Viral RNA annealing activities of the nucleocapsid protein of Moloney murine leukemia virus are zinc independent.

The zinc fingers of retroviral gag nucleocapsid proteins (NC) are required for the specific packaging of the dimeric RNA genome into virions. In vitro, NC proteins activate both dimerization of viral RNA and annealing of the replication primer tRNA onto viral RNA, two reactions necessary for the production of infectious virions. In this study the role of the zinc finger of Moloney murine leukemia virus (MoMuLV) NCp10 in RNA binding and annealing activities was investigated through modification or replacement of residues involved in zinc coordination. These alterations did not affect the ability of NCp10 to bind RNA and promote RNA annealing in vitro, despite a complete loss of zinc affinity. However mutation of two conserved lysine residues adjacent to the finger motif reduced both RNA binding and annealing activities of NCp10. These findings suggest that the complexed NC zinc finger is not directly involved in RNA-protein interactions but more probably in a zinc dependent conformation of NC protein modulating viral protein-protein interactions, essential to the process of viral RNA selection and virion assembly. Then the NC zinc finger may cooperate to select the viral RNA genome to be packaged into virions.     

Sequence homology between Moloney murine sarcoma virus and Moloney leukemia virus RNA.

The Moloney murine sarcoma-leukemia virus [M-MSV (MuLV)], propagated at high multiplicity of infection (MOI), was demonstrated previously to contain a native genome mass of 4 X 10(6) daltons as contrasted to a mass of 7 X 10(6) daltons for Moloney murine leukemia virus (M-MuLV). The 4 X 10(6)-dalton classof RNA from M-MSV (MuLV) was examined for base sequence homology with DNA complementary to the 7 X 10(6)-dalton M-MuLV RNA genome. Approximately 86% of the M-MSV (MuLV) was protected from RNase digestion by hybridization, whereas 95% of M-MuLV was protected under identical conditions. These results indicate that the small RNA class of high-MOI M-MSV (MuLV) contains little (perhaps 10%) genetic information not present in M-MuLV. Virtually all of the 1.8 X 10(6)-dalton subunits of M-MSV (MuLV) RNA contained regions of poly(A) since 94% of the RNA bound to oligo(dT) cellulose in 0.5 M KCl. This suggests that the formation of the 1.8 X 10(6)-dalton subunits occurs before their packaging into virions and does not result from hydrolysis of intact 3.5 X 10(6)-dalton subunits by a virion-associated nuclease.     

Three-dimensional 1H NMR structure of the nucleocapsid protein NCp10 of Moloney murine leukemia virus

Summary The nucleocapsid protein of Moloney murine leukemia virus (NCp10) is a 56-amino acid protein which contains one zinc finger of the CysX₂CysX₄HisX₄Cys form, a highly conserved motif present in most retroviruses and retroelements. At pH5, NCp10 binds one zinc atom and the complexation induces a folding of the CysX₂CysX₄HisX₄Cys box, similar to that observed for the zinc-binding domains of HIV-1 NC protein. The three-dimensional structure of NCp10 has been determined in aqueous solution by 600 MHz ¹H NMR spectroscopy. The proton resonances could be almost completely assigned by means of phase-sensitive double-quantum-filtered COSY, TOCSY and NOESY techniques. NOESY spectra yielded 597 relevant structural constraints, which were used as input for distance geometry calculations with DIANA. Further refinement was performed by minimization with the program AMBER, which was modified by introducing a zinc force field. The solution structure is characterized by a well-defined central zinc finger (rmsd of 0.747±0.209 Å for backbone atoms and 1.709±0.187 Å when all atoms are considered), surrounded by flexible N- and C-terminal domains. The Tyr²⁸, Trp³⁵, Lys³⁷, Lys⁴¹ and Lys⁴² residues, which are essential for activity, lie on the same face of the zinc finger, forming a bulge structure probably involved in viral RNA binding. The significance of these structural characteristics for the various biological functions of the protein is discussed, taking into account the results obtained with various mutants.     

Sequence-specific binding of DNA by the Moloney murine leukemia virus integrase protein.

Genetic studies have indicated that integration of retroviral DNA into the host genome depends on the presence of the inverted repeats at the free termini of the long terminal repeats on the unintegrated DNA and on the product of the 3' end of the pol gene (the integrase [IN] protein). While the precise function of the Moloney murine leukemia virus IN protein is uncertain, others have shown that it is a DNA-binding protein and functions in the processing of the inverted repeats prior to integration. By using site-directed mutagenesis, we cloned and expressed the IN protein in Escherichia coli. Crude extracts of total cellular protein were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose filters, denatured in guanidine, renatured, and incubated with oligonucleotide probes. Single- and double-stranded oligonucleotides corresponding to the termini of unintegrated linear viral DNA were specifically bound by the IN protein in this assay. These data suggest that the role of the Moloney IN protein in the early steps of integration involves sequence-specific recognition of the DNA sequences found at the ends of the long terminal repeats.     

