Milan V. Nermut

Evidence that the amantadine-induced, M2-mediated conversion of influenza A virus hemagglutinin to the low pH conformation occurs in an acidic trans golgi compartment

Amantadine treatment of cells infected with H7 strains of influenza A viruses causes an M2 protein-mediated conversion of hemagglutinin (HA) from its native to its low pH conformation. Immunofluorescence and electron microscopic observations showed that the structural alteration and hence drug action occur shortly after HA exits from the Golgi complex during its passage through the strans Golgi region. Using the DAMP/anti-DNP pH probe it is evident that virus infection causes increased acidity of the trans Golgi region and that vesicles containing low pH HA in amantadine-treated virus-infected cells are particularly acidic. These results indicate therefore that the alteration in HA is the direct consequence of exposure to an adverse low pH and provide further support for the conclusion that the M2 protein, the target of amantadine action, is involved in regulating vesicular pH, a function important for the correct maturation of the HA glycoprotein.

Structural Analyses of Purified Human Immunodeficiency Virus Type 1 Intracellular Reverse Transcription Complexes

ABSTRACT Retroviruses copy their RNA genome into a DNA molecule, but little is known of the structure of the complex mediating reverse transcription in vivo. We used confocal and electron microscopy to study the structure of human immunodeficiency virus type 1 (HIV-1) intracellular reverse transcription complexes (RTCs). Cytoplasmic extracts were prepared 3, 4, and 16 h after acute infection by Dounce homogenization in hypotonic buffer. RTCs were purified by velocity sedimentation, followed by density fractionation in linear sucrose gradients and dialysis in a large pore cellulose membrane. RTCs had a sedimentation velocity of approximately 350 S and a density of 1.34 g/ml and were active in an endogenous reverse transcription assay. Double labeling of nucleic acids and viral proteins allowed specific visualization of RTCs by confocal microscopy. Electron microscopy revealed that RTCs are large nucleoprotein structures of variable shape consisting of packed filaments ca. 6 nm thick. Integrase and Vpr are associated with discrete regions of the 6-nm filaments. The nucleic acids within the RTC are coated by small proteins distinct from nucleocapsid and are partially protected from nuclease digestion.

The NB protein is an integral component of the membrane of influenza B virus

The results of biochemical and immunoelectron microscopic studies provide evidence that the NB protein is an integral component of the influenza B virion. Its glycosylation and orientation in the membrane were shown to be equivalent to that of NB in the plasma membrane of virus-infected cells. Sensitivity to proteinase K showed that the N terminus is exterior to the virion and gold immunolabelling of freeze-fractured replicas showed that the C terminus is located in the interior of the virion. The similarities between NB of influenza B and M2 of influenza A viruses in structural features, their presence in the virion and possession of an ion channel activity suggest that, by analogy with the M2 protein, NB may also have a role in virus entry.

Gag-Gag interactions in the C-terminal domain of human immunodeficiency virus type 1 p24 capsid antigen are essential for Gag particle assembly.

Seven internal deletions within the p24 domain of the human immunodeficiency virus type 1 Gag precursor have been assessed for their effect on Gag particle formation following their expression using recombinant baculoviruses. In addition, each deleted molecule was assessed for its ability to bind soluble p24 antigen in vitro. The mutants fell into three different phenotypic groups: (i) three mutants that had no effect on either p24 binding or Gag particle assembly, (ii) three mutants that abolished both features and (iii) one mutant that bound p24 in vitro but failed to assemble particles. Mutations that abolished both in vitro p24 binding and particle assembly mapped to the C terminus of p24 confirming this region as critical for virion assembly. We suggest the division of virion assembly into at least two distinct phases and suggest a model in which the critical sequences mapped to date are combined with available structural information.

Analysis of Mason-Pfizer Monkey Virus Gag Domains Required for Capsid Assembly in Bacteria: Role of the N-Terminal Proline Residue of CA in Directing Particle Shape

ABSTRACT Mason-Pfizer monkey virus (M-PMV) preassembles immature capsids in the cytoplasm prior to transporting them to the plasma membrane. Expression of the M-PMV Gag precursor in bacteria results in the assembly of capsids indistinguishable from those assembled in mammalian cells. We have used this system to investigate the structural requirements for the assembly of Gag precursors into procapsids. A series of C- and N-terminal deletion mutants progressively lacking each of the mature Gag domains (matrix protein [MA]-pp24/16-p12-capsid protein [CA]-nucleocapsid protein [NC]-p4) were constructed and expressed in bacteria. The results demonstrate that both the CA and the NC domains are necessary for the assembly of macromolecular arrays (sheets) but that amino acid residues at the N terminus of CA define the assembly of spherical capsids. The role of these N-terminal domains is not based on a specific amino acid sequence, since both MA-CA-NC and p12-CA-NC polyproteins efficiently assemble into capsids. Residues N terminal of CA appear to prevent a conformational change in which the N-terminal proline plays a key role, since the expression of a CA-NC protein lacking this proline results in the assembly of spherical capsids in place of the sheets assembled by the CA-NC protein.

