Ts27l virion was first described as containing non functional heamagglutinin and reduced neuraminidase activities when grown at non permissive temperature (Portner et

a1.,1974; Portner et

al.,

1975).

HN

was not detected under restrictive conditions either

by iodination of

the

infected cell

surface proteins

(Tuffereau et al.,

1985),

or in

the virus particles after 35S-methionine labelling

(Markwell

al.,

1985). This latter

point

is emphasized

in figure I

and

Table

^1.

Figure

1 panel

A,

shows a comparison

of

the

35s-methionine labelled viral

polypeptides

found in wild type (wt) or mutant

^(ts)

virions,

grown under permissive (31 oC) _{or non}permissive (39 oC) conditions.

At 3l

oC, the amount

of

^HN1r271incorporated

in

the virus particles

is

about

twofold lower

than that of HNye1, when the amounts of

HN

are normalized to the amounts of the other membrane

viral protein M (for clarity

see

HN

and

M in

the tracks

lx,31

^oC,

figure

panel

A,

and table

I, 31'C

^lines).

At

39 oC, however,

HN

appears barely detectable

in

the mutant virions, while its amount in the

wild

type virus is not reduced relative to the other

viral

^proteinsand

in particular

M (figure

1 panel

A tracks 3x,39 ^oC;

table

I

39oC lines). The data in table

I

indicate that at 39oC, HNts271 is reduced

by

about

15-20 folds

compared

to HNwt,

and

by

folds

compared

to

HN1s271 at

31oc,

when

normalized to M. However,

these numbers must

reflect an over estimation of

the

HNts271

incorporated

in

the

virions

at 39oC due

to

a non specific radioactive signal,

since HN was not

detected

by specific HN

antibodies

in the

W'estern

blot of

the samples (tracks ts 39

oC, lx, 3x, figure

1 panel

B).

Therefore,

within

the

limits of sensitivity of

the Western

blot

method, the mutant

virions grown

at non permissive

temperature

appears

free of HN. Furthermore, this massive reduction of HN

incorporation

in virions

appears to take place

with

significant

impairment

of viral

budding, since productions

of

virus particles

for wt

and ts271 at 39 oC are equivalent (see labelled virus figure panel

A).

The previous data, which

confirm

the conclusions reached in previous reports (Portner et

al.,

1974; Portner et

al.,

1975: Tuffereau et

al.,

1985;

Markwell

al.,

1985), could

still not

exclude the presence

in

the

virions of

the

HN

transmembrane-cytoplasmic

portion.

HNts271

is efficiently

synthesized at the non permissive temperature, but is degraded before reaching

the cell

surface (Tuffereau

et al., l9S5). HN

degradation

could therefore leave the HN-transmembrane-cytoplasmic portion in place. An

antiserum was therefore raised against the N-terminal twenty amino acids representing

two-thirds of

the

HN

cytoplasmic

tail

^(Blumberget

al.,

1985),

by

three subcutaneous

injections of

the peptide

coupled to keyholelimpet

hemocyanin.

As

expected, this antiserum does not react

with

the

HN

ectodomain expressed at the

cell

surface (figure 2, lanes

I

^andZ),^asdoes an antiserum raised against the whole

viral

particle (Rab-vir,

figure

2, lanes 3 and

4). Under

these conditions,

Rab-vir

recognizes

mainly

the two glycoproteins since they are the only one expressed at the cell surface. In contrast, after

cell

disruption, under conditions where Rab-vir now reacts also

with

the intracellular

viral

proteins (lanes

7

and 8),

Rab-HNtail, now,

gains access to the cytoplasmic tails, and specifically precipitates

HN

(figure 2, lanes 5 and 6).

To verify that

Rab-HN1ai1

could

detect a

putative HN

transmembrane-cytoplasmic

portion, purified wild

type virus particles were digested

with

bromelain to remove the glycoproteins ectodomains. These bald particles were then analyzed

by'Westem blot using

Rab-HN1a11.

Figure

3 panel

A ^(wt 31 oC

lanes) shows

that upon bromelain

digestion the

HN

^proteinis degraded

into

smaller products. The

X

bands appear to be intermediates in the digestion (they completely disappear with more extensive digestion, not shown),

whilst

TCP represents the

final

digestion product. Its migration relative to bovine

insulin

⁽²¹¹³⁰amino acids) is consistent

with

a polypeptide of about 60 residues

which

constitutes the

minimal

size

for the HN

transmembrane-cytoplasmic domain

(Blumberg et al.,

1985). The same result

was

obtained

with SV-wt grown at

39 oC

(not shown). V/hen a similar treatment was applied to ts271 particles grown

at permissive temperature (ts 31 oC lanes), the smaller digestion product of

HN

migrating at the

position of

the

wt-HN

intermediates

(X)

was observed. This product, however, was

likely to

represent the

final

digestion product containing the

HN

transmembrane cytoplasmic part, since, upon further treatment

with

bromelain,

it

was never cleaved

into a smaller polypeptide (figure 3,

^panel

B). This

presumably

reflects a different susceptibilily of

the mutant

HN

to bromelain. The analysis of the virus ts271 particles

produced at non

permissive temperature

(lanes ts 39 oC) clearly

showed

that

the absence

of HN

was

not

compensated

for by the

presence

of

bromelain

resistant polypeptide of any size that could represent a residual transmembrane portion.