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.     

Interaction of Moloney murine leukemia virus matrix protein with IQGAP

The matrix protein (MA) of the Moloney murine leukemia virus (M-MuLV) was found to interact with IQGAP1, a prominent regulator of the cytoskeleton. Mutational studies defined residues of MA critical for the interaction, and tests of viruses carrying MA mutations revealed a near-perfect correlation between binding and virus replication. The replication-defective mutants showed defects in both early and late stages of the life cycle. Four viable second-site revertant viruses were isolated from three different replication-defective parental mutants, and in all cases the interaction with IQGAP1 was restored by the suppressor mutations. The interaction of MA and IQGAP1 was readily detected in vitro and in vivo. Virus replication was potently inhibited by a C-terminal fragment of IQGAP1, and impaired by RNAi knockdown of IQGAP1 and 2. We suggest that the IQGAPs link the virus to the cytoskeleton for trafficking both into and out of the cell.     

Bipartite signal for genomic RNA dimerization in Moloney murine leukemia virus

Retroviral virions each contain two identical genomic RNA strands that are stably but noncovalently joined in parallel near their 5' ends. For certain viruses, this dimerization has been shown to depend on a unique RNA stem-loop locus, called the dimer initiation site (DIS), that efficiently homodimerizes through a palindromic base sequence in its loop. Previous studies with Moloney murine leukemia virus (Mo-MuLV) identified two alternative DIS loci that can each independently support RNA dimerization in vitro but whose relative contributions are unknown. We now report that both of these loci contribute to the assembly of the Mo-MuLV dimer. Using targeted deletions, point mutagenesis, and antisense oligonucleotides, we found that each of the two stem-loops forms as predicted and contributes independently to dimerization in vitro through a mechanism involving autocomplementary interactions of its loop. Disruption of either DIS locus individually reduced both the yield and the thermal stability of the in vitro dimers, whereas disruption of both eliminated dimerization altogether. Similarly, the thermal stability of virion-derived dimers was impaired by deletion of both DIS elements, and point mutations in either element produced defects in viral replication that correlated with their effects on in vitro RNA dimerization. These findings support the view that in some retroviruses, dimer initiation and stability involve two or more closely linked DIS loci which together align the nascent dimer strands in parallel and in register.     

Identification of a subdomain in the Moloney murine leukemia virus envelope protein involved in receptor binding.

We have mutated amino acids within the receptor-binding domain of Moloney murine leukemia virus envelope in order to identify residues involved in receptor binding. Analysis of mutations in the region of amino acids 81 to 88 indicates that this region is important for specific envelope-receptor interactions. None of the aspartate 84 (D-84) mutants studied bind measurably, although they are efficiently incorporated into particles. D-84 mutants have titers that correspond to the severity of the substitution. This observation suggests that D-84 may provide a direct receptor contact. Mutations in the other charged amino acids in this domain (R-83, E-86, and E-87) yield titers similar to those of wild-type envelope, but the affinity of the mutant envelope in the binding assay is decreased by nonconservative substitutions in parallel to the severity of the change. These other amino acids may either provide secondary receptor contacts or assist in maintaining a structure in the domain that favors efficient binding. We also studied other regions of high hydrophilicity. Our initial characterization indicates that amino acids 106 to 111 and 170 to 188 do not play a major role in receptor binding. Measurements of relative binding affinity and titer indicate that most mutations in the region of amino acids 120 to 131 did not significantly affect receptor binding. However, SU encoded by mutants H123V, R124L, and C131A as well as C81A could not be detected in particles and therefore did not bind measurably. Therefore, the region encompassed by amino acids 81 to 88 appears to be directly involved in receptor binding.     

An additional dimer linkage structure in Moloney murine leukemia virus RNA.

We have identified an additional dimerization linkage structure in the genome of Moloney murine leukemia virus (MoMLV). Retroviral genomes have long been known to be linked at their 5' ends to form dimers. In MoMLV, a hairpin loop functioning as a dimer linkage structure (DLS) has previously been identified at nucleotides 278-303. Here, we describe RNA dimers formed from sections of the MoMLV 5' untranslated region that do not contain the previously described MoMLV DLS. These dimers exhibit the distinctive characteristics previously described for whole genome dimers. We have mapped this novel region to nucleotides 199-243. This sequence contains a stem-loop structure (nucleotides 204-227) much like the 278-303 region. We describe the chemical and thermal stability of dimers containing the 204-227 stem-loop as well as kinetics and salt-dependence of dimer formation. Our results show that dimerization of MoMLV RNA can be nucleated at multiple sites and suggest that the 5' untranslated region may contain separately folding and dimerizing domains.     

cis-Elements involved in expression of unspliced RNA in Moloney murine leukemia virus.

