The spacer peptide between human immunodeficiency virus capsid and nucleocapsid proteins is essential for ordered assembly and viral infectivity.

Morphogenesis of retroviruses involves ordered assembly of the structural Gag- and Gag-Pol polyproteins, with subsequent budding from the plasma membrane and proteolytic cleavage by the viral proteinase (PR). Two cleavage sites exist between the capsid (CA) and nucleocapsid (NC) domains of the human immunodeficiency virus (HIV) type 1 Gag polyprotein which are separated by a 14-amino-acid spacer peptide of unknown function. To analyze the role of the two cleavage sites and the spacer peptide, both sites were individually mutated and a deletion mutation that precisely removes the spacer peptide was constructed. Following transfection of proviral DNA carrying the point mutations, mutant polyproteins were synthesized and assembled like wild-type polyprotein, and release of particles was not significantly altered. Both mutations abolished cleavage at the respective site and reduced or abolished viral infectivity. Deletion of the spacer peptide severely affected ordered assembly and reduced particle release. The extracellular particles that were released exhibited normal density but were heterogeneous in size. Electron micrographs revealed large electron-dense plaques underneath the plasma membrane of transfected cells which appeared like confluent ribonucleoprotein complexes arrested early in the budding process. Extracellular particles exhibited very aberrant and heterogeneous morphology and were incapable of inducing viral spread. These particles may correspond to membrane vesicles sequestered by the rigid structures underneath the cell membrane and not released by a regular budding process.

Stoichiometry of mitochondrial transcripts and regulation of gene expression by mitochondrial transcription factor A.

The steady state concentration of cytochrome c oxidase subunit I mRNA and 12 S rRNA, respectively, measured by a quantitative reverse transcription/polymerase chain reaction method, was 4 and 15 molecules per molecule of mt DNA in rat liver and 2 and 9 molecules in rat muscle, respectively. These results imply that in the mitochondrial compartment, the molar concentration of all thirteen mRNAs by far exceeds the concentration of ribosomes, a situation fundamentally different from the cytosolic compartment. Following thyroid hormone treatment, both mitochondrial transcripts increased, in parallel with the mRNA encoding mitochondrial transcription factor A. We conclude that this transcription factor might be the rate limiting factor for mitochondrial transcription in vivo, at least under these conditions.

A large deletion in the matrix domain of the human immunodeficiency virus gag gene redirects virus particle assembly from the plasma membrane to the endoplasmic reticulum.

Morphogenesis of retroviruses involves assembly of the structural Gag and Gag-Pol polyproteins with subsequent budding of the virus particle from the plasma membrane and proteolytic cleavage by the viral proteinase. The matrix (MA) domain, representing the N-terminal segment of Gag, plays a critical role in this process. We constructed an in-frame deletion in the MA coding region (lacking codons 16 to 99) of the human immunodeficiency virus (HIV) type 1 gag gene. Following transient transfection of the complete proviral DNA carrying the deletion, the mutant polyprotein was synthesized and proteolytically processed like the wild-type polyprotein. However, release of virus particles was reduced approximately 10-fold. The extracellular particles that were released did not contain viral glycoproteins and were noninfectious. Electron micrographs revealed budding of virus particles into the endoplasmic reticulum (ER) of transfected cells and large numbers of particles within the ER. These particles were all immature and morphologically indistinguishable from intracisternal A-type particles, a class of murine endogenous retrovirus elements. Budding structures at the plasma membrane were rarely seen and only a few extracellular particles were observed, but in contrast to those in the ER, these particles had the morphology of mature particles, similar to that of wild-type HIV, except for the lack of surface projections.

Cell-free translation and proteolytic processing of the hepatitis A virus polyprotein.

Virus-encoded proteinase activity of hepatitis A virus (HAV) was studied in vitro. Genomic regions coding for segments of the viral polyprotein were expressed by in vitro transcription and translation in rabbit reticulocyte lysates. Polyproteins translated from synthetic transcripts encoding P1-P2 or delta VP1-P2 were not processed indicating that no proteolytic activity is encoded within P2 of HAV, in contrast to other picornaviruses. Proteinase activity was, however, detected in the genomic region encoding 3C. Mutant transcripts (mu) which encode an alanine in place of the cysteine residue at amino acid position 172 of 3C did not yield proteolytic activity, consistent with the hypothesis that proteinase 3C is a cysteine-containing trypsin-like proteinase. Processing products 3ABC and P3 were identified by immunoprecipitation, providing evidence that proteolytic cleavage occurs at the 2C/3A and less frequently at the 3C/3D junction. For cleavages at either site, the complete 3D moiety was not required. In general, analysis of cleavage products was made difficult by the presence of polypeptides which were translated from internal start sites, predominantly within the P3 region. Since only small amounts of the full-length products P1-P2-P3 or P2-P3 were translated, possible cleavage of P1 and P2 by 3C could not be resolved in this system. Furthermore, no intermolecular cleavage could be detected when in vitro translated polypeptides of the P3 region were incubated with P1, P1-P2 or P2-P3 mu as substrates.

