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1.
Nucleic Acids Res ; 35(15): 5141-53, 2007.
Article in English | MEDLINE | ID: mdl-17660191

ABSTRACT

HIV-1 Vif (viral infectivity factor) is associated with the assembly complexes and packaged at low level into the viral particles, and is essential for viral replication in non-permissive cells. Viral particles produced in the absence of Vif exhibit structural defects and are defective in the early steps of reverse transcription. Here, we show that Vif is able to anneal primer tRNA(Lys3) to the viral RNA, to decrease pausing of reverse transcriptase during (-) strand strong-stop DNA synthesis, and to promote the first strand transfer. Vif also stimulates formation of loose HIV-1 genomic RNA dimers. These results indicate that Vif is a bona fide RNA chaperone. We next studied the effects of Vif in the presence of HIV-1 NCp, which is a well-established RNA chaperone. Vif inhibits NCp-mediated formation of tight RNA dimers and hybridization of tRNA(Lys3), while it has little effects on NCp-mediated strand transfer and it collaborates with nucleocapsid (NC) to increase RT processivity. Thus, Vif might negatively regulate NC-assisted maturation of the RNA dimer and early steps of reverse transcription in the assembly complexes, but these inhibitory effects would be relieved after viral budding, thanks to the limited packaging of Vif in the virions.


Subject(s)
Gene Products, vif/metabolism , HIV-1/genetics , Molecular Chaperones/metabolism , RNA, Viral/metabolism , Reverse Transcription , Capsid Proteins/metabolism , DNA, Single-Stranded/biosynthesis , Dimerization , Gene Products, gag/metabolism , RNA, Transfer, Amino Acyl/metabolism , Viral Proteins/metabolism , gag Gene Products, Human Immunodeficiency Virus , vif Gene Products, Human Immunodeficiency Virus
2.
J Mol Biol ; 312(5): 985-97, 2001 Oct 05.
Article in English | MEDLINE | ID: mdl-11580244

ABSTRACT

Retroviral reverse transcriptases use host cellular tRNAs as primers to initiate reverse transcription. In the case of human immunodeficiency virus type 1 (HIV-1), the 3' 18 nucleotides of human tRNA(Lys,3) are annealed to a complementary sequence on the RNA genome known as the primer binding site (PBS). The HIV-1 nucleocapsid protein (NC) facilitates this annealing. To understand the structural changes that are induced upon NC binding to the tRNA alone, we employed a chemical probing method using the lanthanide metal terbium. At low concentrations of NC, the strong terbium cleavage observed in the core region of the tRNA is significantly attenuated. Thus, NC binding first results in disruption of the tRNA's metal binding pockets, including those that stabilize the D-TPsiC tertiary interaction. When NC concentrations approach the amount needed for complete primer/template annealing, NC further destabilizes the tRNA acceptor-TPsiC stem minihelix, as evidenced by increased terbium cleavage in this domain. A mutant form of NC (SSHS NC), which lacks the zinc finger structures, is able to anneal tRNA(Lys,3) efficiently to the PBS, and to destabilize the tRNA tertiary core, albeit less effectively than wild-type NC. This mutant form of NC does not affect cleavage significantly in the helical regions, even when bound at high concentrations. These results, as well as experiments conducted in the presence of polyLys, suggest that in the absence of the zinc finger structures, NC acts as a polycation, neutralizing the highly negative phosphodiester backbone. The presence of an effective multivalent cationic peptide is sufficient for efficient tRNA primer annealing to the PBS.


Subject(s)
HIV-1 , Nucleic Acid Conformation , Nucleocapsid/chemistry , Nucleocapsid/metabolism , RNA, Transfer, Lys/metabolism , RNA/metabolism , Zinc Fingers/physiology , Amino Acid Sequence , Base Sequence , Binding Sites , Humans , Lysine-tRNA Ligase/metabolism , Models, Molecular , Molecular Chaperones/chemistry , Molecular Chaperones/genetics , Molecular Chaperones/metabolism , Molecular Sequence Data , Mutation/genetics , Nucleic Acid Hybridization , Nucleocapsid/genetics , Polylysine/genetics , Polylysine/metabolism , Protein Binding , RNA/chemistry , RNA/genetics , RNA, Transfer, Lys/chemistry , RNA, Transfer, Lys/genetics , Templates, Genetic , Terbium/metabolism , Zinc Fingers/genetics
3.
Proc Natl Acad Sci U S A ; 98(11): 6121-6, 2001 May 22.
Article in English | MEDLINE | ID: mdl-11344257

