J. gen. Virol. (1989), 70, 2097-2109. Printed in Great Britain
2097
Key words: influenza virus/neutratization/uncoating
IgG-neutralized Influenza Virus Undergoes Primary, but Not Secondary
Uncoating in riro
By R. J. R I G G , t
A. S. C A R V E R AND N. J. D I M M O C K *
Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, U.K.
(Accepted 20 April 1989)
SUMMARY
Even when neutralized by saturating amounts of monoclonal IgG directed against
the haemagglutinin, influenza virus attaches to cells with kinetics similar to those of
infectious virus. It then enters those cells and is uncoated; its R N A becomes localized
within the nucleus and its lipid envelope and associated proteins remain in the
cytoplasm. In this report we show that despite the apparent normality of these early
stages of virus-cell interaction, neutralized virus underwent no detectable primary
transcription. In contrast, there was only a slight inhibition of transcription by
neutralized virus in vitro which was insufficient to account for the loss in infectivity,
despite using m R N A to measure the production of capped oligonucleotides or to prime
the elongation step. To test whether the absence of primary transcription in vivo
resulted from non-accessibility of the genome rather than an effect on the transcriptase
complex itself, we examined the susceptibility to RNase of virion R N A after
inoculation of cells with neutralized virus. Data clearly show that, unlike R N A of
infectious virus, RNA of neutralized virus did not become sensitive to RNase and we
conclude that neutralization of influenza virus by IgG results in failure of virus to
undergo a secondary uncoating process which is necessary for the activity of the virion
transcriptase complex. Finally we show that by treatment of virions in vitro with
detergent it is possible to produce a core structure which is stable and has some of the
properties expected of a structure resulting from primary uncoating.
INTRODUCTION
The initial stages of infection by influenza virus are attachment, uptake, uncoating and
transport of the virion RNA and associated proteins to the nucleus (Stephenson & Dimmock,
1975; Stephenson et al., 1978; Hudson et al., 1978). Then follow primary and secondary
transcription and replication, all of which take place in the nucleus (Shapiro et al., 1987). For
virus neutralized with saturating amounts of IgG, attachment and entry into CEF and BHK
cells occur with approximately normal kinetics and to the same extent as infectious virus (Possee
& Dimmock, 1981 ; Possee et al., 1982; Dimmock et aL, 1984; Taylor & Dimmock, 1985b). In
contrast virus neutralized with IgM or secretory IgA does not enter cells at all although about
50% attaches (Taylor & Dimmock, 1985a, b). These data also prove that IgG-neutralized virus
enters cells since they were all obtained using the same techniques. Attachment of both
infectious and neutralized virus is sensitive to pretreatment of ceils with neuraminidase
indicating that they use the same cell receptors (N. J. Dimmock, unpublished data). After entry
into ceils IgG-neutralized virus is uncoated as demonstrated by the presence of 94% of envelope
choline radiolabel in the cytoplasm while virion RNA accumulates normally in the nucleus
(Possee et al., 1982). However, there was no secondary transcription in cells inoculated with
neutralized influenza virus indicating that neutralizing antibody interrupts some stage before
this event (Possee et al., 1982). In order to locate the loss of function primarily responsible for
neutralization we have examined the possibility that neutralized virus directs primary
transcription but fails to make the transition to secondary transcription. To this end we have
Present address: ZMBH, Universit~itHeidelberg, D-6900 Heidelberg, F.R.G.
0000-8904 © 1989 SGM
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2098
R . J . RIGG, A. S. CARVER AND N. J. DIMMOCK
used cycloheximide to block the synthesis of proteins necessary for secondary transcription (Hay
et al., 1977; Barrett et al., 1979) and a radiolabelled probe sensitive enough to detect the small
quantities of m R N A synthesized. The latter is necessary as primary transcription of m R N A
represents only 5 to 15 % of that ( + ) R N A synthesis which occurs in the absence of inhibitors of
protein synthesis (Mark et al., 1979).
