Importance of the N terminus of rous sarcoma virus protease for structure and enzymatic function.

All retrovirus proteases (PRs) are homodimers, and dimerization is essential for enzymatic function. The dimer is held together largely by a short four-stranded antiparallel beta sheet composed of the four or five N-terminal amino acid residues and a similar stretch of residues from the C terminus. We have found that the enzymatic and structural properties of Rous sarcoma virus (RSV) PR are exquisitely sensitive to mutations at the N terminus. Deletion of one or three residues, addition of one residue, or substitution of alanine for the N-terminal leucine reduced enzymatic activity on peptide and protein substrates 100- to 1,000-fold. The purified mutant proteins remained monomeric up to a concentration of about 2 mg/ml, as determined by dynamic light scattering. At higher concentrations, dimerization was observed, but the dimer lacked or was deficient in enzymatic activity and thus was inferred to be structurally distinct from a wild-type dimer. The mutant protein lacking three N-terminal residues (DeltaLAM), a form of PR occurring naturally in virions, was examined by nuclear magnetic resonance spectroscopy and found to be folded at concentrations where it was monomeric. This result stands in contrast to the report that a similarly engineered monomeric PR of human immunodeficiency virus type 1 is unstructured. Heteronuclear single quantum coherence spectra of the mutant at concentrations where either monomers or dimers prevail were nearly identical. However, these spectra differed from that of the dimeric wild-type RSV PR. These results imply that the chemical environment of many of the amide protons differed and thus that the three-dimensional structure of the DeltaLAM PR mutant is different from that of the wild-type PR. The structure of this mutant protein may serve as a model for the structure of the PR domain of the Gag polyprotein and may thus give clues to the initiation of proteolytic maturation in retroviruses.

PR domain of rous sarcoma virus Gag causes an assembly/budding defect in insect cells.

While baculovirus expression of Gag proteins from numerous retroviruses has led reliably to production of virus-like particles (VLPs), we observed that expression of Rous sarcoma virus Gag failed to produce VLPs. Transmission and scanning electron microscopy analysis revealed that the Gag protein reached the plasma membrane but was unable to correctly form particles. Addition of a myristylation signal had no effect on the budding defect, but deletion of the PR domain of Gag restored normal budding. The resulting VLPs were morphologically distinct from human immunodeficiency virus type 1 VLPs expressed in parallel.

Characterization of Rous sarcoma virus Gag particles assembled in vitro.

Purified retrovirus Gag proteins or Gag protein fragments are able to assemble into virus-like particles (VLPs) in vitro in the presence of RNA. We have examined the role of nucleic acid and of the NC domain in assembly of VLPs from a Rous sarcoma virus (RSV) Gag protein and have characterized these VLPs using transmission electron microscopy (TEM), scanning TEM (STEM), and cryoelectron microscopy (cryo-EM). RNAs of diverse sizes, single-stranded DNA oligonucleotides as small as 22 nucleotides, double-stranded DNA, and heparin all promoted efficient assembly. The percentages of nucleic acid by mass, in the VLPs varied from 5 to 8%. The mean mass of VLPs, as determined by STEM, was 6.5 x 10(7) Da for both RNA-containing and DNA oligonucleotide-containing particles, corresponding to a stoichiometry of about 1,200 protein molecules per VLP, slightly lower than the 1,500 Gag molecules estimated previously for infectious RSV. By cryo-EM, the VLPs showed the characteristic morphology of immature retroviruses, with discernible regions of high density corresponding to the two domains of the CA protein. In spherically averaged density distributions, the mean radial distance to the density corresponding to the C-terminal domain of CA was 33 nm, considerably smaller than that of equivalent human immunodeficiency virus type 1 particles. Deletions of the distal portion of NC, including the second Zn-binding motif, had little effect on assembly, but deletions including the charged residues between the two Zn-binding motifs abrogated assembly. Mutation of the cysteine and histidine residues in the first Zn-binding motif to alanine did not affect assembly, but mutation of the basic residues between the two Zn-binding motifs, or of the basic residues in the N-terminal portion of NC, abrogated assembly. Together, these findings establish VLPs as a good model for immature virions and establish a foundation for dissection of the interactions that lead to assembly.

The organization of mature Rous sarcoma virus as studied by cryoelectron microscopy.

