NMR structure of the HIV-1 nucleocapsid protein bound to stem-loop SL2 of the psi-RNA packaging signal. Implications for genome recognition

The RNA genome of the human immunodeficiency virus type-1 (HIV-1) contains a approximately 120 nucleotide Psi-packaging signal that is recognized by the nucleocapsid (NC) domain of the Gag polyprotein during virus assembly. The Psi-site contains four stem-loops (SL1-SL4) that possess overlapping and possibly redundant functions. The present studies demonstrate that the 19 residue SL2 stem-loop binds NC with affinity (K(d)=110(+/-50) nM) similar to that observed for NC binding to SL3 (K(d)=170(+/-65) nM) and tighter than expected on the basis of earlier work, suggesting that NC-SL2 interactions probably play a direct role in the specific recognition and packaging of the full-length, unspliced genome. The structure of the NC-SL2 complex was determined by heteronuclear NMR methods using (15)N,(13)C-isotopically labeled NC protein and SL2 RNA. The N and C-terminal "zinc knuckles" (Cys-X(2)-Cys-X(4)-His-X(4)-Cys; X=variable amino acid) of HIV-1 NC bind to exposed guanosine bases G9 and G11, respectively, of the G8-G9-U10-G11 tetraloop, and residues Lys3-Lys11 of the N-terminal tail forms a 3(10) helix that packs against the proximal zinc knuckle and interacts with the RNA stem. These structural features are similar to those observed previously in the NMR structure of NC bound to SL3. Other features of the complex are substantially different. In particular, the N-terminal zinc knuckle interacts with an A-U-A base triple platform in the minor groove of the SL2 RNA stem, but binds to the major groove of SL3. In addition, the relative orientations of the N and C-terminal zinc knuckles differ in the NC-SL2 and NC-SL3 complexes, and the side-chain of Phe6 makes minor groove hydrophobic contacts with G11 in the NC-SL2 complex but does not interact with RNA in the NC-SL3 complex. Finally, the N-terminal helix of NC interacts with the phosphodiester backbone of the SL2 RNA stem mainly via electrostatic interactions, but does not bind in the major groove or make specific H-bonding contacts as observed in the NC-SL3 structure. These findings demonstrate that NC binds in an adaptive manner to SL2 and SL3 via different subsets of inter and intra-molecular interactions, and support a genome recognition/packaging mechanism that involves interactions of two or more NC domains of assembling HIV-1 Gag molecules with multiple Psi-site stem-loop packaging elements during the early stages of retrovirus assembly.     

Structure of SL4 RNA from the HIV-1 packaging signal.

The NMR-based structure is described for an RNA model of stem-loop 4 (SL4) from the HIV-1 major packaging domain. The GAGA tetraloop adopts a conformation similar to the classic GNRA form, although there are differences in the details. The type II tandem G.U pairs have a combination of wobble and bifurcated hydrogen bonds where the uracil 2-carbonyl oxygen is hydrogen-bonded to both G,H1 and G,H2. There is the likelihood of a Na(+) ion coordinated to the four carbonyl oxygens in the major groove for these G.U pairs and perhaps to the N7 lone pairs of the G bases as well. A continuous stack of five bases extends over nearly the whole length of the stem to the base of the loop in the RNA 16mer: C15/U14/G13/G5/C6. There is no evidence for a terminal G.A pair; instead, G1 appears quite unrestrained, and A16 stacks on both C15 and G2. Residues G2 through G5 exhibit broadened resonances, especially G3 and U4, suggesting enhanced mobility for the 5'-side of the stem. The structure shows G2/G3/U4 stacking along the same strand, nearly isolated from interaction with the other bases. This is probably an important factor in the signal broadening and apparent mobility of these residues and the low stability of the 16mer hairpin against thermal denaturation.     

Identification of a high affinity nucleocapsid protein binding element within the Moloney murine leukemia virus Psi-RNA packaging signal: implications for genome recognition.

