Stefan Höglund

Structural proteins of adenoviruses

Abstract The penton antigen from adenovirus type 2 has been purified by DEAE chromatography, agarose chromatography and preparative polyacrylamide electrophoresis. The final product is homogeneous by electron microscopy, immunoelectrophoresis, polyacrylamide electrophoresis, and analytical ultracentrifugation. Penton antigen so prepared has a sedimentation coefficient of 10.5 and a molecular weight around 400,000. A minimum of three antigenic specificities can be demonstrated with immunodiffusion. Trypsin selectively inactivates the cytopathic effect of the type 2 penton. At high trypsin concentrations the main part of the vertex capsomer antigenicity and morphology is also lost. Rabbit antisera to pure pentons contain low titers of neutralizing antibodies when assayed by inhibition of plaque formation. The use of the fluorescent focus assay reveals, in contrast, high neutralizing activity in these sera. Treatment of penton antigen with pyridine releases free vertex capsomers which are immunologically and morphologically intact and have the ability to induce cytopathic changes in KB-cell cultures.

Structural proteins of adenoviruses: IV. Sequential degradation of the adenovirus type 2 virion

Abstract The composition of highly purified type 2 adenovirion has been studied by different degradation procedures. Dialysis against Tris-maleate buffers in the pH range 6.0–6.6 was used as an initial step, resulting in a quantitative release of pentons from the virions. At room temperature, these pentonless virions rapidly also release 5 or 6 hexons per original penton unit. The remainder of the capsid is further disintegrated by repeated freezing and thawing, which releases hexons in a nonaggregated form and also a precipitated DNA-protein complex. Two major protein components may be extracted from the latter with mineral acids. Amino acid analysis of these internal DNA complexed proteins reveals that they are highly basic, with an arginine content of 19%. Three or more low-molecular weight antigens distinct from hexon, penton base, fiber, and basic core proteins are also released during this procedure. Pyridine at a concentration of 10% degrades virions by a different pattern; hexons originating from the faces of the adenovirus icosahedron are preserved in groups of nine. Peripentonal hexons, fibers, degraded vertex capsomers, and low molecular weight antigens are also released. The internal core is retained in a suspended state and contains 15% of the total virion protein. The virion appears to be composed of 8 or more antigenically distinct proteins.

ISCOMs and Immunostimulation with Viral Antigens

With the dissection of microorganisms followed by biochemical and immunological characterization, antigens inducing protective immunity became recognized. Early attempts to use these isolated antigens as vaccines, i.e. subunit vaccines, showed that although immunogenic in situ as part of the microorganism, they were not immunogenic as purified antigens. Subsequent studies showed that the formation of antigen into defined multimeric forms such as protein micelles or into liposomes made them considerably more immunogenic. In a way, micelles and liposomes mimic a submicroscopic particle of a microorganism with several copies of surface antigens. By contrast, monomeric forms of antigens, e.g., envelope proteins of parainfluenza-3 virus or Semliki forest virus, not only had a low immunogenicity but had a specific suppressive effect on the immune response as well; this was shown when the monomers were given simultaneously with the same antigen in a micelle (Morein et al., 1982, 1983; Morein and Simons, 1985; Jennings, 1987).

Influenza virus ISCOMs: biochemical characterization.

Immunostimulating complexes (ISCOMs) have been prepared from influenza A virus envelope glycoproteins, i.e. haemagglutinin (HA) and neuraminidase (NA). An ISCOM consists of a matrix, which is the micellar form of the glycoside, Quil A, in hydrophobic interaction with both the envelope glycoproteins (HA/NA). The Quil A bound to the ISCOM amounted to 50 micrograms mg-1 (5%) of ISCOM protein. ISCOMs were morphologically identified as symmetrical cage-like structures of approximately equal to 40 nm in diameter with hexagonal or pentagonal subunits of approximately equal to 12 nm. The sedimentation coefficient was approximately equal to 19 S as compared to 30 S for the glycoprotein micelles. The biological activities of the HA and NA are preserved in both ISCOMs and micelles.