ABSTRACT

The nucleocapsid protein (NC) of HIV type 1 is a nucleic acid chaperone that facilitates the rearrangement of nucleic acids into conformations containing the maximum number of complementary base pairs. We use an optical tweezers instrument to stretch single DNA molecules from the helix to coil state at room temperature in the presence of NC and a mutant form (SSHS NC) that lacks the two zinc finger structures present in NC. Although both NC and SSHS NC facilitate annealing of complementary strands through electrostatic attraction, only NC destabilizes the helical form of DNA and reduces the cooperativity of the helix-coil transition. In particular, we find that the helix-coil transition free energy at room temperature is significantly reduced in the presence of NC. Thus, upon NC binding, it is likely that thermodynamic fluctuations cause continuous melting and reannealing of base pairs so that DNA strands are able to rapidly sample configurations to find the lowest energy state. The reduced cooperativity allows these fluctuations to occur in the middle of complex double-stranded structures. The reduced stability and cooperativity, coupled with the electrostatic attraction generated by the high charge density of NC, is responsible for the nucleic acid chaperone activity of this protein.


Subject(s)
DNA, Viral/metabolism , HIV-1 , Molecular Chaperones/metabolism , Nucleocapsid/metabolism , Zinc Fingers/physiology , Humans
4.
J Virol ; 75(1): 115-24, 2001 Jan.
Article in English | MEDLINE | ID: mdl-11119580

ABSTRACT

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.


Subject(s)
Gene Products, gag/metabolism , Nucleocapsid/physiology , Simian Immunodeficiency Virus/physiology , Virion/physiology , Zinc/metabolism , Amino Acid Sequence , Animals , Chromatography, High Pressure Liquid , Molecular Sequence Data , Nucleocapsid/chemistry , Structure-Activity Relationship , Terminal Repeat Sequences
5.
J Virol ; 74(24): 11935-49, 2000 Dec.
Article in English | MEDLINE | ID: mdl-11090194

ABSTRACT

Molecular clones were constructed that express nucleocapsid (NC) deletion mutant simian immunodeficiency viruses (SIVs) that are replication defective but capable of completing virtually all of the steps of a single viral infection cycle. These steps include production of particles that are viral RNA deficient yet contain a full complement of processed viral proteins. The mutant particles are ultrastructurally indistinguishable from wild-type virus. Similar to a live attenuated vaccine, this approach should allow immunological presentation of a full range of viral epitopes, without the safety risks of replicating virus. A total of 11 Macaca nemestrina macaques were inoculated with NC mutant SIV expressing DNA, intramuscularly (i.m.) in one study and i.m. and subcutaneously in another study. Six control animals received vector DNA lacking SIV sequences. Only modest and inconsistent humoral responses and no cellular immune responses were observed prior to challenge. Following intravenous challenge with 20 animal infectious doses of the pathogenic SIV(Mne) in a long-term study, all control animals became infected and three of four animals developed progressive SIV disease leading to death. All 11 NC mutant SIV DNA-immunized animals became infected following challenge but typically showed decreased initial peak plasma SIV RNA levels compared to those of control animals (P = 0.0007). In the long-term study, most of the immunized animals had low or undetectable postacute levels of plasma SIV RNA, and no CD4(+) T-cell depletion or clinical evidence of progressive disease, over more than 2 years of observation. Although a subset of immunized and control animals were boosted with SIV(Mne) proteins, no apparent protective benefit was observed. Immunization of macaques with DNA that codes for replication-defective but structurally complete virions appears to protect from or at least delay the onset of AIDS after infection with a pathogenic immunodeficiency virus. With further optimization, this may be a promising approach for vaccine development.


