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THEIR COMPONENTS1
WERNER SCHAFER
Max-Planck-Institut fur Virusforschung, Tubingen, Germliany
INTRODUCTION..................................................................................
STRUCTURE OF THE MODEL VIRUSES ............................................................ 1
Maus-Elberfeld Virus ......................................................................... 1
Fowl Plague Virus ........................................................................... 2
Newcastle Disease Virus.......................................................................3
SIGNIFICANCE OF VIRAL COMPONENTS ................. .......................................... 4
Biological Investigation of Isolated Viral Components ...........................................
5
Investigation of Viral Reproduction ............................................................
7
Reproduction of Maus-Elberfeld virus ........................................................
7
Reproduction of fowl plague virus and Newcastle disease virus ................................
8
Summary.................................................................................... 12
APPLICATION OF SOME FEATURES EVALUATED TO PROBLEMS OF PRACTICAL IMPORTANCE ......... 12
CONCLUDING REMARKS ......................................................................... 14
LITERATURE CITED .14
Subunits ?
ofIl
FIG. 8. Hemagglutinin of fowl plague virus, PTA.
I
w~~~I is
-
'lE. 1
FIG 11 Newcastle disease virus, unshadowed.
graphically, using 5-min pulses of tritium-labeled unpublished data). Adding FPA up to 1 hr after
uridine. Normal L cells show, under these condi- infection inhibits the decrease of nuclear RNA
tions, a strong labeling of the nucleus, especially synthesis as well as the increase of cytoplasmic
of the nucleoli (Fig. 19), whereas the cytoplasm RNA synthesis. After addition at 3 hr postinfec-
is practically free of label. In infected cells, how- tion inhibition of RNA synthesis in the nucleus.
ever, nuclear RNA synthesis begins to decrease 2 persists but no synthesis of RNA in the cytoplasm
hr after infection. After 3 to 3.5 hr, only a negligi- occurs. Thus, special virus-induced proteins seem
ble number of cells showed nuclear tritium- to be necessary before the RNA metabolic
uridine incorporation. Instead, increasing changes observed in connection with ME virus
amounts of labeled compound were found in the multiplication can proceed.
cytoplasm starting at about 3 hr after infection Correlating these findings with the observa-
(Fig. 19). Thus, in the RNA metabolism of ME- tions described earlier, it seems reasonable to
infected L cells, at least two different processes believe that the cytoplasmic RNA is mostly viral
seem to occur: first, an inhibition of nuclear RNA RNA. This became somewhat improbable, how-
synthesis, and then a new, rapid RNA synthesis ever, following the chemical characterization of
in the cytoplasm. Corresponding controls insured the RNA synthesized later (4 to 6 hr) in the
that RNA and not an acid-soluble compound was multiplication cycle (Scholtissek, unpublished
concerned. data). The method used was developed by
By using small amounts of p-fluorophenylala- Scholtissek (44-46) and is hereafter referred to
nine (FPA), which still inhibit viral production, simply as "chemical RNA characterization." The
it can be demonstrated that an FPA-sensitive newly synthesized RNA is labeled by P32. After
phase precedes the changes observed (Hausen, digestion of the labeled RNA with ribonuclease
and separation of the various oligonucleotides by
chromatography, the oligonucleotide pattern of
the labeled RNA is determined. This pattern is
much more characteristic than is the simple base
ratio. Examination with this method showed that
the total complement of RNA synthesized at 4
to 6 hr after infection in the cell is not identical
with viral RNA. Nevertheless, the rapidly syn-
thesized cytoplasmic RNA seems to participate in
Ask A M"
It
* some manner in the synthesis of viral protein be-
cause the latter is produced at nearly the same
time at the same cell site. In view of the recently
advanced hypothesis of protein synthesis (2), one
Contra i might speculate that the particular cytoplasmic
RNA has a messenger function. At the present
time, however, this is nothing more than pure
F g t' o ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~4
By It:..AL speculation.