Murine leukemia virus (MLV) is currently the most widely used gene delivery system in gene therapy trials. The simple retrovirus packages two copies of its RNA genome by a mechanism that involves interactions between the nucleocapsid (NC) domain of a virally-encoded Gag polyprotein and a segment of the RNA genome located just upstream of the Gag initiation codon, known as the Psi-site. Previous studies indicated that the MLV Psi-site contains three stem loops (SLB-SLD), and that stem loops SLC and SLD play prominent roles in packaging. We have developed a method for the preparation and purification of large quantities of recombinant Moloney MLV NC protein, and have studied its interactions with a series of oligoribonucleotides that contain one or more of the Psi-RNA stem loops. At RNA concentrations above approximately 0.3 mM, isolated stem loop SLB forms a duplex and stem loops SL-C and SL-D form kissing complexes, as expected from previous studies. However, neither the monomeric nor the dimeric forms of these isolated stem loops binds NC with significant affinity. Longer constructs containing two stem loops (SL-BC and SL-CD) also exhibit low affinities for NC. However, NC binds with high affinity and stoichiometrically to both the monomeric and dimeric forms of an RNA construct that contains all three stem loops (SL-BCD; K(d)=132(+/-55) nM). Titration of SL-BCD with NC also shifts monomer-dimer equilibrium toward the dimer. Mutagenesis experiments demonstrate that the conserved GACG tetraloops of stem loops C and D do not influence the monomer-dimer equilibrium of SL-BCD, that the tetraloop of stem loop B does not participate directly in NC binding, and that the tetraloops of stem loops C and D probably also do not bind to NC. These surprising results differ considerably from those observed for HIV-1, where NC binds to individual stem loops with high affinity via interactions with exposed residues of the tetraloops. The present results indicate that MLV NC binds to a pocket or surface that only exists in the presence of all three stem loops.     

NMR structure of stem-loop SL2 of the HIV-1 psi RNA packaging signal reveals a novel A-U-A base-triple platform

The genome of the human immunodeficiency virus type-1 (HIV-1) contains a stretch of approximately 120 nucleotides known as the psi-site that is essential for RNA packaging during virus assembly. These nucleotides have been proposed to form four stem-loops (SL1-SL4) that have both independent and overlapping functions. Stem-loop SL2 is important for efficient recognition and packaging of the full-length, unspliced viral genome, and also contains the major splice-donor site (SD) for mRNA splicing. We have determined the structure of the 19-residue SL2 oligoribonucleotide by heteronuclear NMR methods. The structure is generally consistent with the most recent of two earlier secondary structure predictions, with residues G1-G2-C3-G4 and C6-U7 forming standard Watson Crick base-pairs with self-complementary residues C16-G17-C18-C19 and A12-G13, respectively. However, residue A15, which is located near the center of the stem, does not form a predicted bulge, and residues A5 and U14 do not form an expected Watson-Crick base-pair. Instead, these residues form a novel A5-U14-A15 base-triple that appears to be stabilized by hydrogen bonds from A15-H61 and -H62 to A5-N1 and U14-O2, respectively; from A5-H61 to U14-O2, and from C16-H42 to U14-O2'. A kink in the backbone allows the aromatic rings of the sequential U14-A15 residues to be approximately co-planar, adopting a stable platform motif that is structurally similar to the A-A (adenosine) platforms observed in the P4-P6 ribozyme domain of the Tetrahymena group I intron. Platform motifs generally function in RNA by mediating long-range interactions, and it is therefore possible that the A-U-A base-triple platform mediates long-range interactions that either stabilize the psi-RNA or facilitate splicing and/or packaging. Residue G8 of the G8-G9-U10-G11 tetraloop is stacked above the U7-A12 base-pair, and the remaining tetraloop residues are disordered and available for potential interactions with either other RNA or protein components.     

Affinities of the nucleocapsid protein for variants of SL3 RNA in HIV-1

Efficient packaging of genomic RNA into new HIV-1 virus particles requires that nucleocapsid domains of precursor proteins bind the SL3 tetraloop (G317-G-A-G320) from the 5'-untranslated region. This paper presents the affinities of 35 RNA variants of SL3 for the mature 55mer NC protein, as measured by fluorescence quenching of tryptophan-37 in the protein by nucleobases. The 1:1 complexes that form in 0.2 M NaCl have dissociation constants ranging from 8 nM (GGUG) to 20 microM (GAUA). The highly conserved (GGAG) sequence for the wild type is not the most stable (K(d) = 28 nM), suggesting that other selective pressures beyond the stability of the complex must be satisfied. The leading requirement for strong interaction is for G320, followed closely by G318. Replacing either with U, A, or C reduces affinity by a factor of 15-120. NC-domains from multiple proteins combine to recognize unpaired G(2)-loci, where two guanines are in close proximity. We have previously measured affinities of the NC protein for the important stem-loops of the major packaging domain [Shubsda, M. F., Paoletti, A. C., Hudson, B. S., and Borer, P. N. (2002) Biochemistry 41, 5276-82]. Comparison with the present work shows that the nature of the stem also modulates NC-RNA interactions. Placing the G(2)-loci from the apical SL2 or SL1 loops on the SL3 stem increases affinity by a factor of 2-3, while placing the SL4 loop on the SL3 stem reduces affinity 50-fold. These results are interesting in the context of RNA-protein interaction, as well as for the discovery of antiNC agents for AIDS therapy.     