These experiments (described below) demonstrate that neutralized virus is unable to direct
primary transcription. We were then concerned to establish whether neutralizing antibody
affected the transcriptase complex per se and investigated the ability of detergent-uncoated
neutralized virus to undergo m K N A - p r i m e d transcription in vitro. In the course of synthesis
of capped viral m R N A influenza virus obtains 5'-capped oligonucleotides from suitable
heterologous m R N A s (Krug, 1985). The transcriptase complex binds to the 5' cap, endonucleolytically cleaves the m R N A 10 to 13 bases from the cap, and adds one nucleotide
(initiation) and by repeated addition (elongation) synthesizes influenza virus m R N A with a
short heterologous sequence making up the 5' end. These processes were only slightly affected by
a neutralizing monoclonal IgG suggesting to us the possibility that although neutralized virus is
uncoated of its lipid envelope, it fails to undergo primary transcription because of the absence of
an event required for activation of the transcriptase enzyme complex.
The process of uncoating of influenza virus is poorly documented. The first stage appears to
occur when the low pH of the endosomal vesicle in which the virus has entered the cell induces a
conformational change in the haemagglutinin (HA) resulting in fusion of the viral and
endosomal membranes and liberation of the subviral particles into the cytoplasm (reviewed by
Patterson & Oxford, 1986). In the presence of low concentrations of the antiviral compound
rimantadine, subviral particles containing membrane protein (M) and nucleoprotein (NP)
[Ml-ribonucleoprotein (RNP)] are found in the nuclear-associated cytoplasm and nucleus
whereas in the absence of the drug, R N P complexes in which only N P is detectable are seen in
the same location (Bukrinskaya et al., 1982). Thus there is a second stage of uncoating involving
loss of M1 protein from subviral particles which results in the release of R N P complexes.
Furthermore when cells are infected, virion R N A becomes sensitive to RNase digestion, but in
the presence of rimantadine R N A remains resistant to RNase degradation (Koff & Knight,
1979). Similarly, rimantadine prevents the loss of light sensitivity seen when neutral red-labelled
virus is inoculated onto cells (Kato & Eggers, 1969). Taken together these data suggest that a
second stage of uncoating concerned with the relaxation of a core structure is necessary before
the viral genome becomes functional.
Below we describe data which support the concept of secondary uncoating and demonstrate
that neutralized virus does not undergo this process.
METHODS
Virus. We used the avian strain of influenza virus A/FPV/Rostock/34 (H7NI) and a reassortant
FPV/Rn :PR/8N (H7NI). These were obtained as infected allantoic fluids from embryonated hens' eggs.
Infectivity was measured by plaque assay on primary chick embryo fibroblast (CEF) monolayers (Morser et al.,
1973) and HA by agglutination of chicken red blood cells. Virus was purified by differential centrifugation
followed by successivebanding on sucrose velocity and density gradients (Kelly & Dimmock, 1974). Radiolabelled
virus was grown in CEF cell monolayers (Dimmock et al., 1977).
Antibodies. We used HC2, a mouse neutralizing anti-HA (FPV/R) monoclonal IgG2a (kindly provided by J. J.
Skehel and A. R. Douglas), an anti-neuraminidase IgG (A/PR/8/34; H 1N 1) 112]10/2R6 (from W. Gerhard) as a
virion-binding, non-neutralizing antibody and a monoelonal antibody (MAb) (185/1) directed against an H3 HA
as a non-specific IgG. Polyclonal IgG against the HA was prepared by H. P. Taylor by intraveous inoculation of
rabbits with virus in phosphate-buffered saline (PBS) on days 0 and 21. Animals were bled on day 28. IgG was
purified by affinity chromatography on Protein A-Sepharose (Sigma) (Ey et al., 1978).
Detection of primary transcription. BHK-21 cell monolayers (107 cells/dish) were inoculated with virus-antibody
mixtures in PBS (equivalent to 100 p.f.u./cell for non-neutralized virus), or mock-inoculated with PBS alone, and
held on ice for 1 h. The cells were washed and incubated in tissue culture medium for 3 h at 37 °C. Cells were
treated for 1 h at 37 °C before infection with cycloheximide at 200 ktg/ml (Avery & Dimmock, 1975).
Cycloheximide was also included at the time of inoculation. This dose of cycloheximide inhibited protein synthesis
by 98.9 %. Cells were harvested and RNA was extracted with guanidinium isothiocyanate according to Maniatis et
al. (1982). RNA (18 Isg per duplicate) was denatured by the method of White & Bancroft (1982), applied to
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Uncoating o f neutralized influenza virus
2099
nitrocellulose (Andemann Chemicals) and hybridized by a method based on that of Maniatis et al. (1982).
Transcripts were detected with virion RNA that had been partially digested with alkali (100 mM-NaCO3 at 50 °C
for 1 h) and 5' end-labelled with [7-32p]ATP and polynucleotide kinase (Bethesda Research Laboratories).