Fine structure of adenovirus type 5: I. Virus capsid

Abstract Advanced techniques of electron microscopy enabled a more accurate determination of the size and organization of the virus capsid of adenovirus type 5. The edge of the virus icosahedron (= a centre-to-centre distance of two pentons) was found to measure 430 A (obtained from freeze-dried and freeze-etched preparations), and the theoretical values calculated therefrom are as follows: The point-to-point distance = 820 A, the face-to-face distance = 662 A and the edge-to-edge distance = 696 A. The “diameter” of a virus particle in a five-fold symmetry orientation would be 731 A. The overall shape of a hexon is close to a “conical triangular prism” with a base of 86 × 98 A (= height × edge of an equilateral triangle) and a narrower top of about 74 A in diameter. It is proposed that the hexon is built up of three subunits in close contact at the bottom but comparatively free and mobile at the top. The average height of hexons was found to be 110 A. The pentons are about 95 A in “diameter” and their fibers 210–340 A long.

Extracellular Fibril Production by Freshwater Algae and Cyanobacteria

In order to study the ability of freshwater algae and cyanobacteria to form extracellular fibrils, a screening test using ruthenium red (RR) staining was carried out on 28 species. Five of these were examined for growth and production of fibrillar material in culture media of different phosphate (P;) contents. RR-staining and uronic acid determinations at various stages of algal growth were complemented by electron microscopy of the cells and of fibrillar material released into the medium. The lower Pi concentrations enhanced growth of Micrasterias radiata, Eremosphaera sp., and Microcystis aeruginosa, and had little or no effect on growth of a Xanthidium sp. and Scenedesmus quadricauda. Extracellular uronic acid production, which was higher in low Pi medium in M. radiata, M. aeruginosa, and Xanthidium sp., could reach levels of 50 mg/liter or more. Algae with high proportions of RR-positive cells (M. radiata, Eremosphaera sp., Xanthidium sp., and M. aeruginosa) produced high levels of slime-like material and distinct fibrils that were often seen attached to the cell surface and only slowly released into the medium. No such material was found in cultures (or supernatants) of Sc. quadricauda, which also produced relatively low amounts of polyuronic acids. Specific types of filaments, often forming “fascicles” with rectangular arrays of globular particles were observed by negative staining electron microscopy of some algal cultures. RR-positive material was also observed in the cytoplasm and on the cell walls and surfaces of M. radiata and M. aeruginosa.

Visualization of cAMP receptor protein-induced DNA kinking by electron microscopy☆

The effect of specific DNA binding of the cAMP . cAMP receptor protein complex to two DNA fragments (301 and 2685 base-pairs in length) containing the lac operon has been investigated by electron microscopy. It is shown that specific DNA binding of the cAMP . cAMP receptor protein complex induces a kink of 30 to 45 degrees in the DNA with the apex of the kink located at the site of protein attachment. These findings lend direct visual support for the kinking hypothesis based on the observation of anomalous electrophoretic mobility of DNA fragments containing specifically bound cAMP receptor protein.

The organization of frog virus 3 as revealed by freeze-etching

A variety of freeze-fracture techniques has been employed in this study with the aim of dissecting the frog virus 3 virion and obtaining further information about its architecture. The icosahedral capsid has a skew symmetry with a triangulation number of 133 or 147. The capsomers are closely packed with a center-to-center spacing of 72 A. The inner membrane contains transmembrane proteins which appear as intra-membranous particles on both fracture faces. Rod-like structures (about 100 A in diameter) are present in the virus interior suggesting that the DNA-protein complex is highly organized.

Ultrastructural and biochemical evidence of the trimeric nature of frog virus 3 (FV3) six-coordinated capsomers

Image analysis of freeze-etch replicas of cylindrical aberrant forms of FV3 provided evidence for three morphological subunits protruding from the six-coordinated capsomers. Negatively stained capsomers displayed both triangular and hexagonal profiles which suggests that their innermost portion is pseudohexagonal. Images from underfocused micrographs of capsomers are indicative of a central channel. The trimeric nature of the capsomer has been established by electrophoresis in the presence of Triton X-100, which showed that the molecular weight of the nondissociated capsomer is about 140,000 whereas that of the polypeptide itself is 48,000. This trimeric association does not occur via disulfide bonds, and inside the capsomers there are no free amino groups accessible to the usual bifunctional reagents. Thus, the chemical nature of the interpolypeptide bonds inside the trimers is still unknown. We have previously estimated the triangulation number (T) of FV3 to be 147 or 133 (Darcy-Tripier et al., 1984). The present study, using optical diffraction of the facets of FV3, allowed a better determination of the angle of skewness and is in favor of T = 133 (h = 9, k = 4, 18 degrees).