Affinities of packaging domain loops in HIV-1 RNA for the nucleocapsid protein

To design anti-nucleocapsid drugs, it is useful to know the affinities the protein has for its natural substrates under physiological conditions. Dissociation equilibrium constants are reported for seven RNA stem-loops bound to the mature HIV-1 nucleocapsid protein, NCp7. The loops include SL1, SL2, SL3, and SL4 from the major packaging domain of genomic RNA. The binding assay is based on quenching the fluorescence of tryptophan-37 in the protein by G residues in the single-stranded loops. Tightly bound RNA molecules quench nearly all the fluorescence of freshly purified NCp7 in 0.2 M NaCl. In contrast, when the GGAG-tetraloop of tight-binding SL3 is replaced with UUCG or GAUA, quenching is almost nil, indicating very low affinity. Interpreting fluorescence titrations in terms of a rapidly equilibrating 1:1 complex explains nearly all of the experimental variance for the loops. Analyzed in this way, the highest affinities are for 20mer SL3 and 19mer SL2 hairpin constructs (K(d) = 28 +/- 3 and 23 +/- 2 nM, respectively). The 20mer stem-UUCG-loop and GAUA-loop constructs have <0.5% of the affinity for NCp7 relative to SL3. Affinities relative to SL3 for the other stem-loops are the following: 10% for a 16mer construct to model SL4, 30% for a 27mer model of the 9-residue apical loop of SL1, and 20% for a 23mer model of a 1 x 3 asymmetric internal loop in SL1. A 154mer construct that includes all four stem-loops binds tightly to NCp7, with the equivalent of three NCp7 molecules bound with high affinity per RNA; it is also possible that two strong sites and several weaker ones combine to give the appearance of three strong sites.     

High affinity nucleocapsid protein binding to the muPsi RNA packaging signal of Rous sarcoma virus

The genomes of all retroviruses contain sequences near their 5' ends that interact with the nucleocapsid domains (NC) of assembling Gag proteins and direct their packaging into virus particles. Retroviral packaging signals often occur in non-contiguous segments spanning several hundred nucleotides of the RNA genome, confounding structural and mechanistic studies of genome packaging. Recently, a relatively short, 82 nucleotide region of the Rous sarcoma virus (RSV) genome, called muPsi, was shown to be sufficient to direct efficient packaging of heterologous RNAs into RSV-like particles. We have developed a method for the preparation and purification of large quantities of recombinant RSV NC protein, and have studied its interactions with native and mutant forms of the muPsi encapsidation element. NC does not bind with significant affinity to truncated forms of muPsi, consistent with earlier packaging and mutagenesis studies. Surprisingly, NC binds to the native muPsi RNA with affinity that is approximately 100 times greater than that observed for other previously characterized retroviral NC-RNA complexes (extrapolated dissociation constant K(d)=1.9 nM). Tight binding with 1:1 NC-muPsi stoichiometry is dependent on a conserved UGCG tetraloop in one of three predicted stem loops, and an AUG initiation codon controvertibly implicated in genome packaging and translational control. Loop nucleotides of other stem loops do not contribute to NC binding. Our findings indicate that the structural determinants of RSV genome recognition and NC-RNA binding differ considerably from those observed for other retroviruses.     