Hybridization was carried out for 24 h at 42 °C and then filters were washed three times for 5 rain at room
temperature in 2 × SSC and three times for 20 rain in 0.25 x SSC at 65 °C, air-dried and autoradiographed at
- 7 0 °C using Fuji Rx X-ray film and an intensifying screen, individual dots were cut from the membrane and
immersed in scintillation fluid (Beckman) to determine the amount of radioactivity bound.
Polyacrylamide gel electrophoresisof proteins. The gel system of Cook et al. (1979) containing 0.I ~ SDS and the
discontinuous buffer system (Laemmli, 1970) were used. Gels were fixed in 50~ methanol/7~ acetic acid, soaked
in Amplify (Amersham), dried and exposed to X-ray film. The intensity of bands on the autoradiograph was
quantified using a Joyce-Loebl Chromoscan 3 densitometer.
Assay of influenza virus transcription in vitro
Elongation activity. The transcriptase activity of antibody-treated virus was assayed using the conditions
described by Bouloy et al. (1979) with 0-25 ~ NP40 to disrupt the viral envelope, but with cytoplasmic polyhedrosis
virus (CPV) mRNA as primer. CPV mRNA was synthesized from CPV according to Smith & Furuichi (1980) but
with 100 gg/ml bentonite in the reaction mixture. After removal of virus particles by centrifugation RNA was
extracted with phenol. Ethanol-precipitated RNA was dissolved in 10 mM-Tris-HCl pH 8.0, 150 msi-LiC1, 2-5
mM-EDTA (TLE) containing 0-1 ~ SDS and purified by chromatography on Sephadex G50.
To measure transcription, mixtures were made up on ice, adding virus last, and the reaction was started by
raising the temperature to 30 °C. Aliquots were assayed for TCA-insoluble [3H]UTP at the times shown.
Consistent with its expected priming activity, we found that RNA synthesis was directly proportional to the
amount of CPV mRNA added (data not shown). Its molar efficiency relative to ApG was 9500, comparing well
with the figure of 7100 quoted by Kawakami & Ishihama (1983).
The transcriptase activity of viral cores isolated from gradients was assayed in the absence or presence of 0-25
NP40. The amount of TCA-insoluble radioactivity produced after 2 h incubation at 30 °C was measured and the
relative transcriptase activity ( - N P 4 0 / + NP40) calculated.
Assays for initiation of influenza virus transcription. Reaction conditions were as described for elongation except
that the only ribonucleoside triphosphate included was [32p]GTP (Plotch et al., 1981) at a concentration of 1 ~t~l
(20 gCi/50 ral reaction mixture) and reactions were incubated at 30 °C for 60 min. At the end of the reaction
50 gl of 2 x TLE solution containing 0.2~ SDS was added. Unincorporated [32p]GTP was removed by spin
column chromatography (Maniatis et al., 1982) using Sephadex G25 in 1 x TLE containing 0.1~ SDS. The
eluate was phenol-extracted and ethanol-precipitated. Equal quantities were loaded onto slab gels of 16~
acrylamide/0-8 ~ bisacrylamide (Sanger & Coulson, 1978) and electrophoresed until the bromophenol blue marker
had migrated 21 cm. Production of radiolabeUed oligonucleotides was dependent upon the presence of both virus
and CPV mRNA.
Assay for RNase sensitivity of influenza virion RNA after inoculation onto BHK-21 cells and in viral cores made in
vitro. 32p-labelled influenza virus was inoculated onto BHK-21 cells held at 4 °C and subsequently incubated for
90 rain at 37 °C. The RNase sensitivity was assayed following the method of Koff & Knight (1979) except that cells
were frozen and thawed once only and further disrupted with ultrasound. The sonicate was incubated with
RNases A (Sigma) (100 gg) and T1 (Sigma) (500 units) for 30 rain at 37 °C and samples were trapped onto filters.
For each sample the fraction of radioactivity resistant to boiling in TCA was determined and subtracted.
Viral cores were produced by incubating virus for 4 rain on ice with a mixture of detergents, usually I gl of 10
NP40 and 0.25 ~ Triton X-100 per 1000 haemagglutination units (HAU) in a volume of 300 gl. Cores were purified
by centrifugation at 112000 g for 16 h at 4 °C on a 10:40:60 ~ w/v sucrose step gradient and were treated with
RNases A and T1 as described above.
