system (A). Membranes were stripped and reprobed with rabbit anti-gp120 and p8NC concomitantly and visualized with ECL as well (B). Molecular mass markers are shown on the left, and immunolabeled proteins are shown on the right.

View larger version (23K): [in this window] [in a new window]   FIG. 6.   RP-HPLC analysis of WT and NC mutant SIV(Mne). Virus was pelleted from 30 ml of clarified culture supernatant of 293T cells transfected with WT or NC mutant expression plasmids and then resuspended in PBS in the presence of 8 M guanidine-HCl, 10% acetonitrile, and 10 mM dithiothreitol. Equivalent volumes were loaded on a microbore RP-HPLC column and eluted as described in Materials and Methods. Chromatographs are grouped according to the ZF mutation for ease of comparison. The acetonitrile concentration is superimposed over the WT chromatograph. Viral protein peaks were determined by Coomassie staining, immunoblotting, and protein sequencing analyses of eluted fractions and are labeled accordingly: NC, nucleocapsid protein; p1/p2, spacer protein 1 and spacer protein 2 of Gag polyprotein; MA, matrix; and CA, capsid. An asterisk indicates contamination of the p6Gag peak with an unidentified cellular protein in the ZF1+2 chromatograph.

View larger version (26K): [in this window] [in a new window]   FIG. 7.   SDS-PAGE and immunoblot analysis of incomplete Gag polyprotein processing. (A) A section of the chromatograph from Fig. 6 (ZF2) showing an example of the NC mutant, ZF2, RP-HPLC, and numbered protein fractions that were fractionated by SDS-PAGE and visualized with Coomassie stain. Four distinct proteins (I to IV) were noted in peaks more prominent in the NC mutant than in the WT SIV(Mne) chromatographs. (B) Immunoblot analyses of fractions for anti-p28CA, anti-p2Gag, anti-p8NC, and anti-p6Gag antibodies. Molecular markers are shown on the left; the positions of labeled proteins I to IV are indicated on the right. Note that the p2Gag antibody only recognizes a cleaved or C-terminal epitope. (C) Identity of incompletely processed Gag proteins I to IV based on Coomassie, immunoblot, and partial amino acid sequence analyses. The same Gag polyproteins I to IV were observed in all NC mutants, only in different amounts.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

While previous studies of SIV have focused on genomic RNA encapsidation and general viral protein production and processing in relation to NC ZFs, we show for the first time that subtle differences in virion morphology can be attributed to the Zn²⁺ coordination of each finger. Specifically, proper core condensation is severely disrupted by mutation of ZF2, whereas mutation of ZF1 disrupts this process to a lesser extent (Table 1 and Fig. 2). We utilized SIV(Mne) NC mutants (Fig. 1) that inhibit zinc binding by either changing specific zinc-coordinating Cys residues to Ser or deleting the first four amino acids in each or both fingers, including the basic linker sequence. Our results support previous reports that the two finger arrays of retroviral NC proteins exert different activities, yet do not function independently (9, 13, 25, 35). However, our analyses of SIV NC ZF function differ from those reported for other retroviruses, such as HIV, murine leukemia virus, and Rous sarcoma virus (16, 41, 44, 45). In agreement with previous reports (12, 40), we found that genomic RNA incorporation (Fig. 3) is less dependent on Zn²⁺ coordination, but is more dependent on the basic charge of NC. However, contrary to observations with HIV-1, we found that it is ZF2 that affects RNA encapsidation more than ZF1.

