Alteration of zinc-binding residues of simian immunodeficiency virus p8(NC) results in subtle differences in gag processing and virion maturation associated with degradative loss of mutant NC.

In all retroviruses analyzed to date (except for the spumaretroviruses), the Zn(2+)-coordinating residues of nucleocapsid (NC) perform or assist in crucial reactions necessary to complete the retrovirus life cycle. Six replication-defective mutations have been engineered in the two NC Zn(2+) fingers (ZFs) of simian immunodeficiency virus [SIV(Mne)] that change or delete specific Zn(2+)-interacting Cys residues and were studied by using electron microscopy, reversed-phase high-performance liquid chromatography, immunoblotting, and RNA quantification. We focused on phenotypes of produced particles, specifically morphology, Gag polyprotein processing, and genomic RNA packaging. Phenotypes were similar among viruses containing a point or deletion mutation involving the same ZF. Mutations in the proximal ZF (ZF1) resulted in near-normal Gag processing and full-length genomic RNA incorporation and were most similar to wild-type (WT) virions with electron-dense, conical cores. Mutation of the distal ZF, as well as point mutations in both ZFs, resulted in more unprocessed Gag proteins than a deletion or point mutation in ZF1, with an approximate 30% reduction in levels of full-length genomic RNA in virions. These mutant virions contained condensed cores; however, the cores typically appeared less electron dense and more rod shaped than WT virions. Surprisingly, deletion of both ZFs, including the basic linker region between the ZFs, resulted in the most efficient Gag processing. However, genomic RNA packaging was approximately 10% of WT levels, and those particles produced were highly abnormal with respect to size and core morphology. Surprisingly, all NC mutations analyzed demonstrated a significant loss of processed NC in virus particles, suggesting that Zn(2+)-coordinated NC is protected from excessive proteolytic cleavage. Together, these results indicate that Zn(2+) coordination is important for correct Gag precursor processing and NC protein stability. Additionally, SIV particle morphology appears to be the result of proper and complete Gag processing and relies less on full-length genomic RNA incorporation, as dictated by the Zn(2+) coordination in the ZFs of the NC protein.

Fluorescence and nucleic acid binding properties of the human T-cell leukemia virus-type 1 nucleocapsid protein.

We used intrinsic tryptophan fluorescence to study the nucleocapsid protein from human T-cell leukemia virus-type one, HTLV-1 p15, an 85-amino-acid protein with two Trp-containing zinc-finger motifs. Fluorescence spectra suggested an interaction between the two zinc fingers and another interaction involving the C-terminal tail and the zinc fingers. Titrations with nucleic acid revealed similar, sub-micromolar affinity for poly(dT) and poly(U) in 1 mM sodium phosphate, pH 7. Double-stranded DNA bound an order of magnitude weaker, suggesting helix-destabilizing activity. Base preference of p15 was T approximately U>I approximately C approximately G>A; affinity spanned about one order of magnitude. HTLV-1 p15 bound weaker and with less variation than reported values for either human or simian immunodeficiency virus homologues. The low affinity of p15 for nonspecific nucleic acids distinguishes it from other nucleocapsid proteins, and may suggest its involvement in additional steps of the virus life cycle other than RNA packaging.

Nucleic acid binding properties of the simian immunodeficiency virus nucleocapsid protein NCp8.

The nucleocapsid protein of simian immunodeficiency virus (SIV) NCp8 has two copies of conserved sequences (termed zinc fingers, ZF) of 14 amino acids with 4 invariant residues (CCHC) that coordinate Zn(II). Each of its two ZFs has a Trp residue. A significant quenching of NCp8 Trp fluorescence was seen in nucleic acid complexes, suggesting stacking of the indole ring with nucleobases and the simultaneous involvement of both ZFs in the binding process. Both ZFs contribute to the nucleic acid binding free energy of NCp8, albeit in a not additive manner. NCp8 exhibited a base preference analogous to that of NCp7: G approximately I > T > U > C > A. Alternating base sequences that bind HIV-1 NCp7 in a sequence-specific manner were also bound selectively by NCp8. Specific sequence recognition required at least five bases and the presence of bound Zn(II). The two ZFs account for the net displacement of 3 out of 4 sodium ions upon binding (2 by the first and one by the second finger), and for most (85%) of the hydrophobic stabilization in complex formation. Based on the sequence and functional similarity of SIV NCp8 and HIV-1 NCp7, and using available structural information for free and oligonucleotide bound NCp7, we propose a structural model for NCp8-oligonucleotide complexes.

Actin-binding cellular proteins inside human immunodeficiency virus type 1.