Comparative structural effects of HIV-1 Gag and nucleocapsid proteins in binding to and unwinding of the viral RNA packaging signal

The major RNA binding region of the HIV-1 Gag polyprotein is the nucleocapsid (NC) domain, which is responsible for the specific capture of the genomic RNA genome during viral assembly. The Gag polyprotein has other RNA chaperone functions, which are mirrored by the isolated NC protein after physiological cleavage from Gag. Gag, however, is suggested to have superior nucleic acid chaperone activity. Here we investigate the interaction of Gag and NC with the core RNA structure of the HIV-1 packaging signal (Ψ), using 2-aminopurine substitution to create a series of modified RNAs based on the Ψ helix loop structure. The effects of 2-aminopurine substitution on the physical and structural properties of the viral Ψ were characterized. The fluorescence properties of the 2-aminopurine substitutions showed features consistent with the native GNAR tetraloop. Dissociation constants (K(d)) of the two viral proteins, measured by fluorescence polarization (FP), were similar, and both NC and Gag affected the 2-aminopurine fluorescence of bases close to the loop binding region in a similar fashion. However, the influence of Gag on the fluorescence of the 2-aminopurine nucleotides at the base of the helix implied a much more potent helix destabilizing action on the RNA stem loop (SL) versus that seen with NC. This was further supported when the viral Ψ SL was tagged with a 5' fluorophore and 3' quencher. In the absence of any viral protein, minimal fluorescence was detected; addition of NC yielded a slight increase in fluorescence, while addition of the Gag protein yielded a large change in fluorescence, further suggesting that, compared to NC, the Gag protein has a greater propensity to affect RNA structure and that Ψ helix unwinding may be an intrinsic step in RNA encapsidation.     

A high affinity binding site for the HIV-1 nucleocapsid protein.

The nucleocapsid protein (NC) of HIV-1 is a small zinc finger protein that contributes to multiple steps of the viral life cycle, including the proper encapsidation of HIV RNA. This is accomplished through an interaction between NC and a region at the 5'-end of the RNA, defined as the Psi element. However, the specificity of NC for Psi or for RNA in general is not well understood. To study this problem, we used SELEX to identify high affinity RNA ligands that bind to NC. A 'winner' molecule (SelPsi), as well as a subregion of Psi RNA, were further characterized to understand the interaction between NC and SelPsi and its relationship to the interaction between NC and Psi. The comparison makes predictions about the sequence and structure of a high affinity binding site within the HIV-1 Psi element.     

Stem of SL1 RNA in HIV-1: structure and nucleocapsid protein binding for a 1 x 3 internal loop

The 5'-leader of HIV-1 RNA controls many viral functions. Nucleocapsid (NC) domains of gag-precursor proteins select genomic RNA for packaging by binding several sites in the leader. One is likely to be a stem defect in SL1 that can adopt either a 1 x 3 internal loop, SL1i (including G247, A271, G272, G273) or a 1 x 1 internal loop (G247 x G273) near a two-base bulge (A269-G270). It is likely that these two conformations are both present and exchange readily. A 23mer RNA construct described here models SL1i and cannot slip into the alternate form. It forms a 1:1 complex with NCp7, which interacts most strongly at G247 and G272 (K(d) = 140 nM). This demonstrates that a linear G-X-G sequence is unnecessary for high-affinity binding. The NMR-based structure shows an easily broken G247:A271 base pair. G247 stacks on both of its immediate neighbors and A271 on its 5'-neighbor; G272 and G273 are partially ordered. A bend in the helix axis between the SL1 stems on either side of the internal loop is probable. An important step in maturation of the virus is the transition from an apical loop-loop interaction to a dimer involving intermolecular interactions along the full length of SL1. A bend in the stem may be important in relieving strain and ensuring that the strands do not become entangled during the transition. A stem defect with special affinity for NCp7 may accelerate the rate of the dimer transformation. This complex could become an important target for anti-HIV drug development, where a drug could exert its action near a high-energy intermediate on the pathway for maturation of the dimer.     

Induced structural changes of 7SL RNA during the assembly of human signal recognition particle

The eukaryotic signal recognition particle (SRP) is a cytoplasmic ribonucleoprotein particle that targets secretory and membrane proteins to the endoplasmic reticulum. The binding of SRP54 to the S domain of 7SL RNA is highly dependent on SRP19. Here we present the crystal structure of a human SRP ternary complex consisting of SRP19, the M domain of SRP54 and the S domain of 7SL RNA. Upon binding of the M domain of SRP54 to the 7SL RNA-SRP19 complex, the asymmetric loop of helix 8 in 7SL RNA collapses. The bases of the four nucleotides in the long strand of the asymmetric loop continuously stack and interact with the M domain, whereas the two adenines in the short strand flip out and form two A-minor motifs with helix 6. This stabilizing interaction is only possible when helix 6 has been positioned parallel to helix 8 by the prior binding of SRP19 to the tetraloops of helices 6 and 8. Hence, the crystal structure of the ternary complex suggests why SRP19 is necessary for the stable binding of SRP54 to the S domain RNA.     