Tripeptide Interference with Human Immunodeficiency Virus Type 1 Morphogenesis

ABSTRACT Capsid assembly during virus replication is a potential target for antiviral therapy. The Gag polyprotein is the main structural component of retroviral particles, and in human immunodeficiency virus type 1 (HIV-1), it contains the sequences for the matrix, capsid, nucleocapsid, and several small polypeptides. Here, we report that at a concentration of 100 μM, 7 of 83 tripeptide amides from the carboxyl-terminal sequence of the HIV-1 capsid protein p24 suppressed HIV-1 replication (>80%). The three most potent tripeptides, glycyl-prolyl-glycine-amide (GPG-NH2), alanyl-leucyl-glycine-amide (ALG-NH2), and arginyl-glutaminyl-glycine-amide (RQG-NH2), were found to interact with p24. With electron microscopy, disarranged core structures of HIV-1 progeny were extensively observed when the cells were treated with GPG-NH2 and ALG-NH2. Furthermore, nodular structures of approximately the same size as the broad end of HIV-1 conical capsids were observed at the plasma membranes of treated cells only, possibly indicating an arrest of the budding process. Corresponding tripeptides with nonamidated carboxyl termini were not biologically active and did not interact with p24.

Structural proteins of adenoviruses: VIII. Characterization of incomplete particles of adenovirus type 3

Abstract Incomplete virus particles were isolated from cells infected with adenovirus type 3. More than 30% of the particles recovered at 72 hr after infection were found to be incomplete. Polyacrylamide electrophoresis revealed that incomplete particles were deficient in the two major core polypeptides, but contained enhanced levels of a polypeptide present in complete virions in only minute amounts. The amino acid composition of incomplete particles showed reduced levels of both arginine and alanine compared to virions. These two amino acids are enriched in the core proteins. The main fraction of the incomplete particles contained little, or no, DNA. A small fraction, however, contained DNA which was smaller (about 16 S) than viral DNA, as determined by neutral and alkaline sucrose gradient centrifugation. DNA-DNA hybridizations revealed that the DNA of the incomplete particles was mainly of viral origin. The incomplete particle preparations contained reduced levels of infectivity compared to complete particles. Infectivity may either be caused by aggregation of complete and incomplete particles or by complementation.

Gradient centrifugation of viruses in colloidal silica

Abstract Density gradients are generated in colloidal silica sol after a short centrifugation at high speed, depending on the large variation in size of the colloidal particles. Tobacco mosaic virus, adenovirus, and poliovirus can be isolated in sharp bands in these gradients. Virus infectivity can be quantitatively recovered from these gradients after precipitation of silica by polyamines provided that chelators and sulfhydryls are included in the gradients. The main advantages of silica gradients are (a) short centrifugation times are required for isodensity banding; (b) gradients can be generated over a large density range (1.0–1.45 g/ml); (c) particles appear to be separated both on the basis of density and the exclusion effect of the virus on the silica particles; and (d) the colloid has low osmotic effect and low viscosity; furthermore (e) gradient centrifugation can be carried out at physiological pH and ionic strength. The mechanism involved in silica gradient separation is discussed.

