RETROVIRUSES
The Recollections of an Electron Microscopist
By Etienne de Harven
Reappraising AIDS Nov./Dec. 1998
For an electron microscopist who spent most of his research career studying
retroviruses associated with murine leukemias and who followed closely their
hypothetical impact on the field of human oncology, it was predictable that
contemporary AIDS research was completely on the wrong track. The following
"Recollections" are presented to explain why.
The importance of electron microscopy in the emergence of modern cell
biology, between 1945 and 1965, is unanimously recognized. Unquestionably,
the relationships between cell structures and cell functions would never
have been elucidated without the high resolving power of the electron
microscope (EM). What is less generally recognized, however, is the role
virus research had in the study of cell ultrastructure. Historically, when
Rüdenberg filed the patent for the electron microscope in 1931 (1), his
motivation stemmed from the hope of visualizing the polio virus! And during
the WW2 years, when electron microscopes started to be accessible to
biologists, attempts to visualize "virus particles" associated with cancer
cells of laboratory animals received first priority. Albert Claude, working
at the Rockefeller Institute, succeeded in demonstrating the Rous sarcoma
virus in chicken fibroblasts (2). And a few years later, Keith Porter et al.
had a similar success in imaging the "milk factor" in murine mammary
adenocarcinomas cells (3). The viral etiology of Rous sarcoma in chickens
and of mammary tumors in mice were well established by microbiological
ultrafiltration experiments, years before the EM pictures were published.
Still, the direct observation of virus particles in these experimental
tumors gave an enormous (today, we would perhaps say excessive!) impetus to
virus research in oncology.
Viral etiology of several murine and avian malignancies had been clearly
demonstrated by ultrafiltration experiments which permitted the calculation
of the approximate diameter of the virus particles. Electron microscopists
knew, therefore, what size particle to look for, frequently around 100 nm.
This facilitated the initial identification of oncogenic viruses by EM,
although it was later observed that many microvesicles or particulate
components of normal cells fall into a similar size range.
The discovery by Charlotte Friend, at the Sloan Kettering Institute in New
York, of a murine erythroleukemia transmissible by cell-free filtrates
illustrates well the research approaches around 1955. Moreover, since I
started working in Dr. Friend's laboratory at that time, the rationale of
our research program is clearly in my mind. For EM, we gave priority to two
types of sample: 1) a variety of tissues from leukemic Swiss mice (spleen,
lymph nodes, thymus and bone marrow), and 2) ultracentrifugation pellets
obtained from leukemic tissue cell-free filtrates which readily transmitted
the disease to adult Swiss and/or DBA/2 mice. We knew from ultrafiltration
experiments that the activity was absent when using filters with an average
pore size diameter smaller than 200 nm. Classic theories on ultrafiltration
indicated that the infective particles were therefore probably in the 100 nm
range. The study of thin sections from plastic embedded leukemic tissue
frequently revealed the presence of particles of approximately that
diameter, closely associated with a variety of cells. The particles were
bound by a single membrane and had a centrally located electron-dense core
or nucleoid. They had a characteristic structure and strikingly constant
diameters. To our knowledge, such particles did not resemble any known
components of normal cells. However, they did resemble particles previously
identified by others in several filterable experimental tumors and
classified by W. Bernhard as "type C" particles (4). More importantly, we
observed identical particles in ultracentrifugation pellets prepared from
cell-free filtrates capable of transmitting the disease to susceptible mice.
On the basis of these data, we hypothesized that these particles indeed
represented the oncogenic virus etiologically related to the Friend
erythroleukemia (5). Somewhat surprisingly, the virus was also found in
close association with cells not apparently related to the leukemic process,
such as bone marrow megakaryocytes, for example. These early EM studies also
indicated that all electron- dense particles averaging 100 nm in diameter
are not viruses and that a rigorous ultrastructural characterization was
essential for adequate differentiation between viruses and "virus-like
particles."