Subject(s)
Macaca nemestrina/immunology , Macaca nemestrina/virology , SAIDS Vaccines/administration & dosage , Simian Acquired Immunodeficiency Syndrome/immunology , Simian Acquired Immunodeficiency Syndrome/prevention & control , Simian Immunodeficiency Virus , Animals , DNA, Viral/genetics , DNA, Viral/immunology , Mutation , Nucleocapsid Proteins/administration & dosage , Nucleocapsid Proteins/genetics , Nucleocapsid Proteins/immunology , SAIDS Vaccines/genetics , SAIDS Vaccines/immunology , Simian Acquired Immunodeficiency Syndrome/virology , Vaccines, DNA/administration & dosage , Vaccines, DNA/genetics , Vaccines, DNA/immunology
6.
J Med Primatol ; 29(3-4): 209-19, 2000 Aug.
Article in English | MEDLINE | ID: mdl-11085583

ABSTRACT

A simian immunodeficiency virus (SIV)(Mne) DNA clone was constructed that produces viruses containing a four amino acid deletion in the second zinc finger of the nucleocapsid (NC) domain of the Gag polyprotein. Viruses produced from this clone, although non-infectious both in vitro and in vivo, complete a majority of the steps in a single retroviral infection cycle. Eight pig-tailed macaques (Macaca nemestrina) were inoculated intramuscularly and subcutaneously three times over the course of 24 weeks with the NC mutant expressing DNA. These macaques, and four controls, were then challenged mucosally (intrarectally) with the homologous virus (SIV Mne CL E11S) and monitored for evidence of infection and clinical disease. Prior to challenge, a measurable humoral immune response was noted in four of eight immunized macaques. After challenge, all 12 macaques became infected, although four immunized animals greatly restricted their viral replication, and one immunized animal that controlled replication remains antibody negative. No disease has been evidence during the 46-week period of monitoring after challenge.


Subject(s)
Antibodies, Viral/blood , Immunity, Mucosal , Nucleocapsid/genetics , SAIDS Vaccines/immunology , Simian Acquired Immunodeficiency Syndrome/immunology , Simian Immunodeficiency Virus/immunology , Vaccines, DNA/immunology , Animals , Antibody Formation , Enzyme-Linked Immunosorbent Assay , Female , Immunoglobulin G/blood , Macaca nemestrina , Nucleocapsid/immunology , Rectum , Simian Acquired Immunodeficiency Syndrome/transmission , Simian Immunodeficiency Virus/genetics , Time Factors , Viral Load , Virion/immunology
7.
J Virol ; 74(19): 8980-8, 2000 Oct.
Article in English | MEDLINE | ID: mdl-10982342

ABSTRACT

The nucleocapsid protein (NC) of human immunodeficiency virus type 1 (HIV-1) has two zinc fingers, each containing the invariant metal ion binding residues CCHC. Recent reports indicate that mutations in the CCHC motifs are deleterious for reverse transcription in vivo. To identify reverse transcriptase (RT) reactions affected by such changes, we have probed zinc finger functions in NC-dependent RT-catalyzed HIV-1 minus- and plus-strand transfer model systems. Our approach was to examine the activities of wild-type NC and a mutant in which all six cysteine residues were replaced by serine (SSHS NC); this mutation severely disrupts zinc coordination. We find that the zinc fingers contribute to the role of NC in complete tRNA primer removal from minus-strand DNA during plus-strand transfer. Annealing of the primer binding site sequences in plus-strand strong-stop DNA [(+) SSDNA] to its complement in minus-strand acceptor DNA is not dependent on NC zinc fingers. In contrast, the rate of annealing of the complementary R regions in (-) SSDNA and 3' viral RNA during minus-strand transfer is approximately eightfold lower when SSHS NC is used in place of wild-type NC. Moreover, unlike wild-type NC, SSHS NC has only a small stimulatory effect on minus-strand transfer and is essentially unable to block TAR-induced self-priming from (-) SSDNA. Our results strongly suggest that NC zinc finger structures are needed to unfold highly structured RNA and DNA strand transfer intermediates. Thus, it appears that in these cases, zinc finger interactions are important components of NC nucleic acid chaperone activity.


Subject(s)
Genome, Viral , HIV-1/genetics , Nucleocapsid Proteins/genetics , Zinc Fingers , Humans
8.
J Virol ; 73(10): 8185-95, 1999 Oct.
Article in English | MEDLINE | ID: mdl-10482569