The ME virus seems to us to be a very inter-
esting model with respect to its reproduction
i,. r, 3.
mechanism. The problems here are based on very
impressive and characteristic changes, and are
therefore relatively easy to attack.
Reproduction of fowl plague virus and New-
castle disease virus. Somewhat more information
is available with regard to the multiplication
process of fowl plague virus (1, 4-6, 17, 19, 22,
ME, 5h P-F
30, 31, 33, 34, 41, 43, 47-49, 53, 54, 56). The fea-
FIG. 19. Incorporation of H3-uridine in L cells.
tures evaluated here are integrated into a sche-
(top) normal cells; (bottom) liE-infected cells, 5 matic picture. I am fully aware that in presenting
hr after infection. this scheme I must neglect somewhat the cate-
voL. 27' 1963' STRUCTURE OF ANIMAL VIRUSES 9
gorical imperative postulated initially. To bring
the scheme into a reasonable form, I must sub-
stitute some missing links by speculation.
As demonstrated in the scheme (Fig. 20), the
fowl plague virion attaches with the spikes of its
shell onto the cell surface. The possible function of
the neuraminidase probably contained in the
spikes during the penetration of viral material into
the cell is still open to discussion. After adsorption
onto the cell, the fowl plague virion goes into
eclipse. The ratio of infective virus to the number
of cells acting as infective centers falls to a value
smaller than 0.01. Investigations with P32-labeled
virus indicated that eclipsing of the virion is paral-
leled by liberation of s antigen from which the 0 2 C 6 0 10
RNA seems to have been released. In brief, these rime of adding FPA
investigations showed that shortly after infection (hours p.i.)
homogenized suspensions of the cells contained a FIG. 21. Influence of FPA ($00 jig/ml) on viral
considerable amount of P32 carriers which could production capacity of chick embryo cells, when
be precipitated by highly specific s antigen anti- added at different times after infection.
serum. This serum does not react with intact
viria. After precipitation of s antigen, labeled the appearance of viral RNA and other viral com-
RNA was still contained in the supernatant. This ponents, as far as they can be identified by the
seems to be viral RNA, since no significant trans- available tests. When FPA was added after the
fer of viral p32 to cell substances was observed. No critical time of about 1 hr postinfectien, a ma-
convincing experimental evidence is available at terial behaving serologically like s antigen and
present as to how the hemagglutinin shell of the containing viral RNA was produced in amounts
virion behaves during the degradation procedure. comparable to those occurring under normal
Before the production of detectable amounts conditions. Further effects of FPA will be con-
of new viral material begins, a substance has to sidered later on.
be formed the occurrence of which could be With regard to the functions of the early oc-
demonstrated only indirectly using FPA as curring FPA-sensitive material, which is very
inhibitor, as in the case of ME virus (Fig. 21). probably protein, no fully convincing explanation
The dose of FPA used did not disturb markedly can be offered at present even in this somewhat
the metabolism of cell RNA, the incorporation more intensively investigated case. Since, how-
of C'4-leucine into cell protein, or the eclipsing of ever, FPA does not markedly disturb synthesis of
fowl plague virus after infection. When added early normal cell RNA and since fowl plague viral
in the multiplication cycle, however, it inhibited RNA does not contain abnormal nucleotides, the
proteins in question may not be particular en-
zymes needed for viral RNA synthesis. Moreover,
the hypothesis comes to mind that in the case of
fowl plague virus the particular protein works in
stabilizing the viral genetic material; once this
has occurred, synthesis of viral RNA and of pro-
tein behaving serologically like s antigen protein
can no longer be stopped by FPA, as the normal
cell RNA metabolism might not be stopped by
FPA, since corresponding stabilized templates
are already established in the cell.