Fortunately, our EM studies of Friend leukemia soon added an important
feature to the structural characterization of oncogenic RNA viruses. It
appeared that virus assembly is a cell surface phenomenon, the surface
membrane of the infected cell directly contributing the future viral
envelope by a multiple step mechanism for which we coined the word "budding"
(6). Viruses are released into intercellular spaces by such a budding
process. EM identification of viruses of that group in other experimental
malignancies became therefore more rigorous, observation of "budding
particles" being required. This probably helped in eliminating thousands of
"virus-like particles" observed in human malignancies and with which
over-enthusiastic electron microscopists attempted to contaminate the
literature! In addition, recognition of budding particles on cell surfaces
clearly allowed us to identify infected single cells, and to observe that
such cells are perfectly viable, with absolutely no ultrastructural evidence
of any cytolytic effect of viral infection. Moreover, typical viruses were
frequently observed in cells undergoing mitotic division (7).
Since obviously human experimentation was out of the question, the eventual
observation in human cancer cells of particles resembling those well
characterized in experimental tumors would have been of great interest,
although by itself not conclusive. Around 1960, many laboratories around the
world, using ever improving EM methodologies, were focusing on that target.
At that time, well before the emergence of molecular biology, EM was the
best we had to identify viruses in cell samples. The crucial role of EM was
well recognized at the Cold Spring Harbor conference of 1962, when Lwoff,
Horne and Tournier proposed classifying all viruses primarily on the basis
of their morphological characteristics demonstrated by electron microscopy
(8).
Continuing our studies on the Friend leukemia virus (FLV), and encouraged
by Dr. J. Beard from Duke University who had considerable experience with
avian leukosis, we oriented our effort toward the demonstration, by EM, of
viremia in leukemic mice. The most efficient initial step in purifying avian
leukosis viruses was to start not from leukemic tissues but from the blood
plasma of leukemic chickens. We wondered if a similar situation could also
prevail in leukemic mice. This was important for us because the efficiency
of our early virus purification method from homogenates of leukemic tissues
such as spleen or lymph nodes was poor. We developed, therefore, a very
simple purification procedure based on two steps of Millipore filtration. A
diluted plasma sample, around 10 ml (i.e. bleeding about 25 leukemic mice),
was first clarified by aspiration filtration through 0.65 um filter;
filtrate #1 was then further filtered through a 0.22 um membrane; filtrate
#2 was then centrifuged at 30,000g for 120 min. The resulting pellet was
extremely small...almost invisible, still worth embedding for EM! Thin
sections of such pellets revealed the presence of a most impressive
population of typical, well preserved virus particles, packed together, and
with very little contaminating debris (9). This was our approach to the
demonstration of viremia in 1965...
Meanwhile, many EM cancer research centers (those lead by Dr. W. Bernhard,
at Villejuif, France, Dr. A.J. Dalton, at NCI, Bethesda Md, Dr. L.
Dmochowski, at MD Anderson, Houston, TX, and us at Sloan Kettering, New
York, NY), were spending a considerable amount of time in attempts to
demonstrate virus particles associated with human cancer cells. "Virus-like
particles" were occasionally reported but convinced nobody. Typical viruses
were never conclusively demonstrated. This was in sharp contrast with the
highly reproducible demonstration, by EM, of viruses in a variety of murine
and avian leukemias and tumors. Very few papers were published to report on
these negative findings in human cancers and leukemias. However, Haguenau,
in 1959 (10), reported on the difficulty of identifying any typical virus
particles in a large series of human mammary carcinomas. Bernhard and
Leplus, in 1964 (11) in an EM survey of cases of Hodgkin's disease,
lymphosarcomes, lymphoid leukemias and metastatic diseases failed to
recognize virus particles associated with these malignant conditions. At
Sloan Kettering in New York, I decided, in 1965, to stop surveying cases of
leukemias and lymphomas by EM for the presence of viruses in view of our
entirely negative results. This was reported at a conference on
Methodological Approaches to the Study of Leukemias held at the Wistar
Institute, in 1965 (12).