Host proteins are incorporated both on and inside human immunodeficiency virus type 1 (HIV-1) virions. To identify cellular proteins inside HIV-1, virion preparations were treated by a protease-digestion technique that removes external host proteins, allowing for the study of the proteins inside the virus. Treated HIV-1 preparations were analyzed by immunoblot, high-pressure liquid chromatography, and protein sequence analyses. These analyses identified several cellular proteins inside HIV-1: elongation factor 1alpha, glyceraldehyde-3-phosphate dehydrogenase, HS-1, phosphatidylethanolamine-binding protein, Pin1, Lck, Nm23-H1, and the C-terminal tail of CD43. Several of these proteins were found as fragments of their full-sized proteins that appear to be generated by our protease treatment of the virions, the HIV-1 protease, or a cellular protease. Recent advances in cell biology and biochemistry have identified some of these proteins as actin-binding proteins. These results support the hypothesis that actin filaments are incorporated into the virion and may provide additional clues for the understanding of the interaction between viral and cellular proteins during assembly and budding.

Binding properties of the human immunodeficiency virus type 1 nucleocapsid protein p7 to a model RNA: elucidation of the structural determinants for function.

HIV-1 nucleocapsid protein (NCp7) is a double zinc-fingered protein that has been traditionally implicated in viral RNA recognition and packaging, in addition to its tight association with genomic RNA and tRNA primer within the virion nucleocapsid. The availability of large quantities of viral or recombinant wild-type NCp7 and mutant p7 has made possible the assignment of the different roles that structural motifs within the protein play during RNA binding. At low ionic strength binding to the homopolymeric fluorescent RNA, poly(epsilonA), is electrostatically driven and four sodium ions are displaced. Arg7 in the flanking N-terminal region, Lys20 and Lys26 in the first zinc finger and one positively charged residue (attributed to Lys41) in the second zinc finger are involved in electrostatic contacts with RNA. The p7 zinc fingers do not function independently but concomitantly. The first zinc finger (both isolated or in the context of the full-length protein) has a more prominent electrostatic interaction than the second one. The second zinc finger dominates the non-electrostatic stabilization of the binding to RNA due to stacking of its Trp residue with nucleic acid bases. Mutations in the highly conserved retroviral Zn-coordinating residues (CCHC) to steroid hormone receptor (CCCC) or transcription factor (CCHH) metal cluster types do not affect RNA binding. In spite of the limited impact in RNA binding affinity in vitro or RNA packaging in vivo that such mutations or structural alterations impart, they impair or abolish virus infectivity. It is likely that such an effect stems from the involvement of NCp7 in crucial steps of the virus life cycle other than RNA binding.

Ubiquitin is covalently attached to the p6Gag proteins of human immunodeficiency virus type 1 and simian immunodeficiency virus and to the p12Gag protein of Moloney murine leukemia virus.

Host proteins are incorporated into retroviral virions during assembly and budding. We have examined three retroviruses, human immunodeficiency virus type 1 (HIV-1), simian immunodeficiency virus (SIV), and Moloney murine leukemia virus (Mo-MuLV), for the presence of ubiquitin inside each of these virions. After a protease treatment to remove exterior viral as well as contaminating cellular proteins, the proteins remaining inside the virion were analyzed. The results presented here show that all three virions incorporate ubiquitin molecules at approximately 10% of the level of Gag found in virions. In addition to free ubiquitin, covalent ubiquitin-Gag complexes were detected, isolated, and characterized from all three viruses. Our immunoblot and protein sequencing results on treated virions showed that approximately 2% of either HIV-1 or SIV p6Gag was covalently attached to a single ubiquitin molecule inside the respective virions and that approximately 2 to 5% of the p12Gag in Mo-MuLV virions was monoubiquitinated. These results show that ubiquitination of Gag is conserved among these retroviruses and occurs in the p6Gag portion of the Gag polyprotein, a region that is likely to be involved in assembly and budding.

Probing the topography of HIV-1 nucleocapsid protein with the alkylating agent N-ethylmaleimide.

Retroviral nucleocapsid (NC) proteins contain one or two zinc fingers (ZFs) consisting of a CCHC peptide motif that coordinates Zn(II). Mutational and biochemical analyses have shown that NC ZFs are directly involved in multiple stages of viral replication, including genomic RNA encapsidation, virus maturation, and the early infection process. The multiple roles of the conserved retroviral ZFs make them attractive targets for antiviral agents. We have previously shown that a variety of chemical compounds can inactivate the whole virus by attacking NC ZFs. For the enhancement of the specificity of antiviral reagents, it is desirable to have a detailed knowledge of the spatial organization of reactive sites on the NC protein in its free and oligonucleotide-bound states. A method has been developed using chemical probes to assess the reactivity of specific Cys residues in the NC protein, and is being used to investigate the topography of ZFs in different contexts. In this study we focus on the reaction mechanism of N-ethylmaleimide (NEM) with free HIV-1 NCp7 protein. Our results show that the conformation of free NCp7 restricts the initial site of attack to Cys-49 (the most distal Cys residue in the second ZF) and that the reactivity of thiols in full-length protein differs from that of the isolated ZF peptides. A moderate to near complete reduction in reaction rate was observed when NCp7 was complexed with different oligonucleotides. These findings provide a set of experimentally determined parameters that can serve to guide computational modeling of the NC protein and will be useful for the rational design of drugs directed against retroviral ZFs.