Analysis of protein expression and virus-like particle formation in mammalian cell lines stably expressing HIV-1 gag and env gene products with or without active HIV proteinase.

Cell lines stably releasing noninfectious virus-like particles containing wild type or mutant gene products represent useful tools for a biochemical, immunological, and structural analysis of virus assembly. Human immunodeficiency virus (HIV) type 1 gag and env gene products were transiently and stably expressed in mammalian cells and the formation of virus-like particles incorporating viral glycoproteins was analyzed. Transient cotransfection of plasmids directing the synthesis of gag and env gene products yielded efficient release of particles but specific incorporation of HIV glycoproteins was not detected. A stable cell line expressing wild type HIV-1 glycoproteins was generated and transient transfection of this cell line with gag-encoding constructs led to the release of virus-like particles incorporating HIV surface and transmembrane glycoproteins. Attempts to establish stable cell lines expressing wild type HIV gag and pol genes were unsuccessful and only highly unstable lines primarily expressing uncleaved precursor polyproteins were obtained. This result appears to be caused by the cytotoxic effects of the viral proteinase since stable lines were readily selected after transfection of constructs either encoding an inactive mutant of the proteinase or a mutated frameshift signal which prevented expression of the pol reading frame. Stable coexpression of uncleaved Gag polyprotein and wild type env gene products yielded efficient release of immature virus-like particles incorporating HIV glycoproteins. Electron micrographs revealed lentiviral budding structures with the typical surface projections of viral glycoprotein oligomers.

Analysis of HIV particle formation using transient expression of subviral constructs in mammalian cells.

Segments of the human immunodeficiency virus (HIV) type 1 gag and pol genes and mutants thereof were transiently expressed in mammalian cells. Expression was dependent on the presence of the rev responsive element in cis and the rev protein in trans and was readily detected by indirect immunofluorescence or Western blotting. Transfection of constructs encoding the entire gag and pol open reading frames yielded efficient release of particles banding at a density of 1.16 g of sucrose per milliliter and consisting mainly of processed gag proteins. In addition, these particles contained the p66/p51 heterodimer of reverse transcriptase (RT), had associated RT activity, and contained RNA. Electron micrographs revealed immature retrovirus-like particles budding primarily from the plasma membrane and extracellular particles with morphological characteristics of HIV. Particle production was independent of the pol open reading frame or an active HIV proteinase (PR) but without active PR, cell-associated and particle-associated proteins remained completely uncleaved and budding occurred primarily into intracellular vacuoles. A mutation preventing myristoylation of the viral polyproteins abolished particle release but did not interfere with polyprotein synthesis and did not prevent processing. Expression of gag and PR in the same reading frame yielded complete processing of polyproteins but no budding and led to increased cell toxicity. A mutation of the PR active site in this construct prevented cytotoxicity and restored particle release indicating that the observed phenotype was caused by the overexpression of PR. These particles were aberrant in size and morphology when analyzed on sucrose density gradients and by electron microscopy. Budding was arrested at an early stage and extracellular particles appeared to be released by a different mechanism. Only short C-terminal extensions were compatible with this release mechanism since expression of a similar mutant construct encoding the entire gag-pol open reading frame did not yield particles.

Characterization of poliovirus 2A proteinase by mutational analysis: residues required for autocatalytic activity are essential for induction of cleavage of eukaryotic initiation factor 4F polypeptide p220.

The poliovirus proteinase 2A is autocatalytically released from the poliovirus polyprotein by cotranslational cleavage at its own amino terminus, resulting in separation of structural and nonstructural protein precursors. Cleavage is a prerequisite for further processing of the structural protein precursor and consequently for poliovirus encapsidation. A second function of 2Apro is in the rapid shutoff of host cell protein synthesis that occurs upon infection with poliovirus. This is associated with proteolytic cleavage of the p220 component of eukaryotic initiation factor eIF-4F, which is induced but not directly catalyzed by 2Apro. We introduced single-amino-acid substitutions in the 2Apro-coding region of larger poliovirus precursors that were subsequently translated in vitro and thus demonstrated that His-20, Asp-38, and Cys-109 (which constitute the putative catalytic triad) are essential for, and that His-117 is an important determinant of, the autocatalytic activity of 2Apro. This is consistent with the proposal that 2Apro is structurally related to a subclass of trypsinlike serine proteinases. Moreover, 2Apro containing a Cys109Ser substitution retained a small but significant autocatalytic activity. Cleavage of p220 was not induced by those mutants that had reduced proteolytic activity, indicating that the cellular factor that cleaves p220 is probably activated by 2Apro-catalyzed proteolytic cleavage.