Of mice and men
Publication of these negative findings failed to discourage fanatical virus
hunters! An explanation for these negative results had to be found
somewhere! Perhaps the technique of EM by the thin section method was not
the best approach? (although it worked perfectly for mice!). Preparing thin
sections was time-consuming and skill- demanding! Who had time for that,
when research funding was getting difficult, and when major pharmaceutical
corporations were starting to finance "crash programs" for speedy
answers?... Why don't we try the negative staining method? It is very easy
and very fast! And, after all, it gave beautiful results with unenveloped
viruses like adenovirus and polyoma. The results were absolute disaster
because fragile RNA tumor viruses (not yet called retroviruses...) are badly
distorted by air-drying during the negative staining procedures; they appear
as particles with a long tail! Unfortunately many cell debris and vesicular
fragments, when air-dried for negative staining, form similar "tailed"
structures. Interpreting "tailed" particles as RNA tumor viruses was
therefore a bonanza for virus hunters! Still, we had demonstrated that
"tailed" virions were preparation artifacts which can be avoided by proper
control of the osmolality and by osmium fixation prior to negative staining
(13), or by critical-point drying (14). The chaos created by reports on
"tailed" particles damaged the credibility of EM in the search for cancer
related viruses. Cow's milk and human milk were being screened for "tailed"
particles and Sol Spiegelman was eloquent on the possible risks of breast
feeding...
A major discovery, which had nothing to do with EM, completely reoriented
ideas on how RNA tumor viruses might work, the discovery by Temin and
Baltimore, in 1970, of reverse transcriptase, RT. How RNA tumor viruses
could induce genomic alterations of the cells they infect suddenly became
comprehensible. Moreover, these viruses remained candidates as possible
oncogens because they were known NOT to be cytolytic. RNA tumor viruses were
given a new name, retroviruses, and the study of their eventual role in
causing human cancer soon received levels of federal funding, after Nixon's
"War Against Cancer Act," which were frighteningly in excess of what could
have been expected from a frequently recurring, perhaps interesting but so
far totally unproven hypothesis...
********
The profile of the research effort changed very drastically after the
discovery of RT in 1970. Somehow, most of the research methods which had
dominated the field of viral oncology from 1950 until 1970 were suddenly
substituted by the most exclusive fashion of molecular biology. I observed
this evolution almost as an outsider, since, in my view, electron microscopy
was no longer a primary contributing method to tackle any further the
hypothetical relationships between retroviruses and human cancer.
The 1970-1980 years were dominated by a series of ideas which would never
have withstood scientific scrutiny 10 or 20 years before. For example:
1. It became acceptable to postulate that when viruses cannot be seen by EM
in cancer cells, biochemical or immunological methods supposedly identifying
viral "markers" were enough to demonstrate viral infection of the cells
under scrutiny. Such markers can be an enzyme (RT), an antigen, various
proteins, or some RNA sequences. Never seeing the viral particles was
conveniently explained by the integration of the viral genome into the
chromosomes of the alleged infected cells. To accommodate such
interpretations implied complete oblivion of all we knew from previous
research on cancer of experimental animals. Admittedly, in these models EM
was only showing terminal steps of viral reduplication, initial phases being
a series of molecular events which escape ultrastructural recognition.
Still, in all the classical models such as murine and avian leukoses,
visible terminal steps of viral replication (i.e. "budding") were always
observed and regarded as essential for the spreading of infection from cell
to cell.
2. Another shortcut with disastrous consequences has been the naive notion
that any material banding at 1.16gm/ml represented retroviruses! Sure
enough, true retroviruses band around that density. But this does not mean
that whatever material bands at 1.16gm/ml is retroviral in nature! In the
1960s I was frequently asked to look at such 1.16gm/ml bands by biochemists:
"Look at this, it forms a sharp band, it should be pure viruses!"
Ultracentrifugation pellets obtained from such "sharp bands" showed, in thin
sections for EM, an extreme variety of microvesicles and proteinaceous
debris, but no retroviruses! Still, this approach has been (and still is!)
used to identify virus "markers!" How sad it is to think that a simple EM
control of such "bands" (which takes about two days, and costs a few hundred
dollars, but has never been done before 1997) could have prevented these
highly misleading interpretations of "markers" on which large budgets have
been simply wasted...
3. Collecting viruses from the supernatant of cultures of virus infected
cells raises other questions. We all remember the discovery, by Epstein (15)
in 1964 of the EB virus in cultures derived from African Burkitt's
lymphomas. This was an EM finding and the virus was immediately and properly
classified as a member of the herpes group. To identify this DNA virus in
cultured cells it was necessary to look into partially degenerating cells
because, most obviously, the virus had a marked cytolytic effect. By
complete contrast, retrovirus-carrying cells maintain excellent viability
and released viruses can be easily recovered in the culture supernatant
without the need to apply any lymphokine or growth factors stimulation to
the cultures.