Murine leukemia virus (MLV) produces the unspliced RNA and the singly spliced RNA at a proper ratio at a time. To identify cis-elements involved in the production of the unspliced RNA, we examined the level of unspliced RNA in a series of mutants derived from a prototype Moloney MLV mutant gag-encoding G3.6. Our present data indicated that nt 1560-1906 region in the CA-encoding region in gag was the negative cis-element and nt 5119-5355 region, which was immediately upstream of the splice acceptor site, was the positive cis-element for expression of the unspliced RNA. It was found that the former element made expression of the unspliced RNA dependent upon the latter. These two elements were functional as discrete elements and their activities were relatively position-independent.     

Electron microscopy of the nucleocapsid from disrupted Moloney murine leukemia virus and of associated type VI collagen-like filaments.

To analyze the constituents of retroviruses, the Moloney murine leukemia virus was disrupted and observed by dark-field electron microscopy. Virus disruption was achieved by several methods: osmotic shock, freezing-thawing cycles, and exposure to urea up to 4 M, to NaCl up to 1 M, and to Triton X-100. Several components associated with broken Moloney murine leukemia virus were repeatedly found in preparations. These components have been described as rings, thick filaments, chain-like filaments, threads covered with proteins, threads with buckles, and naked threads. A quantitative analysis of the occurrence of these components has been carried out. Among them, the thick filaments composed of a compact helical arrangement of small beads 5 nm in diameter were considered to represent the nucleocapsid. The protease-sensitive buckles found on some threads could be a compact form of the viral RNA associated to the nucleocapsid protein NCp10. The RNase-sensitive naked threads are interpreted as the deproteinized viral RNA itself. The ubiquitous chain-like filaments possess a periodic structure identical to that of polymerized type VI collagen. It is proposed that this adhesive protein is associated with the viral envelope taken from the cell membrane during the budding process of retroviruses.     

Fluorescence and nucleic acid binding properties of bovine leukemia virus nucleocapsid protein

Peptidyl-prolyl cis/trans isomerization, observed in the native state of an increasing number of proteins, is of considerable biological significance. The first evidence for an asymmetric transmission along the polypeptide chain of the structural effects of prolyl isomerization is now derived from the statistics of the C(alpha)/C(alpha)-atom distance distributions in the crystal structures of 848 non-homologous proteins. More detailed information on how isomerization affects segments adjacent to proline is obtained from crystal structures of proteins, that are more than 95% homologous, and that exhibit two different states of isomerization at a particular prolyl bond. The resulting 64 cases, which represent 3.8% of the database used, form pairs of coordinates which were analyzed for the existence of isomer-specific intramolecular nonbonded C(alpha)/C(alpha)-atom distances around the critical proline, and for the positional preferences for particular amino acids in the isomeric sequence segment. The probability that a native protein exhibits both prolyl isomers in the crystalline state increases in particular with a Pro at the third position N-terminal to the isomeric bond (-3 position), and with Ser, Gly and Asp at the position preceding the isomeric bond (-1 position). Structural alignment of matched pairs of isomeric proteins generates three classes with respect to position-specific distribution of C(alpha)-atom displacements around an isomeric proline imide bond. In the majority of cases the distribution of these intermolecular isomer-specific C(alpha)-atom distances shows a symmetric behavior for the N-terminal and C-terminal segment flanking the proline residue, and the magnitude did not exceed 1.3+/-0.6 A including the C(alpha) atoms in proximity to the prolyl bond. However, in the remaining 12 protein pairs the structural changes are unidirectional relative to the isomerizing bond whereby the magnitude of the isomer-specific effect exceeds 3.0+/-2.0 A even at positions remote to proline. Interestingly, the magnitude of the intramolecular isomer-specific C(alpha) atom displacements reveals a lever-arm amplification of the isomerization-mediated structural changes in a protein backbone. The observed backbone effects provide a structural basis for isomer-specific reactions of proline-containing polypeptides, and thus may play a role in biological recognition and regulation.     

Functional dissection of the Moloney murine leukemia virus envelope protein gp70.

The envelope protein of Moloney murine leukemia virus (Mo-MLV) is a complex glycoprotein that mediates receptor binding and entry via fusion with cell membranes. By using a series of substitution mutations and truncations in the Mo-MLV external envelope surface protein gp70, we have identified regions important for these processes. Firstly, truncations of gp70 revealed that the minimal continuous receptor-binding region is amino acids 9 to 230, in broad agreement with other studies. Secondly, within this region there are two key basic amino acids, Arg-83 and Arg-95, that are essential for receptor binding and may interact with a negatively charged residue(s) or with the pi electrons of the aromatic ring on a hydrophobic residue(s) in the basic amino acid transporter protein that is the Mo-MLV ecotropic receptor. Finally, we showed that outside the minimal receptor-binding region at amino acids 2 to 8, there is a region that is essential for postbinding fusion events.