ABSTRACT

Mutagenesis studies have shown that retroviral nucleocapsid (NC) protein Zn(2+) fingers (-Cys-X(2)-Cys-X(4)-His-X(4)-Cys- [CCHC]) perform multiple functions in the virus life cycle. Moloney murine leukemia virus mutants His 34-->Cys (CCCC) and Cys 39-->His (CCHH) were able to package their genomes normally but were replication defective. Thermal dissociation experiments showed that the CCHH mutant was not defective in genomic RNA dimer structure. Primer tRNA placement on the viral genome and the ability of the tRNA to function in reverse transcription initiation in vitro also appear normal. Some "full-length" DNA copies of the viral genome were synthesized in mutant virus-infected cells. The CCCC and CCHH mutants produced these DNA copies at greatly reduced levels. Circle junction fragments, amplified from two-long-terminal-repeat viral DNA (vDNA) by PCR, were cloned and characterized. Remarkably, it was discovered that vDNA isolated from cells infected with mutant virions had a wide variety of abnormalities at the site at which the two ends of the linear precursor had been ligated to form the circle (i.e., the junction between the 5' end of U3 and the 3' end of U5). In some molecules, bases were missing from regions corresponding to the U3 and U5 linear vDNA termini; in others, the viral sequences extended either beyond the U5 sequences into the primer-binding site and 5' leader or beyond the U3 sequences into the polypurine tract into the env coding region. Still other molecules contained nonviral sequences between the linear vDNA termini. Such defective genomes would certainly be unsuitable substrates for integration. Thus, strict conservation of the CCHC structure in NC is required for infection events prior to and possibly including integration.


Subject(s)
Capsid/genetics , Leukemia Virus, Murine/physiology , Point Mutation , Retroviridae Infections/virology , Tumor Virus Infections/virology , Virus Replication/genetics , Animals , Mice , Mutagenesis, Site-Directed , Zinc Fingers
9.
J Virol ; 73(8): 6670-9, 1999 Aug.
Article in English | MEDLINE | ID: mdl-10400764

ABSTRACT

Integration of retroviral cDNA involves coupled joining of the two ends of the viral genome at precisely spaced positions in the host cell DNA. Correct coupled joining is essential for viral replication, as shown, for example, by the finding that viral mutants defective in coupled joining are defective in integration and replication. To date, reactions with purified human immunodeficiency virus type 1 (HIV-1) integrase protein in vitro have supported mainly uncoupled joining of single cDNA ends. We have analyzed an activity stimulating coupled joining present in HIV-1 virions, which led to the finding that the HIV-1 nucleocapsid (NC) protein can stimulate coupled joining more than 1,000-fold under some conditions. The requirements for stimulating coupled joining were investigated in assays with mutant NC proteins, revealing that mutations in the zinc finger domains can influence stimulation of integration. These findings (i) provide a means for assembling more authentic integrase complexes for mechanistic studies, (ii) reveal a new activity of NC protein in vitro, (iii) indicate a possible role for NC in vivo, and (iv) provide a possible method for identifying a new class of inhibitors that disrupt coupled joining.


Subject(s)
DNA, Viral , HIV Integrase/metabolism , HIV-1/genetics , Nucleocapsid Proteins/metabolism , Virus Integration , Amino Acid Sequence , DNA, Complementary , Humans , Molecular Sequence Data , Mutagenesis , Viral Proteins/metabolism , Virion
10.
Virology ; 256(1): 92-104, 1999 Mar 30.
Article in English | MEDLINE | ID: mdl-10087230

ABSTRACT

The retroviral nucleocapsid (NC) protein contains highly conserved amino acid sequences (-Cys-X2-Cys-X4-His-X4-Cys-) designated retroviral (CCHC) Zn2+ fingers. The NC protein of murine leukemia viruses contains one NC Zn2+ finger and mutants that were competent in metal binding (CCCC and CCHH) packaged wild-type levels of full-length viral RNA but were not infectious. These studies were extended to human immunodeficiency virus type 1 (HIV-1), a virus with two NC Zn2+ fingers. Viruses with combinations of CCHC, CCCC, and CCHH Zn2+ fingers in each position of HIV-1 NC were characterized. Mutant particles contained the normal complement of processed viral proteins. Four mutants packaged roughly wild-type levels of genomic RNA, whereas the remaining mutants packaged reduced levels. Virions with mutated C-terminal position NC fingers were replication competent. One interesting mutant, containing a CCCC Zn2+ finger in the N-terminal position of NC, packaged wild-type levels of viral RNA and showed approximately 5% wild-type levels of infectivity when examined in CD4-expressing HeLa cells containing an HIV-1 LTR/beta-galactosidase construct. However, this particular mutant was replication defective in H9 cells; all other mutants were replication defective over the 8-week course of the assay. Two long terminal repeat viral DNA species could be detected in the CCCC mutant but not in any of the other replication-defective mutants. These studies show that the N-terminal Zn2+ finger position is more sensitive to alterations than the C-terminal position with respect to replication. Additionally, the retroviral (CCHC) NC Zn2+ finger is required for early infection processes. The evolutionary pressure to maintain CCHC NC Zn2+ fingers depends mainly on its function in infection processes, in addition to its function in genome packaging.