RESULTS
P r e v i o u s w o r k has s h o w n that neutralized influenza virus attaches to cells, enters cells and
u n c o a t s ; the e n v e l o p e r e m a i n s in the c y t o p l a s m and the R N A and associated proteins enter the
nucleus (see Introduction). I n n o n e o f these p a r a m e t e r s could neutralized a n d infectious virus be
distinguished either in the kinetics or final status. T h e s e d a t a are r e c o n f i r m e d in this study in
w h i c h we found t h a t e q u a l a m o u n t s of neutralized or n o n - n e u t r a l i z e d virus a t t a c h e d to B H K - 2 1
cells (not shown), and t h a t a p p r o x i m a t e l y 7 0 ~ of cell-associated virion R N A f r o m either
neutralized or infectious inocula b e c a m e n u c l e a r (Fig. 1 a) w h e r e a s o v e r 8 5 ~ o f cellular R N A
labelled w i t h [3H]U o v e r a 24 h period was c y t o p l a s m i c (Fig. 1 b). T h e q u e s t i o n as to the stage at
w h i c h neutralized virus b e c o m e s unable to c a r r y out the infectious process was addressed here
by d e t e r m i n i n g w h e t h e r or not it could carry out p r i m a r y transcription.
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2100
R. J. RIGG, A. S. CARVER AND N. J. DIMMOCK
100
100
(a)
(b)
75
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75
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Z
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I
-= 50
!
25
25
i
C
N
C
N
N
C
v
Non-neut.
Neut.
Fig. |. (a) Distribution of 32pqabelled virion RNA of non-neutralized (non-neut.) and neutralized
(neut.) virus between the nuclear (N) and cytoplasmic (C) fractions of BHK-21 cells. 32p-labelled
FPV/R was incubated with either neutralizing anti-HA (HC2) or a non-specific (185/1) monoclonal
IgG. The infectivity of the neutralized virus was 99.9~ lower than the control. BHK-21 cells (106
cells/dish) were inoculated with virus (100 ~tl)(see Methods). Each 100 ~tl contained 7.5 x 106 p.f.u. 38
HAU and 6.9 x l0 s c.p.m. After 90 min 52~ virus was cell-associated. (b) Distribution of labelled cell
RNA between nuclear and cytoplasmic fraction of BHK-21 cells. Cells were incubated for 24 h at 37 °C
in tissue culture medium containing [3H]uridine (1 ~tCi/ml), the medium was replaced with medium
lacking the radioactive label and incubation continued for a further 24 h. In both (a) and (b) cells were
fractionated by the nuclear monolayer method (Taylor & Dimmock, 1985b) and the TCA-insoluble
radioactivity was determined. The bars indicate the standard error of the mean.
Failure of neutralized virus to direct primary transcription in vivo
Influenza virus transcription can be divided into two stages on the basis of the requirement for
protein synthesis; only primary transcription occurs when protein synthesis is prevented.
Neutralized virus was inoculated onto BHK-21 cells in the presence of cycloheximide and R N A
extracted after incubation at 37 °C for the times shown in Fig. 2. Dot blot hybridization detected
( + ) R N A produced by primary transcription in cells inoculated with non-neutralized virus after
1 h and this accumulated with time. Detectable amounts of ( + ) R N A were not produced in cells
inoculated with neutralized virus even after 4 h incubation. Thus neutralization directly or
indirectly inhibits p r i m a r y transcription, the first synthetic event of replication.