ATPγS Disrupts Human Immunodeficiency Virus Type 1 Virion Core Integrity

ABSTRACT Heat shock protein 70 (Hsp70) is incorporated within the membrane of primate lentiviral virions. Here we demonstrate that Hsp70 is also incorporated into oncoretroviral virions and that it remains associated with membrane-stripped human immunodeficiency virus type 1 (HIV-1) virion cores. To determine if Hsp70 promotes virion infectivity, we attempted to generate Hsp70-deficient virions with gag deletion mutations, Hsp70 transdominant mutants, or RNA interference, but these efforts were confounded, largely because they disrupt virion assembly. Given that polypeptide substrates are bound and released by Hsp70 in an ATP-hydrolytic reaction cycle, we supposed that incubation of HIV-1 virions with ATP would perturb Hsp70 interaction with substrates in the virion and thereby decrease infectivity. Treatment with ATP or ADP had no observable effect, but ATPγS and GTPγS, nucleotide triphosphate analogues resistant to Hsp70 hydrolysis, dramatically reduced the infectivity of HIV-1 and murine leukemia virus virions. ATPγS-treated virions were competent for fusion with susceptible target cells, but viral cDNA synthesis was inhibited to an extent that correlated with the magnitude of decrease in infectivity. Intravirion reverse transcription by HIV-1, simian immunodeficiency virus, or murine leukemia virus was also inhibited by ATPγS. The effects of ATPγS on HIV-1 reverse transcription appeared to be indirect, resulting from disruption of virion core morphology that was evident by transmission electron microscopy. Consistent with effects on capsid conformation, ATPγS-treated viruslike particles failed to saturate host antiviral restriction activity. Our observations support a model in which the catalytic activity of virion-associated Hsp70 is required to maintain structural integrity of the virion core.

Characterization of the invariable residue 51 mutations of human immunodeficiency virus type 1 capsid protein on in vitro CA assembly and infectivity

BackgroundThe mature HIV-1 conical core formation proceeds through highly regulated protease cleavage of the Gag precursor, which ultimately leads to substantial rearrangements of the capsid (CAp24) molecule involving both inter- and intra-molecular contacts of the CAp24 molecules. In this aspect, Asp51 which is located in the N-terminal domain of HIV-1 CAp24 plays an important role by forming a salt-bridge with the free imino terminus Pro1 following proteolytic cleavage and liberation of the CAp24 protein from the Pr55Gag precursor. Thus, previous substitution mutation of Asp51 to alanine (D51A) has shown to be lethal and that this invariable residue was found essential for tube formation in vitro, virus replication and virus capsid formation.ResultsWe extended the above investigation by introducing three different D51 substitution mutations (D51N, D51E, and D51Q) into both prokaryotic and eukaryotic expression systems and studied their effects on in vitro capsid assembly and virus infectivity. Two substitution mutations (D51E and D51N) had no substantial effect on in vitro capsid assembly, yet they impaired viral infectivity and particle production. In contrast, the D51Q mutant was defective both for in vitro capsid assembly and for virus replication in cell culture.ConclusionThese results show that substitutions of D51 with glutamate, glutamine, or asparagine, three amino acid residues that are structurally related to aspartate, could partially rescue both in vitro capsid assembly and intra-cellular CAp24 production but not replication of the virus in cultured cells.

Glycine-Amide Is an Active Metabolite of the Antiretroviral Tripeptide Glycyl-Prolyl-Glycine-Amide

ABSTRACT The chemically modified tripeptide glycyl-prolyl-glycine-amide (GPG-NH2) inhibits replication of human immunodeficiency virus (HIV) type 1 (HIV-1) in vitro, probably by interfering with capsid formation. The aim of the present study was to determine whether the metabolites glycyl-proline (GP-OH), glycine (G-OH), prolyl-glycine-amide (PG-NH2), proline (P-OH), and glycine-amide (G-NH2) from proteolytic cleavage may inhibit the replication of HIV-1 in vitro. PG-NH2 has previously been shown to have a modest effect on HIV-1 replication. In the present study we show that G-NH2 exhibits a pronounced inhibitory effect on HIV-1. This effect was not due to a decrease in cell proliferation or viability and could not be shown for herpes simplex virus type 1. The G-NH2 concentration that inhibited virus replication by 50% (IC50) was equimolar to that of GPG-NH2 and ranged from 3 to 41 μM. Transmission electron microscopy revealed that the effect of G-NH2 on HIV-1 morphology was equivalent to that of GPG-NH2 and showed disarranged capsid structures, indicating interference with capsid formation. Serial passage of HIV-infected cells with G-NH2 for more than 20 subcultivations did not decrease the susceptibility to the compound. The results from this study suggest that GPG-NH2 might act as a prodrug and that G-NH2 is an active antiretroviral metabolite.