The results from the RP-HPLC analyses (Fig. 6) support those of the immunoblot assays (Fig. 5 and 7): complete proteolytic cleavage of Gag is associated with proper core condensation. The RP-HPLC results go two steps further to indicate that (i) the removal of the Zn²⁺ from the NC protein results in a reduction in processed NC protein when compared to all possible Gag cleavage products in each mutant (Fig. 6, compare relative peak heights of Gag cleavage products containing NC with peaks of other cleavage products that do not contain NC) and (ii) the unprocessed Gag polyproteins occur in four major forms. Gag cleavage is an ordered process that is necessary to produce morphologically mature virions (17, 38, 50). Incomplete processing of Gag due to conformation, protein-protein, and/or protein-nucleic acid interactions resulting from the loss of Zn²⁺ in our NC ZF mutants may be responsible for the inability of virion cores to properly condense as has been suggested previously in HIV studies (28, 32, 33, 47). To our knowledge, Gag-Gag interaction domains (I domains) (10) have not been studied in SIV. While it is possible that the mutations discussed here may alter protein conformation, the number of basic residues of the NC in the ZF1+2 CS mutant is the same as that in the WT NC, yet this mutant produces a significant amount of partially processed and uncleaved Gag with some full-length genomic RNA encapsidation. Therefore, effects on the I domains would have to be due to altered conformation of the protein. An altered conformation could result from (i) a "loose" folding of Gag in the NC domain due to the lack of Zn²⁺ coordination of the ZFs or (ii) a "reconfigured" folding of the NC domain due to (a) possible Zn²⁺ coordination between the remaining His and Cys residues in the deleted ZF and those in the complete ZF and/or (b) possible disulfide bond formation in the single-finger deletion mutants. Disulfide bonds do indeed form when the Zn²⁺ ions are ejected from the ZF of WT NC in HIV and SIV (2, 43).

NC in an altered conformation resulting from the loss of the Zn²⁺ in either or both fingers may be open to extensive proteolytic cleavage, most likely from the viral protease. The significant loss of mutant NC protein observed in the RP-HPLC (Fig. 6 and Table 2) suggests this scenario. Previous studies have demonstrated protease-specific NC cleavage in HIV-1 and equine infectious anemia virus (42, 46) and that the removal of the Zn²⁺ is necessary for this cleavage (51). The destruction of NC early in virus formation would surely have a profound effect on downstream events in the life cycle that are known to require NC, including RNA stability, reverse transcription, and, possibly, integration of the viral genome.

NC protein is present in isolated mature conical HIV-1 cores (49) and has been shown to be required for the condensation of viral protein with nucleic acid to a conical core phenotype in in vitro assembly systems (11, 20). Our results show that genomic RNA incorporation in ZF2 CS and ZF2 is not severely reduced, but relative levels of unprocessed Gag are much higher than those in the ZF1 CS and ZF1 mutants (Fig. 3). Yet there is a striking difference in the number of particles with properly condensed cores between virions with ZF1 and ZF2 mutations, suggesting the differences in core morphology are dictated more by Gag cleavage than full-length genomic RNA encapsidation. However, these associations cannot be stated independently, because at least one other factor, the basic charge of NC, is known to affect virion morphology. Although ZF1+2 virions contained more fully processed Gag, it contained very little detectable full-length genomic RNA by Northern blotting (23), whereas the ZF1+2 CS mutant produced a profile of Gag processing similar to those of ZF2 CS and ZF2 and also had severely reduced levels of virion-associated full-length genomic RNA. Cimarelli et al. (12) demonstrated the relationship between the basic charge of NC, RNA incorporation, and virion morphology in HIV-1. Their HIV-1 mutants BR and M1-2/BR contained little and no viral or cellular RNA, respectively, and produced irregular large and small virions with multiple rod-shaped cores similar to those produced by the ZF1+2 mutant. The requirement for nucleic acid in virion core morphology suggests that mutants lacking viral RNA may substitute nonspecific cellular RNA to achieve a condensed core (5).

Previous studies have only demonstrated that mutations in NC protein inhibit virion maturation (6, 7, 23, 35, 45). The negative effect of the ZF mutations on complete Gag processing may suggest an upstream role for NC in the complete process of virion maturation, since Gag mutations outside of the NC domain that result in improper or incomplete proteolytic processing also affect virion core condensation (1, 15, 22, 34, 47, 50). Indeed, there is a correlation between the presence of p30^Gag (Fig. 5A) and an increase in immature virions (Table 1). Whether the NC protein in this process is active (Zn²⁺ transfer or release) or passive (structural conformation or dimerization) remains to be elucidated.