We have studied the organization of mature infectious Rous sarcoma virus (RSV), suspended in vitreous ice, using transmission electron microscopy. The enveloped virions are spherical in shape, have a mean diameter of 127 nm, and vary significantly in size. Image processing reveals the presence of the viral matrix protein underlying the lipid bilayer and the viral envelope proteins external to the lipid bilayer. In the interior of the virus, the characteristic mature retroviral core is clearly imaged. In contrast to lentiviruses, such as human immunodeficiency virus, the core of RSV is essentially isometric. The capsid, or external shell of the core, has a faceted, almost polygonal appearance in electron micrographs, but many capsids also exhibit continuous surface curvature. Cores are not uniform in size or shape. Serrations observed along the projected faces of the core suggest a repetitive molecular structure. Some isolated cores were observed in the sample, confirming that cores are at least transiently stable in the absence of the viral envelope. Using an approach grounded in geometric probability, we estimate the size of the viral core from the projection data. We show that the size of the core is not tightly controlled and that core size and virion size are positively correlated. From estimates of RNA packing density we conclude that either the RNA within the core is loosely packed or, more probably, that it does not fill the core.

Role of the Rous sarcoma virus p10 domain in shape determination of gag virus-like particles assembled in vitro and within Escherichia coli.

Purified retrovirus Gag proteins can assemble in vitro into virus-like particles (VLPs) in the presence of RNA. It was shown previously that a Rous sarcoma virus Gag protein missing only the protease domain forms spherical particles resembling immature virions lacking a membrane but that a similar protein missing the p10 domain forms tubular particles. Thus, p10 plays a role in spherical particle formation. To further study this shape-determining function, we dissected the p10 domain by mutagenesis and examined VLPs assembled within Escherichia coli or assembled in vitro from purified proteins. The results identified a minimal contiguous segment of 25 amino acid residues at the C terminus of p10 that is sufficient to restore efficient spherical assembly to a p10 deletion mutant. Random and site-directed mutations were introduced into this segment of polypeptide, and the shapes of particles formed in E. coli were examined in crude extracts by electron microscopy. Three phenotypes were observed: tubular morphology, spherical morphology, or no regular structure. While the particle morphology visualized in crude extracts generally was the same as that visualized for purified proteins, some tubular mutants scored as spherical when tested as purified proteins, suggesting that a cellular factor may also play a role in shape determination. We also examined the assembly properties of smaller Gag proteins consisting of the capsid protein-nucleocapsid protein (CA-NC) domains with short N-terminal extensions or deletions. Addition of one or three residues allowed CA-NC to form spheres instead of tubes in vitro, but the efficiency of assembly was extremely low. Deletion of the N-terminal residue(s) abrogated assembly. Taken together, these results imply that the N terminus of CA and the adjacent upstream 25 residues play an important role in the polymerization of the Gag protein.

Structure and self-association of the Rous sarcoma virus capsid protein.

BACKGROUND: The capsid protein (CA) of retroviruses, such as Rous sarcoma virus (RSV), consists of two independently folded domains. CA functions as part of a polyprotein during particle assembly and budding and, in addition, forms a shell encapsidating the genomic RNA in the mature, infectious virus. RESULTS: The structures of the N- and C-terminal domains of RSV CA have been determined by X-ray crystallography and solution nuclear magnetic resonance (NMR) spectroscopy, respectively. The N-terminal domain comprises seven alpha helices and a short beta hairpin at the N terminus. The N-terminal domain associates through a small, tightly packed, twofold symmetric interface within the crystal, different from those previously described for other retroviral CAs. The C-terminal domain is a compact bundle of four alpha helices, although the last few residues are disordered. In dilute solution, RSV CA is predominantly monomeric. We show, however, using electron microscopy, that intact RSV CA can assemble in vitro to form both tubular structures constructed from toroidal oligomers and planar monolayers. Both modes of assembly occur under similar solution conditions, and both sheets and tubes exhibit long-range order. CONCLUSIONS: The tertiary structure of CA is conserved across the major retroviral genera, yet sequence variations are sufficient to cause change in associative behavior. CA forms the exterior shell of the viral core in all mature retroviruses. However, the core morphology differs between viruses. Consistent with this observation, we find that the capsid proteins of RSV and human immunodeficiency virus type 1 exhibit different associative behavior in dilute solution and assemble in vitro into different structures.

Expression of the Naegleria intron endonuclease is dependent on a functional group I self-cleaving ribozyme.

