Hans R. Gelderblom

Fine structure of human immunodeficiency virus (HIV) and immunolocalization of structural proteins.

Ultrathin section and surface replica electron microscopy were applied in combination with immunoelectron microscopy to elucidate the fine structure of HIV. The shell of the tubular core shows p24 antigenicity, while p17 is located at the inner leaflet of the lipid membrane. The virus particle is studded with 70–80 protrusions. These knobs have a diameter of 15 nm, a height of 9 nm, and are probably arranged in a T = 7 I symmetry. The major envelope protein gpl20 is spontaneously shed from the viral surface. A possible role of released gpl20 in pathogenesis is discussed.

Factors underlying spontaneous inactivation and susceptibility to neutralization of human immunodeficiency virus

To determine the factors governing inactivation and neutralization, physical, chemical, and biological assays were performed on a molecular clone of human immunodeficiency type 1 (HIV-1HXB3). This included quantitative electron microscopy, gp120 and p24 enzyme-linked immunosorbent assays, reverse, transcriptase assays, and quantitative infectivity assays. For freshly harvested stocks, the ratio of infectious to noninfectious viral particles ranged from 10(-4) to 10(-7) in viral stocks containing 10(9) to 10(10) physical particles per milliliter. There were relatively few gp120 knobs per HIV particle, mean approximately 10 when averaged over the total particle count. Each HIV particle contained a mean approximately 5 x 10(-17) g of p24 and approximately 2 x 10(-16) g of RNA polymerase, corresponding to about 1200 and 80 molecules, respectively. The spontaneous shedding of gp120 envelope proteins from virions was exponential, with a half-life approximately 30 hr. The loss of RNA polymerase activity in virons was also exponential, with a half-life approximately 40 hr. The physical breakup of virions and the dissolution of p24 core proteins were slow (half-life greater than 100 hr) compared to the gp120 shedding and polymerase loss rates. The decay of HIV-1 infectivity was found to obey superimposed single- and multihit kinetics. At short preincubation times, the loss of infectivity correlated with spontaneous shedding of gp120 from virions. At longer times, an accelerating decay rate indicated that HIV requires a minimal number of gp120 molecules for efficient infection of CD4+ cells. The blocking activity of recombinant soluble CD4 (sCD4) and phosphonoformate (foscarnet) varied with the number of gp120 molecules and number of active RNA polymerase molecules per virion, respectively. These results demonstrate that the physical state of virions greatly influences infectivity and neutralization. The knowledge gained from these findings will improve the reliability of in vitro assays, enhance the study of wild-type strains, and facilitate the evaluation of potential HIV therapeutics and vaccines.

Efficient HIV-1 replication can occur in the absence of the viral matrix protein

Matrix (MA), a major structural protein of retroviruses, is thought to play a critical role in several steps of the HIV‐1 replication cycle, including the plasma membrane targeting of Gag, the incorporation of envelope (Env) glycoproteins into nascent particles, and the nuclear import of the viral genome in non‐dividing cells. We now show that the entire MA protein is dispensable for the incorporation of HIV‐1 Env glycoproteins with a shortened cytoplasmic domain. Furthermore, efficient HIV‐1 replication in the absence of up to 90% of MA was observed in a cell line in which the cytoplasmic domain of Env is not required. Additional compensatory changes in Gag permitted efficient virus replication even if all of MA was replaced by a heterologous membrane targeting signal. Viruses which lacked the globular domain of MA but retained its N‐terminal myristyl anchor exhibited an increased ability to form both extracellular and intracellular virus particles, consistent with a myristyl switch model of Gag membrane targeting. Pseudotyped HIV‐1 particles that lacked the structurally conserved globular head of MA efficiently infected macrophages, indicating that MA is dispensable for nuclear import in terminally differentiated cells.

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.

Morphogenesis and morphology of HIV structure-function relations

SummaryFine structure and antigenic make-up analysis of HIV were combined in a 2D model, from which functional aspects can be deduced. On the envelope 72 probably trimeric surface knobs (gp 120) are connected to the virion via the transmembrane protein gp41. Gp120 is shed during ageing of the virion, but host cell antigens stay firmly anchored to the envelope. Underneath the envelope, p17 forms the matrix protein layer, while the capsid of the double cone shaped core is built up of p24. The relation between biochemical findings and morphogensis and maturation of HIV as well as aspects of pathogenesis and vaccination are discussed.