Subject(s)
HIV-1/physiology , HIV-1/pathogenicity , Nucleocapsid/metabolism , Amino Acid Sequence , Amino Acid Substitution , Base Sequence , Cell Line , Conserved Sequence , DNA Primers , HIV Long Terminal Repeat , HIV-1/genetics , HeLa Cells , Humans , Molecular Sequence Data , Mutagenesis, Site-Directed , Nucleocapsid/chemistry , Nucleocapsid/genetics , Polymerase Chain Reaction , Transfection , Virus Replication , Zinc Fingers
11.
J Mol Biol ; 287(1): 59-75, 1999 Mar 19.
Article in English | MEDLINE | ID: mdl-10074407

ABSTRACT

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.


Subject(s)
Capsid Proteins , Capsid/metabolism , Gene Products, gag/metabolism , HIV-1 , Poly A/metabolism , RNA-Binding Proteins/metabolism , Viral Proteins , Zinc Fingers , Amino Acid Sequence , Binding Sites , Capsid/genetics , Fluorescent Dyes/metabolism , Gene Products, gag/genetics , Molecular Sequence Data , Mutation , Peptide Fragments/metabolism , Protein Binding , RNA-Binding Proteins/genetics , Repetitive Sequences, Amino Acid , Thermodynamics , Zinc Fingers/genetics , gag Gene Products, Human Immunodeficiency Virus
12.
Virology ; 253(2): 259-70, 1999 Jan 20.
Article in English | MEDLINE | ID: mdl-9918884

ABSTRACT

All retroviruses (except the spumaretroviruses) contain a nucleocapsid (NC) protein that encodes one or two copies of the Zn2+-finger sequence -Cys-X2-Cys-X4-His-X4-Cys-. This region has been shown to be essential for recognition and packaging of the genomic RNA during virion particle assembly. Additionally, this region has been shown to be involved in early infection events in a wide spectrum of retroviruses, including mammalian type C [e.g., murine leukemia virus (MuLV)], human immunodeficiency virus type 1 (HIV-1), Rous sarcoma virus, and other retroviruses. Mutations in the two Zn2+-fingers of the NC protein of simian immunodeficiency virus strain Mne [SIV(Mne)] have been generated. The resulting virions contained the normal complement of processed viral proteins with densities indistinguishable from wild-type SIV(Mne). All of the mutants had electron micrograph morphologies similar to those of immature particles observed in wild-type preparations. RNA packaging was less affected by mutations in the NC protein of SIV(Mne) than has been observed for similar mutants in the MuLV and HIV-1 systems. Nevertheless, in vitro replication of SIV(Mne) NC mutants was impaired to levels comparable to those observed for MuLV and HIV-1 NC mutants; replication defective NC mutants are typically 10(5)- to 10(6)-fold less infectious than similar levels of wild-type virus. One mutant, DeltaCys33-Cys36, was also found to be noninfectious in vivo when mutant virus was administered intravenously to a pig-tailed macaque. NC mutations can therefore be used to generate replication defective virions for candidate vaccines in the SIV macaque model for primate lentiviral diseases.


Subject(s)
Mutation , Nucleocapsid Proteins/genetics , Simian Immunodeficiency Virus/genetics , Zinc Fingers , Amino Acid Sequence , Animals , Cell Line, Transformed , Cysteine , Humans , Mice , Molecular Sequence Data , Nucleic Acid Hybridization , Polymerase Chain Reaction/methods , RNA, Viral/analysis , Simian Immunodeficiency Virus/physiology , Simian Immunodeficiency Virus/ultrastructure , Virion , Virus Replication
13.
J Virol ; 71(9): 6940-6, 1997 Sep.
Article in English | MEDLINE | ID: mdl-9261422