Evidence for an extended 7SL RNA structure in the signal recognition particle.

The signal recognition particle (SRP) functions in conjunction with the SRP receptor to target nascent ectoplasmic proteins to the protein translocation machinery of the endoplasmic reticulum membrane. SRP is a ribonucleoprotein consisting of six distinct polypeptides and one molecule of 7SL RNA 300 nucleotides long. SRP has previously been visualized by a variety of electron microscopic techniques as a rod-shaped particle 24 nm long and 6 nm wide. We report here microanalysis by electron spectroscopic imaging which localizes the RNA molecule in SRP to primarily the two ends of the particle. These results suggest that the single 7SL RNA molecule spans the length of the particle. Micrographs from a scanning transmission electron microscope permit visualization of unstained SRP with low electron exposure, as well as the direct measurement of the mol. wt of the particle. These micrographs confirm our earlier suggestion that SRP is divided into three structural domains and allow discrimination of the two ends of the structure. The results of both techniques have been combined in a model for the structure of SRP in which we propose the basic orientation of the 7SL RNA. The structure proposed is consistent with the secondary structure predicted for the RNA and with biochemical data.     

The organization of the 7SL RNA in the signal recognition particle.

Digestion of the signal recognition particle (SRP) of dog pancreas with micrococcal nuclease results in the stepwise cleavage of the 300 nucleotide 7SL RNA moiety producing five major fragments approximately 220 (1), 150 (2), 72 (3), 62 (4) and 45 (5) nucleotides long. The RNA molecule is initially cut once yielding fragments 1 and 3. Further degradation releases fragments 2, 4 and 5. The introduction of the first nick into the 7SL RNA does not alter the structure nor the function of the SRP. Further degradation of the RNA results in disruption and loss of activity of the particle. The sequence of the RNA fragments shows that the nuclease causes discrete cuts in the RNA with minimal nibbling indicating that only few sites are accessible to the action of the enzyme. The five major products of nuclease digestion together span almost the entire length of the 7SL RNA. Nicking occurs mainly around the boundary region between the central S sequence and the flanking Alu sequences constituting the 7SL RNA (1). The S fragment is bound to the four largest polypeptides while the 5' and 3' Alu fragments are associated with the two smallest protein constituents of the SRP.     

Role of HIV-1 RNA and protein determinants for the selective packaging of spliced and unspliced viral RNA and host U6 and 7SL RNA in virus particles

HIV-1 particles contain RNA species other than the unspliced viral RNA genome. For instance, viral spliced RNAs and host 7SL and U6 RNAs are natural components that are non-randomly incorporated. To understand the mechanism of packaging selectivity, we analyzed the content of a large panel of HIV-1 variants mutated either in the 5'UTR structures of the viral RNA or in the Gag-nucleocapsid protein (GagNC). In parallel, we determined whether the selection of host 7SL and U6 RNAs is dependent or not on viral RNA and/or GagNC. Our results reveal that the polyA hairpin in the 5'UTR is a major packaging determinant for both spliced and unspliced viral RNAs. In contrast, 5'UTR RNA structures have little influence on the U6 and 7SL RNAs, indicating that packaging of these host RNAs is independent of viral RNA packaging. Experiments with GagNC mutants indicated that the two zinc-fingers and N-terminal basic residues restrict the incorporation of the spliced RNAs, while favoring unspliced RNA packaging. GagNC through the zinc-finger motifs also restricts the packaging of 7SL and U6 RNAs. Thus, GagNC is a major contributor to the packaging selectivity. Altogether our results provide new molecular insight on how HIV selects distinct RNA species for incorporation into particles.     

Diversity of 7 SL RNA from the signal recognition particle of maize endosperm.