NaSSU1 is a complex nuclear group I intron found in several species of Naegleria, consisting of a large self-splicing group I ribozyme (NaGIR2), which itself is interrupted by a small, group I-like ribozyme (NaGIR1) and an open reading frame (ORF) coding for a homing endonuclease. The GIR1 ribozyme cleaves in vitro transcripts of NaSSU1 at two internal processing sites about 400 nt downstream of the 5' end of the intron, proximal to the endonuclease ORF. Here we demonstrate that self-cleavage of the excised intron also occurs in vivo in Naegleria gruberi, generating an ORF-containing RNA that possesses a short leader with a sequence element likely to be involved in gene expression. To assess the functional significance of self-cleavage, we constructed a genetic system in Saccharomyces cerevisiae. First, a mutant yeast strain was selected with a mutation in all the rRNA genes, rendering the rDNA resistant to cleavage by the Naegleria endonuclease. Active endonuclease, which is otherwise lethal, could be expressed readily in these cells. Endonuclease activity also could be detected in extracts of yeast harboring plasmids in which the endonuclease ORF was embedded in its native context in the intron. Analysis of the RNA from these yeast cells showed that the excised intron RNA was processed as in N. gruberi. A mutant intron constructed to prevent self-cleavage of the RNA failed to express endonuclease activity. These results support the hypothesis that the NaGIR1-catalyzed self-cleavage of the intron RNA is a key event in expression of the endonuclease.

Functional alpha-fragment of beta-galactosidase can be expressed from the mobile group I intron PpLSU3 embedded in yeast pre-ribosomal RNA derived from the chromosomal rDNA locus.

PpLSU3, a mobile group I intron found in the ribo-somal RNA genes of Physarum polycephalum, encodes the I-PpoI homing endonuclease. This enzyme represents one of the rare cases in nature where a protein is expressed from an RNA polymerase I transcript. Our previous results showed that the full length intron, but not a further processed species, is the messenger for I-PpoI, implying a role of the untranslated region (UTR) in gene expression. To study the function of the 3'-UTR in expression of the endonuclease and in splicing of the intron, we replaced the I-PpoI gene in PpLSU3 with the gene for the alpha-fragment of Escherichia coli beta-galactosidase, and then integrated this chimeric intron into all the chromosomal rDNA repeats of yeast. The resulting cells synthesized functional alpha-fragment, as evidenced by a complementation assay analogous to that used in E.coli. The beta-galactosidase activity thus provides an unusual and potentially valuable readout for Pol I transcription from chromosomal rDNA. This is the first example in which a eucaryotic homing endonuclease gene has been successfully replaced by a heterologous gene. Using deletion mutagenesis and a novel randomization approach with the alpha-fragment as a reporter, we found that a small segment of the 3'-UTR dramatically influences both splicing and protein expression.

Mass determination of rous sarcoma virus virions by scanning transmission electron microscopy.

The internal structural protein of retroviruses, Gag, comprises most of the mass of the virion, and Gag itself can give rise to virus-like particles when expressed in appropriate cells. Previously the stoichiometry of Gag in virions was inferred from indirect measurements carried out 2 decades ago. We now have directly determined the masses of individual particles of the prototypic avian retrovirus, Rous sarcoma virus (RSV), by using scanning transmission electron microscopy. In this technique, the number of scattered electrons in the dark-field image integrated over an individual freeze-dried virus particle on a grid is directly proportional to its mass. The RSV virions had a mean mass of 2.5 x 10(8) Da, corresponding to about 1,500 Gag molecules per virion. The population of virions was not homogeneous, with about one-third to two-thirds of the virions deviating from the mean by more than 10% of the mass in two respective preparations. The mean masses for virions carrying genomes of 7.4 or 9.3 kb were indistinguishable, suggesting that mass variability is not due to differences in RNA incorporation.

I-PpoI, the endonuclease encoded by the group I intron PpLSU3, is expressed from an RNA polymerase I transcript.

PpLSU3, a mobile group I intron in the rRNA genes of Physarum polycephalum, also can home into yeast chromosomal ribosomal DNA (rDNA) (D. E. Muscarella and V. M. Vogt, Mol. Cell. Biol. 13:1023-1033, 1993). By integrating PpLSU3 into the rDNA copies of a yeast strain temperature sensitive for RNA polymerase I, we have shown that the I-PpoI homing endonuclease encoded by PpLSU3 is expressed from an RNA polymerase I transcript. We have also developed a method to integrate mutant forms of PpLSU3 as well as the Tetrahymena intron TtLSU1 into rDNA, by expressing I-PpoI in trans. Analysis of I-PpoI expression levels in these mutants, along with subcellular fractionation of intron RNA, strongly suggests that the full-length excised intron RNA, but not RNAs that are further cleaved, serves as or gives rise to the mRNA.

In vivo selection of Rous sarcoma virus mutants with randomized sequences in the packaging signal.