ABSTRACT

In an early step in the retroviral infectious process, reverse transcriptase copies the genomic RNA of the virus into complementary minus-strand DNA. The primer for this synthetic event is a molecule of cellular tRNA, which is annealed by its 3' 18 nucleotides to a region of the genomic RNA termed the primer-binding site (PBS); the sequence of the PBS and hence the identity of the tRNA depend upon the retrovirus species. In addition to the primer tRNA, retrovirus particles contain a substantial number of other tRNA molecules. The latter tRNA population is enriched for the tRNA species which serves as primer for the virus. While there is considerable evidence that the enrichment for the primer species can be attributed to the pol gene product, nothing is known regarding mechanisms of annealing the primer to the PBS. We have analyzed pol- mutants of avian leukosis virus (ALV) and murine leukemia virus (MuLV) for the presence of primer at the PBS in virion genomic RNA. Remarkably, the results were different for the two viruses: the PBS was substantially occupied by primer in MuLV but not in ALV. Previous data indicates that the Pol-dependent enrichment of the primer within the virion is much greater in ALV than in MuLV. We therefore propose that the absence of primer at the PBS in pol- ALV is due to the deficiency of the primer species within the particle. The results suggest that, at least in MuLV, the tRNA is unwound by either the Gag protein or a cellular protein for annealing to the PBS. Further, the C-terminal 17 amino acids of Gag are unnecessary for this function in MuLV.


Subject(s)
Avian Leukosis Virus/genetics , Genes, pol , Moloney murine leukemia virus/genetics , RNA, Transfer , RNA, Viral , Animals , Binding Sites , Chick Embryo , Gene Deletion , Gene Expression , RNA
14.
AIDS Res Hum Retroviruses ; 13(7): 533-43, 1997 May 01.
Article in English | MEDLINE | ID: mdl-9135871

ABSTRACT

HIV-1 nucleocapsid, p7, contains two retroviral zinc fingers, which are both necessary for efficient packaging of genomic RNA and infectivity. The nucleocapsid protein is bound tightly to genomic RNA in the mature virion. In this study, the effect of p7 on polymerization of nascent cDNA by viral reverse transcriptase (RT) was examined. An 874-base RNA of HIV-1 was synthesized and used as a template in RT assays with varying concentrations of intact p7, mutants of p7 that have transposed or repeated zinc fingers, and several different peptides that represent various structural regions of p7. Results indicate that at greater than or equal to 50% saturation of p7-binding sites, with p7, there is up to a 90% reduction in total cDNA synthesis, as measured by nucleotide incorporation. However, the cDNA products that are made are almost exclusively full length. Three zinc finger mutants exhibited effects similar to those of wild-type p7. N-terminal and C-terminal halves of p7 inhibited total nucleotide incorporation, but also inhibited synthesis of long cDNA products by RT. In the absence of p7 an array of short transcripts (< 200 bases) was produced by RT. These studies show that full-length p7 is necessary to increase the proportion of long cDNA transcripts produced by RT. The relative position of the two zinc fingers is not critical for this effect.


Subject(s)
Capsid Proteins , Capsid/genetics , DNA, Complementary/metabolism , Gene Products, gag/genetics , HIV Reverse Transcriptase/metabolism , HIV-1/genetics , HIV-1/metabolism , Amino Acid Sequence , DNA Transposable Elements , Electrophoresis, Agar Gel , Molecular Sequence Data , Plasmids , Polymerase Chain Reaction , RNA, Viral/metabolism , Repetitive Sequences, Nucleic Acid , Sequence Alignment , Transcription, Genetic , Viral Proteins/analysis , Zinc Fingers/genetics , gag Gene Products, Human Immunodeficiency Virus
15.
Virology ; 230(1): 134-44, 1997 Mar 31.
Article in English | MEDLINE | ID: mdl-9126269

ABSTRACT

Identification and quantitation of cellular proteins associated with HIV-1 particles are complicated by the presence of nonvirion-associated cellular proteins that copurify with virions. Many cellular proteins are associated with nonviral particles that bud from the surface of cells called microvesicles. Microvesicles band in sucrose gradients in a range of densities that includes the same density as retroviruses. To characterize these microvesicles, HIV-1-infected and uninfected human T-cell lines were propagated and virus and microvesicles were purified from clarified cell culture supernatants by sucrose density gradient centrifugation or centrifugation through 20% sucrose pads. Microvesicles were found to contain various proteins, including HLA DR and beta 2-M, and a substantial amount of RNA and DNA. The concentrations of HIV-1 p24CA, HLA DR and beta 2-microglobulin (beta 2-M) were determined by radioimmunoassay. The ratios of HIV-1 p24CA to HLA DR and beta 2-M were found to vary with respect to the HIV-1 isolate, host cell, and other factors. Electron microscopic analysis of microvesicles revealed that they consisted of particles of various sizes and morphologies. Although HIV-1 particles are known to contain some cellular proteins, microvesicles from HIV-1 infected H9 cells appeared to contain little or no HIV-1 gp120SU.