An 11 S ribonucleoprotein particle was isolated from maize endosperm and shown to be functionally and structurally equivalent to the mammalian signal recognition particle. However, unlike animal cells which apparently contain a single 7 SL RNA species, maize endosperm contains a heterogeneous population of 7 SL RNA. To investigate this diversity, we have cloned and sequenced a number of the maize endosperm 7 SL RNAs isolated from functionally active SRP preparations. Some maize 7 SL RNAs are strikingly similar, differing by single base changes or short deletions; surprisingly, others share less than 70 percent sequence homology. Despite differences in primary sequence, nearly identical secondary structures can be suggested for all maize 7 SL RNAs, consistent with a proposed functional role in protein translocation for each of these RNAs. The amount of new available sequence data enabled us to define two conserved regions of presumed functional importance: A conserved sequence -G-N-A-R- in the center of a variable region which forms a well defined stem-loop and possibly is involved in an interaction with the 19 kDa protein of the SRP. Secondly, three short nucleotide stretches located in the central domain of 7 SL RNA may form part of a dynamic RNA-switch structure.     

Opening of the TAR hairpin in the HIV-1 genome causes aberrant RNA dimerization and packaging

Background

Developing a quantitative understanding of viral kinetics is useful for determining the pathogenesis and transmissibility of the virus, predicting the course of disease, and evaluating the effects of antiviral therapy. The availability of data in clinical, animal, and cell culture studies, however, has been quite limited. Many studies of virus infection kinetics have been based solely on measures of total or infectious virus count. Here, we introduce a new mathematical model which tracks both infectious and total viral load, as well as the fraction of infected and uninfected cells within a cell culture, and apply it to analyze time-course data of an SHIV infection in vitro.

Results

We infected HSC-F cells with SHIV-KS661 and measured the concentration of Nef-negative (target) and Nef-positive (infected) HSC-F cells, the total viral load, and the infectious viral load daily for nine days. The experiments were repeated at four different MOIs, and the model was fitted to the full dataset simultaneously. Our analysis allowed us to extract an infected cell half-life of 14.1 h, a half-life of SHIV-KS661 infectiousness of 17.9 h, a virus burst size of 22.1 thousand RNA copies or 0.19 TCID₅₀, and a basic reproductive number of 62.8. Furthermore, we calculated that SHIV-KS661 virus-infected cells produce at least 1 infectious virion for every 350 virions produced.

Conclusions

Our method, combining in vitro experiments and a mathematical model, provides detailed quantitative insights into the kinetics of the SHIV infection which could be used to significantly improve the understanding of SHIV and HIV-1 pathogenesis. The method could also be applied to other viral infections and used to improve the in vitro determination of the effect and efficacy of antiviral compounds.     

Conservation of a packaging signal and the viral genome RNA packaging mechanism in alphavirus evolution

Alphaviruses are a group of small, enveloped viruses which are widely distributed on all continents. In infected cells, alphaviruses display remarkable specificity in RNA packaging by encapsidating only their genomic RNA while avoiding packaging of the more abundant viral subgenomic (SG), cellular messenger and transfer RNAs into released virions. In this work, we demonstrate that in spite of evolution in geographically isolated areas and accumulation of considerable diversity in the nonstructural and structural genes, many alphaviruses belonging to different serocomplexes harbor RNA packaging signals (PSs) which contain the same structural and functional elements. Their characteristic features are as follows. (i) Sindbis, eastern, western, and Venezuelan equine encephalitis and most likely many other alphaviruses, except those belonging to the Semliki Forest virus (SFV) clade, have PSs which can be recognized by the capsid proteins of heterologous alphaviruses. (ii) The PS consists of 4 to 6 stem-loop RNA structures bearing conserved GGG sequences located at the base of the loop. These short motifs are integral elements of the PS and can function even in the artificially designed PS. (iii) Mutagenesis of the entire PS or simply the GGG sequences has strong negative effects on viral genome packaging and leads to release of viral particles containing mostly SG RNAs. (iv) Packaging of RNA appears to be determined to some extent by the number of GGG-containing stem-loops, and more than one stem-loop is required for efficient RNA encapsidation. (v) Viruses of the SFV clade are the exception to the general rule. They contain PSs in the nsP2 gene, but their capsid protein retains the ability to use the nsP1-specific PS of other alphaviruses. These new discoveries regarding alphavirus PS structure and function provide an opportunity for the development of virus variants, which are irreversibly attenuated in terms of production of infectious virus but release high levels of genome-free virions.