Retrovirus genomes contain a sequence at the 5' end which directs their packaging into virions. In Rous sarcoma virus, previous studies have identified important segments of the packaging signal, Psi, and support elements of a secondary-structure prediction. To further characterize this sequence, we used an in vivo selection strategy to test large collections of mutants. We generated pools of full-length viral DNA molecules with short stretches of random sequence in Psi and transfected each pool into avian cells. Resulting infectious virus was allowed to spread by multiple passages, so that sequences could compete and the best could be selected. This method provides information on the kinds of sequences allowed, as well as those that are most fit. Several predicted stem-loop structures in Psi were tested. A stem at the base of element O3 was highly favored; only sequences which maintained base pairing were selected. Two other stems, at the base and in the middle of element L3, were not conserved: neither base pairing nor sequence was maintained. A single mutation, G213U, was seen upstream of the randomized region in all selected L3 stem mutants; we interpret this to mean that it compensates for the defects in L3. Randomized mutations adjacent to G213 maintained the wild-type base composition but not its sequence. The kissing-loop sequence at end of L3, postulated to function in genome dimerization, was not required for infectivity but was selected for over time. Finally, a deletion of L3 was constructed and found to be poorly infectious.

Genetic determinants of Rous sarcoma virus particle size.

The Gag proteins of retroviruses are the only viral products required for the release of membrane-enclosed particles by budding from the host cell. Particles released when these proteins are expressed alone are identical to authentic virions in their rates of budding, proteolytic processing, and core morphology, as well as density and size. We have previously mapped three very small, modular regions of the Rous sarcoma virus (RSV) Gag protein that are necessary for budding. These assembly domains constitute only 20% of RSV Gag, and alterations within them block or severely impair particle formation. Regions outside of these domains can be deleted without any effect on the density of the particles that are released. However, since density and size are independent parameters for retroviral particles, we employed rate-zonal gradients and electron microscopy in an exhaustive study of mutants lacking the various dispensable segments of Gag to determine which regions would be required to constrain or define the particle dimensions. The only sequence found to be absolutely critical for determining particle size was that of the initial capsid cleavage product, CA-SP, which contains all of the CA sequence plus the spacer peptides located between CA and NC. Some regions of CA-SP appear to be more important than others. In particular, the major homology region does not contribute to defining particle size. Further evidence for interactions among CA-SP domains was obtained from genetic complementation experiments using mutant deltaNC, which lacks the RNA interaction domains in the NC sequence but retains a complete CA-SP sequence. This mutant produces low-density particles heterogeneous in size. It was rescued into particles of normal size and density, but only when the complementing Gag molecules contained the complete CA-SP sequence. We conclude that CA-SP functions during budding in a manner that is independent of the other assembly domains.

Crystals of Rous sarcoma virus capsid protein show a helical arrangement of protein subunits.

Crystals of Rous sarcoma virus (RSV) capsid protein diffract X rays to 3.5 A resolution and belong to the monoclinic space group C2 with unit cell parameters a = 374.4 A, b = 128.1 A, c = 200.2 A, and beta = 121.8 degrees. One asymmetric unit of the crystal may contain between 28 and 35 molecules, based on reasonable crystal density assumptions. A self-rotation function and Patterson synthesis suggest that RSV capsid protein crystallizes as a helical array. The determinants of the viral particle morphology are not encoded in the capsid alone. The assembly of a helical array in the crystal reflects the absence of any conformational switching. However, it is expected that the subunit interactions seen in the crystal will be preferred and will relate to those found in the immature or mature virion.

Cryo-electron microscopy reveals ordered domains in the immature HIV-1 particle.

BACKGROUND: Human immunodeficiency virus type 1 (HIV-1) is the causative agent of AIDS and the subject of intense study. The immature HIV-1 particle is traditionally described as having a well ordered, icosahedral structure made up of uncleaved Gag protein surrounded by a lipid bilayer containing envelope proteins. Expression of the Gag protein in eukaryotic cells leads to the budding of membranous virus-like particles (VLPs). RESULTS: We have used cryo-electron microscopy of VLPs from insect cells and lightly fixed, immature HIV-1 particles from human lymphocytes to determine their organization. Both types of particle were heterogeneous in size, varying in diameter from 1200-2600 A. Larger particles appeared to be broken into semi-spherical sectors, each having a radius of curvature of approximately 750 A. No evidence of icosahedral symmetry was found, but local order was evidenced by small arrays of Gag protein that formed facets within the curved sectors. A consistent 270 A radial density was seen, which included a 70 A wide low density feature corresponding to the carboxy-terminal portion of the membrane attached matrix protein and the amino-terminal portion of the capsid protein. CONCLUSIONS: Immature HIV-1 particles and VLPs both have a multi-sector structure characterized, not by an icosahedral organization, but by local order in which the structures of the matrix and capsid regions of Gag change upon cleavage. We propose a model in which lateral interactions between Gag protein molecules yields arrays that are organized into sectors for budding by RNA.

