Characterization of the block in replication of nucleocapsid protein zinc finger mutants from moloney murine leukemia virus.

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.

Strict conservation of the retroviral nucleocapsid protein zinc finger is strongly influenced by its role in viral infection processes: characterization of HIV-1 particles containing mutant nucleocapsid zinc-coordinating sequences.

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.

Nucleocapsid protein zinc-finger mutants of simian immunodeficiency virus strain mne produce virions that are replication defective in vitro and in vivo.

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.

Wild-type and mutant HIV type 1 nucleocapsid proteins increase the proportion of long cDNA transcripts by viral reverse transcriptase.

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.

Microvesicles are a source of contaminating cellular proteins found in purified HIV-1 preparations.

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.

Human immunodeficiency virus type 1 nucleocapsid protein reduces reverse transcriptase pausing at a secondary structure near the murine leukemia virus polypurine tract.

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.

HIV-1 nucleocapsid protein induces "maturation" of dimeric retroviral RNA in vitro.

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.

Transforming growth factor-beta isoform expression in insulin-like growth factor stimulated myogenesis.

Transforming growth factor betas (TGF-beta s) are the defining members of a super-family of small proteins that are involved in the regulation of development and morphogenesis in a wide array of systems. Previous studies have demonstrated that TGF-beta s both inhibit and, under specialized conditions, induce the differentiation of myoblasts. TGF-beta have been shown to be secreted by mouse C2C12 myoblast cultures undergoing differentiation. Insulin-like growth factors (IGFs) have also been shown to be secreted by myoblasts and to induce myogenesis. This study characterizes the effect of IGF treatment on the expression and secretion of TGF-beta s in the IGF-sensitive L6A1 myoblast line. IGF downregulated the expression of TGF-beta 3 in a concentration-dependent manner at 24 and 48 hours; TGF-beta 1 was not sensitive to IGF treatment at 24 hours but was downregulated by IGFs at 48 hours. This downregulation was mediated by the type 1 IGF receptor and modulated by IGF binding proteins secreted by the myoblasts. Some reexpression of TGF-beta 1 and TGF-beta 3 mRNAs was observed after extensive morphological differentiation had occurred. These results support the hypothesis that IGFs act through the IGF type I receptor as part of a concerted mechanism to modulate expression of the TGF-beta genes, as part of a coordinated set of changes associated with terminal myogenic differentiation.