Immature Virus-like Particles Research Articles

Further Evidence for Hexagonal Organization of HIV gag Protein in Prebudding Assemblies and Immature Virus-like Particles

The fullerene-like model for the organization of HIV gag encoded precursor pr55gag was based on the study of prebudding assemblies at the plasma membrane of cells infected with a recombinant baculovirus expressing HIV-1 gag protein. The objective of the present study was to support the model by image processing of virus-like particles (VLP). In this work we used VLP purified by density gradient centrifugation, which caused partial or occasionally complete loss of the lipid bilayer in some VLP without the use of detergent. In addition more plasma membrane-associated pr55gag protein assemblies were processed. Image processing of negatively stained specimens revealed the presence of threefold symmetry and a hexagonal network of rings with a resolution of 29 A in VLP and better than 25 A in membrane associated assemblies. The center-to-center spacing of the rings was 67 A in VLP and 70 A in membrane assemblies. Patches of gag protein oligomers at the plasma membrane were usually round and varying in size, but some of them were triangular. Indication of triangular-shaped gag protein assemblies was also seen in partly dissociated VLP. Since the hexagonal network is formed by the uncleaved gag polyprotein, we conclude that the threefold symmetry applies to all domains including p24gag. The presence of threefold symmetry and the hexagonal network in VLP are consistent with the hypothesis that immature HIV particles possess icosahedral symmetry.

Characterization of deletion mutations in the capsid region of human immunodeficiency virus type 1 that affect particle formation and Gag-Pol precursor incorporation

The core of human immunodeficiency virus type 1 is derived from two precursor polyproteins, Pr55gag and Pr160gag-pol. The Gag precursor can assemble into immature virus-like particles when expressed by itself, while the Gag-Pol precursor lacks particle-forming ability. We have shown previously that the Gag precursor is able to "rescue" the Gag-Pol precursor into virus-like particles when the two polyproteins are expressed in the same cell by using separate simian virus 40-based plasmid expression vectors. To understand this interaction in greater detail, we have made deletion mutations in the capsid-coding regions of Gag- and Gag-Pol-expressing plasmids and assayed for the abilities of these precursors to assemble into virus-like particles. When we tested the abilities of Gag-Pol precursors to be incorporated into particles of Gag by coexpressing the precursors, we found that mutant Gag-Pol precursors lacking a conserved region in retroviral capsid proteins, the major homology region (MHR), were excluded from wild-type Gag particles. Mutant precursors lacking MHR were also less efficient in processing the Gag precursor in trans. These results suggest that the MHR is critical for interactions between Gag and Gag-Pol molecules. In contrast to these results, expression of mutated Gag precursors alone showed that deletions in the capsid region, including those which removed the MHR, reduced the efficiency of particle formation by only 40 to 50%. The mutant particles, however, were clearly lighter than the wild type in sucrose density gradients. These results indicate that the requirements for Gag particle formation differ from the ones essential for efficient incorporation of the Gag-Pol precursor into these particles.

Proteolytic processing of particle-associated retroviral polyproteins by homologous and heterologous viral proteinases.

Retroviral proteinase(PR)-catalyzed cleavage of the viral Gag and Gag-Pol polyproteins within the nascent virus particle is required for productive viral infection. Kinetic characterization and specificity analyses have been reported for several retroviral PR using oligopeptide substrates. In this study, we performed a comparative analysis of PR from avian, bovine, simian and human retroviruses using polyproteins of human immunodeficiency virus (HIV) type 1 or avian leukosis virus as substrates. Polyproteins were derived from immature virus-like particles purified from culture medium of transfected or recombinant baculovirus-infected cells. Specific cleavage to the correct size intermediate and end products occurred in the presence of detergent and homologous PR. HIV-1 PR cleaved its Gag precursor to completion at a concentration of approximately 25 nM but cleaved the Gag-Pol precursor incompletely even at fourfold higher PR concentration. In contrast to the requirement for high ionic strength for peptide cleavage reported previously, we found that Gag protein cleavage by HIV-1 PR proceeded best at low ionic strength, for both of the protein substrates tested. HIV-2 PR was approximately sixfold less active than HIV-1 PR. PR from avian myeloblastosis-associated virus (MAV) yielded efficient cleavage of the HIV-1 polyprotein only at concentrations above 1 microM. Both enzymes were stimulated by high salt and their cleavage products were identical or very similar to those of HIV-1 PR. A mutant of MAV PR engineered to cleave HIV-1 peptide substrates did not cleave the HIV-1 polyprotein at a concentration of 0.4 microM. The PR of Mason Pfizer monkey virus cleaved this polyprotein very poorly, whereas PR of bovine leukemia virus cleaved it, albeit at different sites.

Retrovirus-like particles produced by vaccinia viruses expressing gag-pro-pol region genes of bovine leukaemia virus

Processing and assembly of bovine leukaemia virus-like particles were studied in African green monkey kidney cells using recombinant vaccinia viruses (rVVs) expressing regions of the bovine leukaemia virus genome. Unprocessed gag precursor protein (Pr44) was detected in immunoblot analysis of lysed cells and particles sedimented from culture supernatants after infection with a rVV carrying the gag and truncated protease (pro) gene. Processing of Pr44 was observed after infection of cells with a rVV carrying the gag and pro gene or a rVV expressing the gag, pro and polymerase (pol) gene. Reverse transcriptase activity was detected only in association with particles produced by gag-, pro- and pol-expressing recombinants. Thin section electron microscopic analysis of infected cells and pelleted particles revealed that Pr44 and processed gag proteins assembled at the cell membrane. Pr44 was released into the cell culture media as immature virus-like particles, whereas processed gag proteins from rVVs expressing gag and pro or gag, pro and pol formed mature particles.

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.

Intranuclear crystal formation in picornavirus-infected cells

Cells infected with echovirus or poliovirus were incubated at a suboptimal temperature (28 degrees C). When the cells were examined by electron microscopy, not only were crystalline arrays of mature virus particles observed in the cytoplasm but crystals composed of immature virus-like particles were also evident in the less electron-dense central region of the nucleus. Immunofluorescent and autoradiographic studies revealed the presence of viral proteins and RNA in the nucleus. These findings strongly suggest that the intranuclear crystals are composed of incomplete virus particles and that crystal formation at such a low temperature may be a remarkable feature common to picornaviruses. Possible mechanisms for the intranuclear synthesis of viral constituents are discussed.