In

the case

of VSV

ts045, however, the G C-terminal cytoplasmic

tail

was

difficult

detect due to

aggregation.

It

^was

in fact only identified in the stacking gel after electrophoresis of

^spikeless

virions under nonreducing conditions (Metsikkô

and

Simons,

1986). Such conditions

of

analysis were therefore used

in

the present study

(figure 3,panel C). In

^contrast

to VSV, the

electrophoresis

of ^ts271

^nonreduced

samples

gives results similar to

those

obtained

under reducing

conditions, i.e.

evidence

for

the presence HN-transmembrane

cytoplasmic portion

was obtained

in

particles produced at restrictive temperature. Neither band

X,

nor any aggregate

in

the stacking gel, specific for ts 39 oC was identifîed (figure 3C).

The data presented here indicate that the HN glycoprotein, which contains the essential

function for virion

attachment,

is not

essential

for virus particle formation. Even

a possible role

for

the transmembrane-cytoplasmic

portion of

the protein was excluded.

Lack of HN participation

in virion

budding was moreover supported by the fact that the extensive

reduction of HN incorporation in virus particles

appears

not to impair

budding efficiency. These conclusions contrast

with

the results reported for the

VSV

G protein, the counterpart

of SV-HN. VSV

G was

judged

_essential

for virus

buclcling,

since its

transmembrane-cytoplasmic

tail was found

present

in

spikeless

virions

(Metsikkô _andSimons, 1986).

In both

Sendai

and VSV, the M protein plays a central

strategic

role in budding

(Yoshida

et al.,

1976; Tuffereau and

Roux,

1988;

Knipe et

ar. 1977).

However,

the

two

viruses

differ in

that

VSV

only contains one glycoprotein, whereas Sendai

virus

contains

HN

and

Fo. If

one assumes that the anchor

portion of

one

glycoprotein

_is essential

for

budding,

G would

not be dispensable

in

the case

of VSV,

whereas,

in

absence of

HN,

Fo could

fulfîll

this function

in

Sendai virus.

It is

relevant

to this

question that a Newcastle disease

virus

temperature sensitive

mutant in the fusion protein, is equally capable of budding in

^absence

of

^Fo

(Matsumura et al.

1990).

V/hether

these

virions contain the Fo

transmembrane-cytoplasmic

portion

has not been reported. For Sendai

virus,

initially

cconsidered Fo as having an accessory function

in

virus budding, based

mainly

on the observation

that its stability

and

function

appeared unaffected

in situations of restricted virus

budding. In these situations, however,

instability

and/or malfunction of

SV-HN

and

M

were always

observed

(Tuffereau

and

Roux,

1988;

Roux et al.,

1985;

Roux

and

Waldvogel,

1982; Roux

et al.,

1984; Yoshida

et al.,

1979).

we

therefore postulated

that a preferential interaction between

HN

and

M

(rather than

Fo/M)

would be essential

for efficient

virus budding. The present data rather support that

M function (i.e ability to

organize a

bud)

does

not

depend on

HN,

and that

HN,

although

it may normally

interact

with M,

is not an essential component

in

the budding process. Therefore,

if

glycoprotein is

needed

in paramyxovirus budding, Fo

has

to

become

this

essential

Dans le document Etude de l'assemblage et du bourgeonnement du virus de Sendai (Page 43-47)

Ts27l virion was first described as containing non functional heamagglutinin and reduced neuraminidase activities when grown at non permissive temperature (Portner et

al.,

HN

by iodination of

infected cell

(Tuffereau et al.,

or in

(Markwell

al.,

point

in figure I

Table

Figure

A,

of

35s-methionine labelled viral

found in wild type (wt) or mutant

virions,

At 3l

of

in

is

twofold lower

HN

viral protein M (for clarity

HN

M in

lx,31

figure

A,

I, 31'C

At

HN

in

wild

viral

in particular

M (figure

A tracks 3x,39 oC;

I

I

by

15-20 folds

to HNwt,

by

folds

to

31oc,

normalized to M. However,

reflect an over estimation of

HNts271

in

virions

to

since HN was not

by specific HN

in the

blot of

oC, lx, 3x, figure

B).

within

limits of sensitivity of

blot

virions grown

temperature

free of HN. Furthermore, this massive reduction of HN

in virions

with

significant

of viral

of

for wt

A).

confirm

al.,

al.,

al.,

Markwell

al.,

still not

A tracks 3x,39 ^oC;

A ^(wt 31 oC