Design of sensitive fluorogenic substrates for human cathepsin D.

Cathepsin D is a lysosomal aspartic proteinase that has been implicated in several pathological processes such as breast cancer and Alzheimer's disease. We designed and synthesized a number of quenched fluorogenic substrates with P2 variations in the series AcEE(EDANS)KPIXFFRLGK(DABCYL)E-NH2, where X=cysteine, methylcysteine, ethylcysteine, tert-butylcysteine, carboxymethylcysteine, methionine, valine or isoleucine. Most of the fluorogenic substrates exhibited greater k(cat)/Km ratios than the best cathepsin D substrates described so far. Differences in kinetic constants, which were rationalized using structure-based modeling, might make certain substrates useful for particular applications, such as active site titrations or initial velocity determination using a fluorescent plate reader.

Cytoskeletal proteins inside human immunodeficiency virus type 1 virions.

We have identified three types of cytoskeletal proteins inside human immunodeficiency virus type 1 (HIV-1) virions by analyzing subtilisin-digested particles. HIV-1 virions were digested with protease, and the treated particles were isolated by sucrose density centrifugation. This method removes both exterior viral proteins and proteins associated with microvesicles that contaminate virion preparations. Since the proteins inside the virion are protected from digestion by the viral lipid envelope, they can be isolated and analyzed after treatment. Experiments presented here demonstrated that this procedure removed more than 95% of the protein associated with microvesicles. Proteins in digested HIV-1(MN) particles from infected H9 and CEM(ss) cell lines were analyzed by high-pressure liquid chromatography, protein sequencing, and immunoblotting. The data revealed that three types of cytoskeletal proteins are present in virions at different concentrations relative to the molar level of Gag: actin (approximately 10 to 15%), ezrin and moesin (approximately 2%), and cofilin (approximately 2 to 10%). Our analysis of proteins within virus particles detected proteolytic fragments of alpha-smooth muscle actin and moesin that were cleaved at sites which might be recognized by HIV-1 protease. These cleavage products are not present in microvesicles from uninfected cells. Therefore, these processed proteins are most probably produced by HIV-1 protease digestion. The presence of these fragments, as well as the incorporation of a few specific cytoskeletal proteins into virions, suggests an active interaction between cytoskeletal and viral proteins.

Phosphorescence and optically detected magnetic resonance investigation of the binding of the nucleocapsid protein of the human immunodeficiency virus type 1 and related peptides to RNA.

The RNA and DNA complexes of nucleocapsid protein p7.Zn (NCp7.Zn) of the human immunodeficiency virus type 1 (HIV-1) are studied by phosphorescence and optically detected magnetic resonance (ODMR). The single tryptophan, Trp37, which is located on the C-terminal zinc finger domain is used as an intrinsic probe. Reductions in the triplet state zero-field splitting (zfs) D parameter of Trp37 upon complex formation with poly(I) and poly(U) are observed. These results, in conjunction with the phosphorescence red-shifts and triplet state lifetime reductions that are observed, suggest the presence of aromatic stacking interactions between NCp7.Zn and the bases of the RNA polymers. An alteration of the intersystem crossing pattern upon complex formation, in addition to the above mentioned spectroscopic shifts, also is consistent with previously observed tryptophans that undergo stacking interactions with DNA bases [Zang, L.-H., Maki, A.H., Murphy, J.B., & Chase, J.W. (1987) Biophys. J. 52, 867-872. Tsao, D.H.H., Casas-Finet, J.R., Maki, A.H., & Chase, J.W. (1989) Biophys. J. 55, 927-936]. These conclusions support those from a recent ODMR study [Lam, W.-C., Maki, A.H., Casas-Finet, J.R., Erickson, J.W., Sowder, R.C., II, & Henderson, L.E. (1993) FEBS Lett. 328, 45-48] of NCp7.Zn binding to 5-mercurated polyuridylic acid [poly(5-HgU)] in which stacking interactions between the RNA and NCp7.Zn are inferred from the observation of an external heavy atom effect induced on Trp37.(ABSTRACT TRUNCATED AT 250 WORDS)