Subject(s)
HIV-1/isolation & purification , Proteins/analysis , HIV Core Protein p24/analysis , HIV Envelope Protein gp120/analysis , HLA-DR Antigens/analysis , Humans , Leukocytes, Mononuclear/virology , Organelles/chemistry , T-Lymphocytes/chemistry , T-Lymphocytes/virology , Tumor Cells, Cultured
16.
J Virol ; 70(10): 7132-42, 1996 Oct.
Article in English | MEDLINE | ID: mdl-8794360

ABSTRACT

In an earlier study on minus-strand DNA synthesis catalyzed by murine leukemia virus reverse transcriptase, we described a prominent pause site near the polypurine tract (J. Guo, W. Wu, Z. Y. Yuan, K. Post, R. J. Crouch, and J. G . Levin, Biochemistry 34:5018-5029, 1995). We now report that pausing at this site is due to a stem-loop structure in the RNA template, formed by interaction of a number of bases in the polypurine tract, including the six G's, and a 3' sequence which includes four C's. Addition of human immunodeficiency virus type 1 (HIV-1) nucleocapsid (NC) protein to reverse transcriptase reactions reduces pausing by approximately 8- to 10-fold and stimulates synthesis of full-length DNA. Thus, NC functions as an accessory protein during elongation of minus-strand DNA and increases the efficiency of DNA synthesis, in this case, by apparently destabilizing a region of secondary structure in the template. Since NC is associated with genomic RNA in the viral core and is likely to be part of a viral replication complex, these results suggest that NC may also promote efficient DNA synthesis during virus replication. Mutational analysis indicates that the features of HIV-1 NC which are important for reduction of pausing include the basic amino acids flanking the first zinc finger, the zinc fingers, and the cysteine and aromatic amino acids within the fingers. These findings suggest that reverse transcription might be targeted by drugs which inactivate the zinc fingers of HIV-1 NC.


Subject(s)
Capsid/metabolism , HIV Reverse Transcriptase/metabolism , HIV-1/metabolism , Leukemia Virus, Murine/metabolism , Amino Acid Sequence , Animals , Base Sequence , Binding Sites , DNA Mutational Analysis , Humans , Mice , Molecular Sequence Data , Protein Structure, Secondary , Sequence Analysis
17.
Proc Natl Acad Sci U S A ; 93(15): 7577-81, 1996 Jul 23.
Article in English | MEDLINE | ID: mdl-8755517

ABSTRACT

After a retrovirus particle is released from the cell, the dimeric genomic RNA undergoes a change in conformation. We have previously proposed that this change, termed maturation of the dimer, is due to the action of nucleocapsid (NC) protein on the RNA within the virus particle. We now report that treatment of a 345-base synthetic fragment of Harvey sarcoma virus RNA with recombinant or synthetic HIV-1 NC protein converts a less stable form of dimeric RNA to a more stable form. This phenomenon thus appears to reproduce the maturation of dimeric retroviral RNA in a completely defined system in vitro. To our knowledge, maturation of dimeric RNA within a retrovirus particle is the first example of action of an "RNA chaperone" protein in vivo. Studies with mutant NC proteins suggest that the activity depends upon basic amino acid residues flanking the N-terminal zinc finger and upon residues within the N-terminal finger, including an aromatic amino acid, but do not require the zinc finger structures themselves.


Subject(s)
HIV-1/metabolism , Harvey murine sarcoma virus/genetics , Nucleocapsid/metabolism , RNA, Double-Stranded/biosynthesis , RNA, Viral/biosynthesis , Amino Acid Sequence , Harvey murine sarcoma virus/metabolism , Kinetics , Molecular Sequence Data , Mutagenesis, Site-Directed , Nucleocapsid/biosynthesis , Nucleocapsid/chemistry , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Thermodynamics , Transcription, Genetic , Zinc Fingers
18.
J Virol ; 70(4): 2593-7, 1996 Apr.
Article in English | MEDLINE | ID: mdl-8642691

ABSTRACT

The effect of changing zinc (Zn2+)-coordinating residues in the nucleocapsid protein of Moloney murine leukemia virus was investigated by introducing a His-34-to-Cys or Cys-39-to-His mutation into the putative Zn2+ finger. Mutant virions contained normal levels of properly processed Gag and Env proteins and wild-type levels of full-length viral RNA. However, the specific infectivity of the mutants was approximately 4 x 10(-4) that of wild-type particles. They were probably noninfectious because of the inability of the particles to synthesize cDNA transcripts, since full-length viral DNA could not be detected in Hirt supernatants of NIH 3T3 cells infected with the CCCC or CCHH virus. These mutants will provide an extremely valuable tool for analysis of the role of retroviral Zn2+ fingers in infection processes, independent of viral RNA recognition and packaging.