Failure of neutralizing antibodies to inhibit mRNA-primed transcriptase activity in vitro
Possee et al. (1982) reported that the elongation part of the transcriptase reaction was
inhibited in vitro by neutralizing polyclonal antibody directed against the H A of strain F P V / R or
by M A b 171/7 against the H A of A/X49 (H3N2) but not by non-neutralizing antibodies against
the neuraminidase. However, the extent of inhibition (up to 89 ~ ) was much less than the extent
of neutralization of infectivity ( > 99.9 ~o). In addition to an elongation activity, the polymerase
enzyme complex of influenza virus possesses activities which recognize and endonucleolytically
remove 5'-capped oligonucleotides from heterologous m R N A s (Krug, 1985). Thus, if antibody
were inhibiting the cap recognition or endonuclease reactions that yield the primer, infectivity
could be reduced without m a r k e d inhibition of the in vitro elongation activity. Fig. 3 shows that
9 9 ~ neutralization by HC2 I g G was accompanied by an inhibition of m R N A - p r i m e d
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Uncoating of neutralized influenza virus
(a)
N
2101
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Time (h)
(b)
I
I
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Fig. 2. Neutralized virus does not direct primary transcription in vivo. This is shown by analysis of
influenza (+) RNA extracted from BHK-21 cell monolayers which had been pretreated with
cycloheximide and inoculated with antibody-treated virus in the presence of cycloheximide. The
reassortant FPV/R :PR/8 (1440 HAU) was incubated with 15 Ilg neutralizing (N; 0) anti-HA IgG
(HC2) or an equivalent amount ~g) of non-neutralizing (C: O) anti-NA IgG (2R6) at 25 °C for 1 h. MI,
mock-infected cells. After inoculation cells were incubated at 37 °C for the times shown. RNA was then
extracted and viral (+) RNA was detected as a dot blot (a) by hybridization to fragmented 5' endradiolabelled virion ( - ) RNA. Dots were excised and radioactivity was counted (b).
transcription of less than 50~. In an experiment where the utilization of capped m R N A was
tested directly, neutralization had no detectable effect upon the generation and addition of the
first nucleotide (32p-labelled GTP) to primer oligonucleotides derived from CPV m R N A (Fig.
4). Similar results were obtained using IgG from a polyclonal anti-HA IgG (data not shown).
Insensitivity of the genorne of neutralized virus to RNase in vivo
Others have shown that, after infection in vivo, the R N A of infectious influenza virus becomes
progressively more sensitive to digestion by RNase thus reflecting its involvement in active
transcription (Koff & Knight, 1979). This gives an ideal system to determine whether
neutralized virus, which we know loses its lipid envelope in vivo with its R N A becoming nuclear
(Possee et al., 1982; Taylor & Dimmock, 1985 a; this study, data not shown), enters that RNasesensitive state which is presumably a necessary preliminary to, or corollary of, primary
transcription. We first confirmed the data of Koff & Knight (1979) by showing that after
inoculation onto cells on ice for 90 min (Fig. 5, right-hand hatched column) the virion R N A of
infectious (non-neutralized) virus was still essentially RNase-resistant but that after warming to
37 °C for 90 min, the majority (58~) of the genome of non-neutralized virus became R N a s e
sensitive, an increase of over sevenfold (Fig. 5, right-hand open column). In contrast, when
neutralized virus was inoculated virion R N A remained RNase-resistant even though, as stated
above, such virus loses its lipid envelope and the genome becomes located in the nucleus. The
technique is relatively insensitive but the differences are significant (P = 0.05 with n - 1 degrees
of freedom). At the three highest concentrations of antibody (all neutralizing to at least 99.9 ~o)
virion R N A remained resistant to RNase even after incubation of inoculated cells at 37 °C, at a
level which did not differ significantly from non-neutralized virus inoculated onto cells and held
on ice. The R N A of 7 9 ~ neutralized virus, however, was more susceptible to RNase, but still
less susceptible than that of non-neutralized virus. There is thus a good correlation between
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2102
R. J. RIGG,
1.7
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A . S. C A R V E R
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Fig. 3. Neutralizing antibody does not inhibit mRNA-primed transcription in vitro. The time courses
of mRNA-primed elongation reactions of non-neutralized and neutralized virus in vitro are shown.
Each reaction mixture (200 ktl) contained 3.5 x 103 HAU of FPV/R :PR/8 which had been incubated
with either neutralizing (O) anti-HA IgG (HC2) or non-neutralizing (O) anti-NA IgG (2R6) (both 16
ktg]103 HAU). The infectivity of neutralized virus (N) was 99.99~ lower than that of non-neutralized
virus (U). CPV mRNA (7.5 ~tg/reaction) was added as primer. Duplicate samples (15 ~tl) were
withdrawn at the times shown and the TCA-insoluble radioactivity determined. Assays were
performed in duplicate and the mean is plotted. The ratio of transcriptase activity for nonneutralized/neutralized virus is inserted at the top of the figure.