Diagnostic electron microscopy is still a timely and rewarding method

Abstract Background: Parallel to its technical development starting in the 1930s, electron microscopy (EM) became an important tool in basic and clinical virology. First utilized in the rapid diagnosis of smallpox, it developed to a diagnostic routine in the early 1960s using the negative staining technique. EM was applied to infected cell-cultures and also to ‘dirty’ specimens including urine, feces, vesicle fluid, liquor. With the implementation of molecular biological and genetic techniques, the use of diagnostic EM decreased. Objectives: (1) To give a perspective on future indications and possible uses by discussing the past and the present of diagnostic EM, (2) To describe the system of External Quality Assessment on EM virus diagnosis (EQA-EMV) established in 1994 by our laboratory and its achievements. Study design: EQA-EMV is run to evaluate, to confirm and to improve the quality of diagnostic EM. Two different types of specimen are sent out: (1) prepared grids to assess and train the diagnostic skills of the participants, (2) stabilized virus particle suspensions to assess preparation efficiency. Results: Diagnostic EM differs from other diagnostic tests in its rapidity and its undirected ‘open view’. To emphasize these advantages, the indications for diagnostic EM are discussed, fundamental for a continuing future adaptation. Besides appropriate techniques, quality control measures are required to achieve and keep high diagnostic standards. The results from 6 years of EQA-EMV are presented. Conclusions: In the history of diagnostic EM in virology, a change in use has been seen. Starting in the 1990s and coincident with the broad introduction of ‘modern’ diagnostic techniques, the number of EM diagnostic labs has decreased considerably—in spite of the obvious advantages of this technique. To guarantee the continuing performance of diagnostic EM in the future, EQA runs have to be performed as with other techniques in the diagnostic armament. The growing number of participants and participating countries indicates an interest in as well as a need for this program.

Quaternary structure of the immunostimulating complex (iscom)

Proteins of either HIV-1, hepatitis B, or rabies virus were incorporated with the adjuvant substance Quil A and cholesterol into the immunostimulating complex: iscom. Formation and symmetry of this regular complex were analyzed by electron microscopy. Micellar structures with a diameter of about 12 nm, occasionally with a 7-nm stain-filled center, were formed in a 0.03% water suspension of Quil A. Cavities or holes appeared in the smooth structures of cholesterol upon the addition of Quil A, and after mixing Quil A and cholesterol 1:1 fragile and flattened structures of matrix were produced with a diameter of about 40 nm. By freeze-drying the matrix was preserved as a cage-like, isometric particle. Stable iscom particles composed of Quil A, cholesterol, and selected viral proteins had an approximate diameter of 32 nm. The particles had an uniform, cage-like structure, exhibiting icosahedral symmetry, irrespective of the viral proteins incorporated. Tilting experiments and rotational image analysis indicated that the iscoms were composed of 20 morphological subunits assembled in a pentagonal dodecahedron with a hole on each of the 12 pentagonal faces. The symmetrical shape of the iscom might explain both its remarkable stability and its capacity to efficiently present antigens to the immune system.

The organization of the envelope projections on the surface of HIV

SummaryThe organization of envelope projections (knobs) of four different isolates of the human immunodeficiency virus types 1 and 2 (HIV-1 and -2) was studied using surface replica and thin section electron microscopy (EM) in combination with rotational image enhancement. All HIV strains show an identical organization of knobs on the virus envelope. The surface of an “ideal”, well-preserved HIV particle is studded with 72 knobs arranged in a T=7 laevo symmetry. The role of the p17 protein, which is coating the inner leaflet of the viral envelope, is discussed as a matrix protein functioning as a scaffold for the envelope and its projections during morphogenesis as well as with mature virions.

Morphological characterization of chicken anaemia agent (CAA)

SummaryChicken anaemia agent (CAA) was characterized as a virion with 25nm in diameter, with a buoyant density in CsCl of 1.36–1.37 g/cm3, and containing a circular, single-stranded DNA genome. The virus is composed of 32 hollow morphological units representing a regular T=3 icosahedron.

New human adenovirus (candidate adenovirus 36), a novel member of subgroup D.

SummaryA new adenovirus (candidate Ad36) was isolated from the feces of a girl with enteritis. It has the biological and biophysical properties of an adenovirus, with a particle diameter of 750±10 Å in thin sections and with a fiber length of 190 Å. The virus belongs to subgroup D. It is distinct both in neutralization and hemagglutination-inhibition from all other human adenoviruses; it also shows a unique DNA restriction pattern.