Subject(s)
Capsid/physiology , Moloney murine leukemia virus/physiology , Viral Core Proteins/physiology , Zinc Fingers/physiology , Zinc/physiology , 3T3 Cells , Amino Acid Sequence , Animals , Base Sequence , Capsid/genetics , Cell Line , DNA, Viral , Humans , Mice , Molecular Sequence Data , Moloney murine leukemia virus/genetics , Moloney murine leukemia virus/pathogenicity , Mutagenesis, Site-Directed , RNA, Viral/metabolism , Viral Core Proteins/genetics , Virus Replication/genetics , Virus Replication/physiology , Zinc Fingers/genetics
19.
J Virol ; 68(9): 6124-9, 1994 Sep.
Article in English | MEDLINE | ID: mdl-8057495

ABSTRACT

We have analyzed RNA packaging by a series of mutants altered in the nucleocapsid (NC) protein of Moloney murine leukemia virus (Mo-MuLV). We found that mutants lacking residues 8 through 11 or 44 through 60 of NC package Mo-MuLV RNA with virtually the same efficiency as wild-type Mo-MuLV. In contrast, point mutants altered at the conserved cysteines in the cysteine array (residues 26 and 29) and a mutant lacking residues 16 through 23 packaged Mo-MuLV RNA with approximately 1% of the efficiency of wild-type Mo-MuLV. The deficiency in packaged RNA was observed not only in Northern (RNA) analysis but also in an RNA-PCR assay, which would detect degraded as well as intact RNA. One of the cysteine array mutants was also shown to be defective with respect to encapsidation of hygromycin phosphotransferase mRNA containing a Mo-MuLV packaging signal. We suggest that a central region of NC, consisting of the cysteine array and flanking basic residues, is required for RNA packaging in Mo-MuLV.


Subject(s)
Capsid/metabolism , Moloney murine leukemia virus/growth & development , Viral Core Proteins/metabolism , Virus Replication , Amino Acid Sequence , Capsid/chemistry , Macromolecular Substances , Molecular Sequence Data , RNA, Viral/metabolism , Sequence Deletion , Structure-Activity Relationship , Viral Core Proteins/chemistry , Zinc Fingers
20.
J Virol ; 68(8): 5013-8, 1994 Aug.
Article in English | MEDLINE | ID: mdl-8035501

ABSTRACT

We have characterized the dimeric genomic RNA in particles of both wild-type and protease (PR)-deficient human immunodeficiency virus type 1 (HIV-1). We found that the dimeric RNA isolated from PR- mutant virions has a lower mobility in nondenaturing gel electrophoresis than that from wild-type virions. It also dissociates into monomers at a lower temperature than the wild-type dimer. Thus, the dimer in PR- particles is in a conformation different from that in wild-type particles. These results are quite similar to recent findings on Moloney murine leukemia virus and suggest that a postassembly, PR-dependent maturation event is a common feature in genomic RNAs of retroviruses. We also measured the thermal stability of the wild-type and PR- dimeric RNAs under different ionic conditions. Both forms of the dimer were stabilized by increasing Na+ concentrations. However, the melting temperatures of the two forms were not significantly affected by the identity of the monovalent cation present in the incubation buffer. This observation is in contrast with recent reports on dimers formed in vitro from short segments of HIV-1 sequence: the latter dimers are specifically stabilized by K+ ions. K+ stabilization of dimers formed in vitro has been taken as evidence for the presence of guanine quartet structures. The results suggest that guanine quartets are not involved in the structure linking full-length, authentic genomic RNA of HIV-1 into a dimeric structure.


Subject(s)
HIV Protease/metabolism , HIV-1/genetics , RNA, Viral/chemistry , Biopolymers , HIV-1/enzymology , HeLa Cells , Humans , Nucleic Acid Conformation , Sodium Chloride , Temperature , Virion/enzymology , Virion/genetics
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