Fig. 4. Neutralizing antibody does not inhibit the generation of primer oligonucleotidesin vitro. PAGE
of the products of initiationassays(I) of non-neutralized and neutralized virus. FPV/R :PR/8 (2-5 × 103
HAU/assay) was incubated at 25 °C for 1h with neutralizing monoclonalanti-HA IgG (HC2), anti-NA
IgG (2R6) (both 25 ktg/103 HAU) or PBS. Initiationassays (50 ~tl)contained 5 ~tgof CPV mRNA. After
incubation for 1 h at 30 °C the RNA was extracted and analysed on a 16~ polyacrylamide gel. The
labelled products were detected by autoradiography at - 70 °C with an intensifying screen. Lane 1, 5'labelled oligo(dT)4_22ladder (Bethesda Research Laboratories); lane 2, virus incubated with PBS; lane
3, neutralized virus (99.99~ neutralization); lane 4, non-neutralized virus incubated with 2R6 IgG;
BPB marks the position of bromophenol blue dye.
neutralization and absence of an increase in RNase sensitivity of the virion genome suggesting
that neutralization prevents events which in the normal infectious process lead to relaxation of
structure(s) associated with, and necessary for, the expression of virion R N A .
Properties o f influenza virus cores produced in vitro
Data above suggest that after removal of the lipid envelope in vivo, the virion genome resides
in a core structure in which it is inaccessible to RNase. Such structures have not been
unequivocally demonstrated to exist although it is widely assumed from electron microscopic
evidence that the M1 protein forms a layer surrounding the R N P (Apostolov & Flewett, 1969;
Bachi et al., 1969; C o m p a n s & Dimmock, 1969) and structures such as these have been seen
under the electron microscope after various delipifying treatments (Nermut, 1970; Skehel, 1971 ;
Schulze, 1972; Reginster & Nermut, 1976). Brief exposure of unlabelled virus to an NP40/Triton
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Uncoating o f neutralized influenza virus
T
60
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3
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99.94
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Fig. 5. Virion RNA of neutralized virus remains resistant to RNase in vivo. The effects of different
dilutions of neutralizing antibody on the RNase sensitivity of the genome of influenza virus after
inoculation onto BHK cells are shown. 32p-labelled FPV/R was incubated for 1 h at 25 °C with dilutions
of neutralizing monoclonal IgG (HC2), which lowered infectivity as indicated, or with non-specific IgG
at the relative concentration shown. Virus-antibody mixtures were cooled and 100 Ixl was inoculated
onto six replicate monolayers of BHK-21 cells (6 x l06 cells/dish). Each 100 ~tl initially contained
6.8 × 107 p.f.u., 2.7 x 102 HAU and 2.1 × l0 s c.p.m. Two monolayers were sampled after 90 rain on ice
(hatched sections of bars) and the remaining four dishes were transferred to 37 °C (unshaded) and
incubated in tissue culture medium for a further 90 rain before RNase sensitivity was assayed. The
ordinate shows the mean values and a bar indicates the standard error of the mean (P = 0-05 with n - 1
degrees of freedom). Values shown along the abscissa are as follows: (i) neutralizing antibody, (ii)
1/relative antibody concentration, (iii) percentage neutralization.
X-100 mixture and centrifugation on a sucrose density gradient produced a uniform band,
visible by eye, which had a higher density than virions (Fig. 6a). W h e n analysed for
polypeptides, cores produced from [3SS]methionine-labeUed virus had the characteristics
expected for a core structure being composed of the majority of N P and M1 present in the
gradient, but with only traces of H A and N A (Fig. 6b). P proteins were not visible at this
exposure even in whole virions, nevertheless cores contained 76 ~o of the m a x i m u m transcriptase
activity (Fig. 6c). Only traces of lipid (defined here as TCA-soluble 32p) were associated with
cores, most of it being found at the top of the gradient (data not shown). Cores and virus banded
at approximate densities of 1.35 and 1.22 g/ml, respectively. Glycoproteins form a discrete b a n d
of lower density than the cores at the position m a r k e d in Fig. 6 (a) although this is never visible
by eye. The procedure was much more efficient than treatment of virus with deoxycholate
(Skehel, 1971) which converted only a minority of virus into cores with the rest being reduced to
constituent components. The cores are stable at 4 °C and can be r e b a n d e d without loss. W i t h a
longer treatment at × 1 detergent concentration the cores disintegrate.