4. As far as scientific policy is concerned, research on potentially
oncogenic viruses was dominated by the retrovirus hypothesis. Federal
funding took the same direction, amplified by the incredibly naive idea that
success was primarily a matter of money! Unusually large levels of federal
support resulted in the creation of a retrovirus research establishment.
Large numbers of research jobs were created in this venture. The
intellectual freedom to think along other avenues of cancer research was
rapidly dwindling, especially when major pharmaceutical companies started to
offer tantalizing contracts to support polarized retrovirus research... The
top priority was to demonstrate, at any cost, that retroviruses had
something to do with human cancer, an hypothesis, however, which didn't
receive the slightest support throughout the 1970s. Such a misdirected
research effort would have been relatively inconsequential as long as public
health was not involved. Unfortunately, the emergence of acquired
immunodeficiency syndromes (AIDS) in 1981 gave the retrovirus establishment
an opportunity to transform what could have been only an academic flop into
a public health tragedy.
********
What happened after 1981 is so well known to all the readers of RA that I
hesitate to elaborate any further. The events which have lead to today's
crisis have been reviewed and analyzed most convincingly by Peter Duesberg
(16). I must say that I read Duesberg's book with great attention but
basically with no surprise, because the way research had been conducted on
retroviruses in the 1970s had so dangerously set the stage for "Impure
Science" (17)...
Soon after the first cases of "Gay related immune deficiency" were
described by Michael Gottlieb it was obvious for all observers that Gallo
and his associates were going to jump on the new syndrome as a Godsent
opportunity to attempt to justify the lavish federal budgets they had
consumed on retroviruses over the past 10 years. In 1980, the scientific
community was getting more and more concerned about the absence of results
in "The War against Cancer" based on retrovirus hunting. The minor episode
of HTLV 1 was not enough, by far, to calm the fears of grossly misdirected
federal research funds. The fact that the syndrome, soon tactically renamed
"AIDS", had nothing to do with cancer was apparently of little embarrassment
for Gallo. Frequent association with Kaposi sarcoma helped to blur the
difference in the eyes of the public.
Dominated by the media, by special pressure groups and by the interests of
several pharmaceutical companies, the AIDS establishment efforts to control
the disease lost contact with open-minded, peer-reviewed medical science
since the unproven HIV/AIDS hypothesis received 100% of the research funds
while all other hypotheses were ignored. The general public and the medical
community were made to believe that the presence of circulating antibodies
is diagnostic of this disease, that Koch's postulates were outdated, that
90% of all cases of an infectious disease can be observed in males, that
viremia can be measured by PCR enhancement of RNA fragments even when viral
particles are not demonstrable, etc., etc...
Most conveniently, it was totally forgotten that heroin addicts were known
for many decades to expose themselves to immuno- deficiencies, that nitrite
inhalants have multiple toxic effects, that the extreme toxicity of AZT was
known for over 20 years, that known retroviruses never have any cytolytic
effects, etc., etc...
And to ensure that the AIDS establishment could profitably continue to
flourish, research on any dissenting (i.e. non-HIV) hypothesis was carefully
prevented by tight control of research funding and by the extreme difficulty
of publishing anywhere any dissenting views... In the late 1980s, I was
considering adding to my research program in Toronto more EM observations on
samples from AIDS patients. Unfortunately, by that time the media and the
CDC had so perfectly orchestrated the panic of a plague-like epidemic that I
was quickly made to understand that my assistants would all transfer out of
the lab if I had insisted to activate such a program... The HIV
seropositivity test was still at that time regarded as providing reliable
diagnostic data. Since then, Papadopulos and the Australian team have
demonstrated that this is very far from the truth...(18).
Since my retirement in France, I take any opportunity to speak out, as
openly as I can, along the lines of this little note. I am proud to be part
of the "Group for the Re-appraising of the HIV-AIDS hypothesis" and I
sincerely hope that the combined activities of "The Group" will soon
contribute to a complete re-appraisal of the etiology of AIDS for the best
interest of patient's care, and for a revival of scientific integrity in
medical science!
Copyright 1998 by Prof. Etienne de Harven, MD; "Le Mas Pitou," 2879 Route de
Grasse, 06530 Saint Cézaire sur Siagne, FRANCE. <Pitou.Deharven@wanadoo.fr>
References
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