Naegleria nucleolar introns contain two group I ribozymes with different functions in RNA splicing and processing.

We have characterized the structural organization and catalytic properties of the large nucleolar group I introns (NaSSU1) of the different Naegleria species N. jamiesoni, N. andersoni, N. italica, and N. gruberi. NaSSU1 consists of three distinct RNA domains: an open reading frame encoding a homing-type endonuclease, and a small group I ribozyme (NaGIR1) inserted into the P6 loop of a second group I ribozyme (NaGIR2). The two ribozymes have different functions in RNA splicing and processing. NaGIR1 is an unusual self-cleaving group I ribozyme responsible for intron processing at two internal sites (IPS1 and IPS2), both close to the 5' end of the open reading frame. This processing is hypothesized to lead to formation of a messenger RNA for the endonuclease. Structurally, NaGIR2 is a typical group IC1 ribozyme, catalyzing intron excision and exon ligation reactions. NaGIR2 is responsible for circularization of the excised intron, a reaction that generates full-length RNA circles of wild-type intron. Although it is only distantly related in primary sequence, NaSSU1 RNA has a predicted organization and function very similar to that of the mobile group I intron DiSSU1 of Didymium, the only other group I intron known to encode two ribozymes. We propose that these twin-ribozyme introns define a distinct category of group I introns with a conserved structural organization and function.

In vitro assembly of virus-like particles with Rous sarcoma virus Gag deletion mutants: identification of the p10 domain as a morphological determinant in the formation of spherical particles.

Retroviruses are unusual in that expression of a single protein, Gag, leads to budding of virus-like particles into the extracellular space. We have developed conditions under which virus-like particles are formed spontaneously in vitro from fragments of Rous sarcoma virus (RSV) Gag protein purified after expression in Escherichia coli. The CA-NC fragment of Gag was shown previously to assemble into hollow cylinders (S. Campbell and V. M. Vogt, J. Virol. 69:6487-6497, 1995). We have now extended these studies to larger Gag proteins. In every case examined, assembly into regular structures required RNA. A nearly full-length Gag missing only the C-terminal PR domain, as well as similar proteins missing in addition the N-terminal half of MA, the C-terminal half of MA, the entire MA sequence, or the entire p2 sequence, all assembled into spherical particles resembling RSV in size. By contrast, proteins missing p10 assembled into cylindrical particles like those formed by CA-NC alone. Thin section electron microscopy showed that each of these Gag proteins formed in the expressing E. coli cells particles similar in shape to those seen in vitro. We conclude from these results that neither the sequences required for membrane binding in vivo, near the N terminus of Gag, nor the sequences required for a late step in budding, in the p2 portion of Gag, are essential for formation of virus-like particles in this system. Furthermore, we postulate the existence of a shape-determining sequence in p10, which provides or facilitates interactions required for the growing particle to be constrained to a spherical shape.

Analysis of cleavage site mutations between the NC and PR Gag domains of Rous sarcoma virus.

In retroviruses, the viral protease (PR) is released as a mature protein by cleavage of Gag, Gag-Pro, or Gag-Pro-Pol precursor polypeptides. In avian sarcoma and leukemia viruses (ASLV), PR forms the C-terminal domain of Gag. Based on the properties of a mutation (cs22) in the cleavage site between the upstream NC domain and the PR domain, the proteolytic liberation of PR previously was inferred to be essential for processing of Gag and Pol proteins. To study this process in more detail, we have analyzed the effects that several mutations at the NC-PR cleavage site have on proteolytic processing in virus-like particles expressed in COS and quail cells. Mutant Gag proteins carrying the same mutations also were synthesized in vitro and tested for processing with purified PR. In both types of studies, N-terminal sequencing of the liberated PR domain was carried out to exactly identify the site of cleavage. Finally, synthetic peptides corresponding to the mutant proteins were assessed for the ability to act as substrates for PR. The results were all consistent and led to the following conclusions. (i) In vivo, if normal processing between NC and PR is prevented by mutations, limited cleavage occurs at a previously unrecognized alternative site three amino acids downstream, i.e., in PR. This N-terminally truncated PR is inactive as an enzyme, as inferred from the global processing defect in cs22 and a similar mutant. (ii) In Gag proteins translated in vitro, purified PR cleaves this alternative site as rapidly as it does the wild-type site. (iii) Contrary to previously accepted rules describing retroviral cleavage sites, an isoleucine residue placed at the P1 position of the NC-PR cleavage site does not hinder normal processing. (iv) A proline residue placed at the P2 position in this cleavage site blocks normal processing.