The R N a s e sensitivity of the R N A in cores produced using a range of detergent concentrations was assayed and was c o m p a r e d to that in structures obtained from neutralized virus
in vivo as described above. A relative detergent concentration of 0.3 removed the lipid (Fig. 7a)
but the cores remained RNase-resistant (Fig. 7 b). A threefold higher concentration of detergent
was required before any sensitivity to R N a s e was seen, suggesting that this much was required
to modify the structure of the core particle. In parallel experiments, m R N A - p r i m e d
transcriptase activity and the protein composition of the cores were determined. Transcriptase
activity of cores was measured without adding more detergent or with the addition of 0-25 ~o
NP40 which gives a m a x i m u m value. As the quantity of detergent used to prepare cores was
increased the transcriptase activity of the cores in the absence of any free detergent also
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2104
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Uncoating o f neutralized influenza virus
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o
0.1
0
I
Virus
0-3
1.0
3.0
I
I
Core
Relative detergent concentration
Fig. 7. RNase sensitivity and transcriptase activity of cores purified by sucrose density gradient
centrifugation of virus treated with increasing amounts of detergent. In (a) and (b) 3:p-labelled
influenza virus was treated with detergent for 4 min on ice and centrifuged as in Fig. 6. A relative
detergent concentration of 3-0 contains 0-1~ NP40 and 0-0025 Triton X-100. The gradients were
fractionated and the total amount of 32p at the top of the gradient (representing mainly viral lipid) is
plotted in (a) and the RNase sensitivity of cores is presented in (b). (c) The transcriptase activity of cores
produced by detergent treatment of unlabelled influenza virus. These were assayed in triplicate for
transcriptase activity using CPV mRNA as primer either without the further addition of detergent or in
the presence of 0-25~ NP40 which gives a maximum value, and expressed as a percentage of the
maximum value.
increased (Fig. 7 c). Analysis of cores by SDS-PAGE (Fig. 8) followed by quantitative scanning
of the resulting autoradiograph showed that the proportion of M 1 relative to NP decreased by
over twofold as more detergent was used in their preparation. At this exposure P proteins could
not be seen although longer exposures showed that they were not diminished at any detergent
concentration.
DISCUSSION
Previous data have shown that saturating amounts of neutralizing IgG specific for influenza
virus HA do not significantly affect any of the early stages of infection in vivo including
attachment, penetration, removal of the envelope or accumulation of the viral genome plus
associated proteins in the nucleus (Possee & Dimmock, 1981 ; Possee et al., 1982; Dimmock et
al., 1984; Taylor & Dimmock, 1985b; Taylor et al., 1987). However, the virus was unable to
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2106
(a)
R" J" RIGG, A" S' CARVER AND N. J. DIMMOCK
1
2
3
4
5
(b)
t
I
I
I
I
I
I
I
NP
"a
~rrr
~'b
I
.
--
r
----M
Fig. 8. (a) SDS-PAGE analysis of the polypeptide composition of cores produced by detergent
treatment of [35S]methionine-labelled virus as described in Fig. 5 showing the progressive loss of M1
protein relative to NP with increasing detergent concentration (concentrations are 0.1, 0.3, 1 and 3 in
lanes 2 to 5, respectively). Lane 1, untreated virus. A scan of autoradiograph tracks showing virus
treated with the lowest (a) and highest (b) relative detergent concentrations is shown in (b).
undergo secondary transcription (Possee et al., 1982). In this report we show that virus
neutralized with monoclonal anti-HA IgG undergoes no primary transcription in vivo. Is this an
effect on the transcriptase activity directly or the result of some indirect influence? To answer
this question we returned to the in vitro system in which Possee et al. (1982) showed that there
was some inhibition of transcriptase by neutralizing antibody, although always at a level over
100-fold lower than the amount of neutralization. One possible explanation was that
neutralization did not affect the elongation part of the transcriptase reaction but the initial
stages of cap recognition and endonucleolytic cleavage of the m R N A primer (Krug, 1985).
However, we have been unable to see any substantial inhibition of mRNA-primed transcription
or capped oligonucleotide production after neutralization with the HC2 MAb or with a
neutralizing polyclonal rabbit IgG (data not shown). Nonetheless as Shimizu et al. (1985) have
found, the result might be different with monoclonal antibodies directed against other antigenic
sites on the HA. [HC2 produces escape mutants with a single mutation in HA1 (Gly 133~Glu)
in site A (J. McCauley, A. S. Carver, H. P. Taylor and N. J. Dimmock, unpublished data).] This
is equivalent to residue 143 and antigenic site A on the H3 structure (Wiley et al., 1981).