During the normal viral multiplication cycle,
new viral RNA and s-antigen protein can be first
FIG. 20. Scheme of fowl plague virus reproduc- detected 2 to 3 hr after infection, just after estab-
tion. lishment of the FPA-sensitive factor. The over-
10 SCHAFER BACTERIOL. Rimv.
synthesis of viral RNA, which is, according to the myxoviruses, lipids-probably of cellular
earlier experiments of Wecker (53), produced origin-serve to hold the viral components
de novo. Be&sides viral RNA and normal cell together in the completed virion.
RNA, no further RNA was observed to be pro- More thorough investigation of the viral
duced during the infectious cycle, not even during multiplication process led to the detection of some
that period when hemagglutinin appears. Thus factors which do not belong to the equipment of
it is not very probable that a RNA different from the virion itself. They seem to be, however,
viral RNA is engaged in the production of essential for establishing the virus-synthesizing
fowl plague viral protein. Unfortunately, chick capacity inside the host cell. First, there may be
embryo cells were unsuitable for autoradio- one particular substance of the host cell which
graphic studies which we would like to perform undresses the virion initially in order to liberate
to settle this topic somewhat more. its nucleic acid. That an undressing of the virion
The contribution of our group to the under- precedes its reproduction process could be rather
standing of NDV reproduction is still of a pre- convincingly shown with fowl plague virus.
liminary nature. It may be mentioned briefly Furthermore, virus systems as different as ME
that a material resembling the viral inner com- and fowl plague produce, apparently early in
ponent (24), and something like viromicrosomes the reproduction cycle, particular proteins which
(Rott and Reda, unpublished data), have been are reminiscent of the "early protein" of phages.
isolated. Therefore, one might suggest that at According to Levintow and co-workers (18), such
least some parallels exist in the reproduction early protein seems to occur also in the polio
mechanism of fowl plague and ND viruses. system. With regard to the function of these
proteins in the animal viral systems, no more is
Summary known at present than that they are necessary
The observations described with respect to for the normal progression of viral reproduction.
the significance of the various virus components A more thorough study of them might, however,
support the concept, already widely accepted, shed some light on the early steps which lead to
that the viral nucleic acid which is situated in the realization of viral information inside the
the interior of the viria possesses all the informa- cell.
tion the host cell needs to produce new virus. A most interesting problem is posed, finally,
With ME virus, this could be proved rather by the drastic RNA metabolic changes observed
convincingly. With fowl plague virus, at least by our group in ME virus-infected cells and by
some indication was obtained in this direction. Franklin and Rosner (7) in Mengo virus-infected
The main tasks of the materials surrounding cells. Of special interest is the establishment of
the nucleic acid are to protect this component an efficient RNA-synthesizing system in the
and to facilitate its penetration into the cell. cytoplasm at a time when viral protein appears
Thereby the "foreign gene" viral nucleic acid there. It may well be that the rapidly synthesized
becomes a "strolling gene" and therewith an cytoplasmic RNA is concerned with the produc-
infectious agent. The surface material contains, tion of virus protein.
on the other hand, those components of the virus
which induce immunity in the organism, and the APPLICATION OF SOME FEATURES
sites where neutralizing antibodies attack. EVALUATED TO PROBLEMS OF PRACTICAL
During the multiplication procedure, the viral IMPORTANCE
nucleic acid or the nucleic acid containing Although the survey presented offers many
inner component and the coating material seems more problems than well-established facts, some
to be generally produced separately and to be of the latter may lead to implications of practical
assembled later to form the new virion. importance.
This principle is well-established now in the The finding that the immunizing capacity of
case of fowl plague virus and can also be sug- the fowl plague virus resides in its hemagglutinin
gested from the results with ME virus. Further shell material led to the proposal to use, instead
evidence for such a concept is delivered by the of inactivated total virus, the isolated hemag-
genetic studies of Hirst (15), who found "pheno- glutinins of influenza and possibly other myxo-
typic mixing" with several animal viruses. In viruses as vaccines. The potency of such a
VOL. 27, 1963 STRUCTURE OF ANIMAL VIRUSES 13
vaccine may be demonstrated only by one result TABLE 2. Behavior of NH20H-treated fowl plague
(3). An injection of 0.003 mg of purified swine virus in the plaque test*
influenza hemagglutinin, prepared with Freund Dilutiont
adjuvant (10), initiated in a rabbit after 6 Time
weeks a hemagglutination-inhibition titer of of
treat- 10-1 102- 10 10-4 10- 10-6 10| 10"
1:32,000. With such vaccines, the occurrence mnent (1.1 (1.1 (1.1 (1.1 (1.1 (1.1 (1.1 (1.1)
x x x x x x x
of undesirable reactions would certainly fall to a 107) 106) 105) 104) 10') 102) 10)
minimum, since in contrast to intact virus iso- hr
lated hemagglutinin is practically free of normal o 110 18 3
host-cell components. 1.5 35 4 0 0
Where the isolation of the immunizing viral 3 64 2 0 0
shell material is not yet possible, one should 6 48 0 0
inactivate the respective viria for vaccine pro- 7.5 159 3 0
duction with a chemical which does not change 9 251 4 0
the antigenic material but acts exclusively on * Treatment was with 0.2 M NH20H. Results
the viral genetic substance, the nucleic acid. In are expressed as the number of plaques formed
this respect, hydroxylamine is clearly superior per culture.
to the still commonly used formaldehyde. NH20H t The numbers in parentheses correspond to
seems to react exclusively with the nucleic acid, the number of virus particles (original PFU's)
where, according to Schuster (50), it changes present in each dilution. Number of cells per cul-
the pyrimidine bases as indicated in Fig. 27. ture: -106.
Small RNA-containing viruses, like iIE,
polio, and EE, as well as the larger influenza
viruses, could be inactivated by this compound.
The deoxyribonucleic acid-containing Herpes
RNA chain (wnh cytidytic- and uridylec acid) -
Purines do not react wiih NH2 OH
NH2 0
NI L I I~~~~~~
IV
CN H
oCCN/N~CH
- --Phosphate
I
Ribose - Phoqpate- Ribose
~~~~~~I hsat
Phosphate - -
0 2 4t 6' 06'
Hours of treatsnt
I 2MolsNH2 OH
FIG. 28. Inactivation of M1E virus by NH2OH. 0,
NH 2-C CH
0.1 mu; 0, 0.5 .if 2H20H, pH 7.0, 20 C.
I
C\
HIt7 ~CH2 NH2 0CN N,.
virus and, surprisingly, the NDV and mumps
(5'-Jsoxazotoo)
N~~~~N virus strains examined proved to be resistant
-- -phosphate- Ribose Phosphate- Ribose - Phosphate- - -
(8, 36), although the latter viruses are similar in
their over-all structure to the influenza agents. In
this connection it was of some interest that TMV
probable further rea-uin
2 H20
was also resistant, but extracted TMIV-RNA
sensitive to NH20H. Thus, differences in coating
O *NH3
C
of viral RNA may play a role in causing differ-
HMt CH2 NH2 ences in their behavior to NH20H.
The inactivation kinetics (36) of ME virus with
O \N' S~ ( Urea)
NH20H were strictly exponential over the course
- - -Phosphate- Ribose - Pate- Ribose - Phosphate - - - examined (Fig. 28). An irregularity was observed
FIG. 27. Reaction of NH20H with RNA with fowl plague virus. After inoculating tissue
(Schuster). cultures with multiplicities higher than one of
14 SCHAFER BACTERIOL. REV.
f
* -------
incompletely inactivated fowl plague virus -a
i -0 - T Em
samples, more plaques appeared than were to be I/0 E m ..... 0 - 0
m |
expected from the number of those appearing /a0
,/nw *
at higher dilutions (Table 2). In this connection, ;/v *E Om0
O
multiplicity is referred to the plaque-forming *-
m mmmo