Thus as the in vitro system does not appear to reflect accurately the situation in vivo we have reexamined the uncoating of neutralized virus in vivo to determine whether this was affected by
neutralization. Since earlier work had shown that the lipid envelope, HA and N A of neutralized
virus were found in the cytoplasm whereas virion R N A accumulated in the nucleus (Possee et
al., 1982; A. S. Carver & N. J. Dimmock, unpublished data), it was thought that uncoating of
neutralized virus occurred normally. However, when we examined the susceptibility of viral
R N A to digestion by RNase (Fig. 5) we found that neutralization inhibited the extent to which
virion R N A became RNase-sensitive. Thus it seems that there is a second stage of uncoating
which occurs after the lipid and associated envelope proteins have been removed, an event
which is suggested by studies on the inhibition of uncoating by rimantadine (Koff & Knight,
1979; Bukrinskaya et al., 1982). It follows that the stage of infection compromised by
neutralization occurs after removal of the viral envelope and at, or prior to, secondary
uncoating. We now postulate that neutralization prevents secondary uncoating which in turn
results in failure of the virus to direct transcription. Thus transcription may not take place
because the transcriptase complex is inaccessible to small molecules needed for R N A synthesis.
Alternatively it may be that continued presence of M1 is directly inhibitory to transcription
(Zvonarjev & Ghendon, 1980; Mikheyeva & Ghendon, 1983; Kato et al., 1985; Ye et al., 1987).
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Uncoating o f neutralized influenza virus
2107
In an effort to gain some understanding of the nature of the de-enveloped, RNase-insensitive,
transcriptionally inactive particle produced by neutralization with IgG we treated virus with
critical amounts of detergent. Unlike a previous study with deoxycholate (Skehel, 1971) this
produced a stable 'core' of greater density than virus and which lacked the majority of lipid and
envelope proteins. Of particular interest was the fact that with a relative detergent concentration
of 0.3, even though there was maximum release of 32p-labelled presumptive lipid, virion RNA
was almost totally resistant to degradation by RNases, a situation which exactly parallels that of
neutralization. Increasing the detergent concentration rendered the genome RNase-sensitive in
a manner analogous to secondary uncoating. Failure to obtain RNase sensitivity of greater than
50~ probably reflects the fact that we used TCA precipitation as a measure of the integrity of
virion RNA rather than, for example, by running it on a denaturing gel. In general there was also
good correlation between the transcriptase activity of viral cores taken from their characteristic
position on the sucrose density gradient and the amount of detergent used to release them from
virions. However the fact that their activity could always be increased by adding detergent
showed that they were not maximally activated. The slightly greater transcriptase activity (30~
of maximum) of cores prepared at a detergent concentration of 0.3 compared with RNase
sensitivity ( 6 ~ of maximum) may reflect the greater permeability of the cores to RNA
precursors than to RNases or indicate that RNA is being precipitated by TCA in association
with other core components.
Although we must be cautious in comparing cores prepared with detergent to structures which
result from secondary uncoating of virus in vivo, our data lead us to suggest that the early events
of infection are as follows. Primary uncoating in vivo consists of the removal of the lipid envelope
and associated glycoproteins, resulting in a spheroidal core structure which is bounded by M 1
protein and contains all the other components of the virion. Further we suggest that this is
transcriptionally inactive until it undergoes the events of secondary uncoating which increase its
permeability before or after it gets to the nucleus.
Much more remains to be done. If cores from neutralized virus are inactive solely because of
an inability to undergo secondary uncoating we would predict that they should be capable of
being activated by detergent in vitro. So far we have failed to obtain an answer to this question.
In summary we see IgG neutralization as the transmission across the viral envelope of a signal
which is initiated by anti-HA IgG as first suggested by Possee et al. (1982). However from the
data described above we conclude that the simplest interpretation of the effect of this signal is
upon the secondary uncoating process rather than the transcriptase activity per se.
We thank J. J. Skehel, A. R. Douglas (National Institute for Medical Research, London, U.K.), R. G. Webster
(St Jude Children's Research Hospital, Memphis, Tenn., U.S.A.) and W. Gerhard (Wistar Institute, Philadelphia
Pa., U.S.A.) for providing monoclonal antibodies, C. C. Payne (AFRC Institute of Horticultural Research,
Littlehampton, U.K.) for the CPV and S. Covey (John Innes Research Institute, Norwich, U.K.) for the scan.
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