Generation of defective interfering particles of vesicular stomatitis virus in Aedes albopictus cells

107, 497-508

VIROLOGY

Generation

(1980)

of Defective

Interfering Particles of Vesicular in Aedes albopictus Cells

STEPHEN Department

of Microbiology,

College

GILLIES

AND

of Medicine Piscataway,

and Dentistry Neu! Jersey

Accepted

VICTOR

August

Stomatitis

Virus

STOLLAR

o.f New

Jersey,

Rutgers

Medical

School,

08854

5, 1980

The serial undiluted passage of clonally purified vesicular stomatitis virus (VSV) in Aedes albopictus cells resulted in a rapidly declining titer of infectious virus. VSV,,,, (a stock derived after two such passages) contained slowly sedimenting truncated (T) particles which were interfering and noninfectious. Whereas a VSV stock produced by three undiluted passages in BHK cells (VSV,,,) interfered with the replication of standard virus (VSV,,,) equally well in A. albopietus and in BHK cells, VSV,,,, interfered efficiently only in the former. These patterns of interference were also observed when gradient purified T particles were tested. When VSV,,, or VSV,,,, was passaged once in the heterologous cell type the pattern of interference was reversed showing that these defective interfering (DI) particles could be phenotypically modified in such a way as to influence their specificity of interference with VSV,,,. When viral RNA synthesis was studied in cells coinfected with VSV,,, and VSV,,, or VSVaPB, the following observations were made: (1) interference was correlated with a decrease in the synthesis of 42 S VSV,,, genome RNA; (2) although VSV,,,, DI particles interfered well in A. albopictus cells but only poorly in BHK cells the DI RNA genomes of VSV,,,, were replicated equally well in both cell types. (3) The major DI particles in VSV,,,, contained “snap-back” RNA composed of covalently linked sequences of positive and negative polarity. By testing the effect of RNase on these RNAs it was concluded that they contained from 90 to 95% selfcomplementary sequences. Snap-back RNA was not readily detectable in the DI particles of VSV,,,. INTRODUCTION

particles during serial undiluted passage in albopictus cells, more recent work (Logan, 1979; King et al., 1979) has shown that alphavirus DI particles can be produced in these cells if a longer incubation time is used. The production of high yields of VSV in cultured mosquito cells (Gillies and Stollar, 1980) has made it possible to generate and study DI particles in these cells. Although VSV T particles have been observed in persistently infected A. albopictus cells by electron microscopy (Artsob and Spence, 1974), there have so far not been any reports or systematic studies of DI particles in any invertebrate cell system. In this report we show that DI particles of VSV are readily produced in A. albopictus cells, and we describe the RNA associated with these particles. We also demonstrate that although DI particles passaged in A.

A.

Several reports (Perrault and Holland, 1972; Holland et al., 19’76; Kang et al., 1978) show that the host cell can markedly influence the generation and replication of vesicular stomatitis virus (VSV) defective interfering (DI) particles. For example, Kang et al. (19’78) demonstrated that a single clonal isolate of VSV produced different size classes of DI particles following serial undiluted passage in four different cell lines. A host cell effect on the replication of DI particles of Sindbis virus (SV) has also been reported (Igarashi and Stollar, 1976). In this case SV DI particles produced in BHK cells failed to replicate or to interfere with standard virus in Aedes albopictus cells. Although in their experiments Igarashi and Stollar were unable to find evidence for the production of SV DI 497

00426822/80/160497-12$02.00/O Copyright All rights

0 1980 by Academic Press. Inc. of reproductmn in any form reservrd.

GILLIES

30

AND

20 IO FRACTION NUMBER

FIG. 1. Sucrose velocity gradient analysis of the progeny particles from A. albopicfm cells infected with VSV,,,,,. A. a~bopictus (C7) monolayer-s in 100. mm plates (approximately 2.0 x 10” cells) \rere infected with undiluted VSV,,,,, (m.o.i. = 2 PFIJicell) and incubated at 28” for 24 hr in the presence of [:‘H]uridine (10 &i/ml). Medium was removed and virus concentrated as described in Materials and Methods. Prior to centrifugation, the virus suspension was sonicated for 15 set in a Raytheon model DFlOl sonicoscillator, immediately layered on a 15 to 30% (w/v) sucrose gradient (.50 mM Tris pH 8.0, 0.1 J4 NaCl, 1 m?4 EDTA, 0.1% bovine serum albumin) and sedimented in a SW41 rotor (4”) for 45 min at 37,000 rpm. Fractions of 0.4 ml were collected and assayed for radioactivity, infectivity in BHK cells. and interfering activity in A. albopictm cells as described in Materials and Methods. For the interference assays, all fractions were diluted I:10 before being tested; the values represent the fold reduction relative to the control yield at 24 hr.

albopictus cells interfere efficiently with VSV,,., in mosquito cells, they interfere very poorly in BHK cells. In contrast DI particles passaged in BHK cells interfere equally well in both cell types. MATERIALS

AND

METHODS

Cells, virus, and infection procedure. The baby hamster kidney (BHK-21) and A. albopictus (LT-C7) cells used in this study have been described (Stollar and Thomas, 1975; Sarver and Stollar, 1977). A stock of VSV, Indiana serotype, originally obtained from Dr. David Bishop was propa-

STOLLAR

gated after three consecutive plaque purifications by low multiplicity infection (Stampfer et al., 1971) of BHK cell monolayers. Unless otherwise stated, cells were infected at an input multiplicity of 10 PFUi cell; adsorption was for 45 min at either 28 or 34” as indicated. Following adsorption, the inoculum was removed and the monolayers rinsed twice with cold phosphate-buffered saline (PBS with Ca” and Mgz+). Prewarmed E medium (Eagle’s minimum essential medium to which were added nonessential amino acids at 0.2 rnM each) supplemented with heat-inactivated fetal calf serum was then added. Purzfication qf DI particles. Medium from suitably infected cultures was clarified by centrifugation, first at 2000 g for 10 min at 4”, then at 8000 g for 20 min at 4”. The supernatant was transferred to SW27 tubes (1” x 3”) and underlaid with 50 ~1 glycerol. Following centrifugation at 26,000 rpm for 2 hr (4”), the supernatant was removed and the virus pellet resuspended overnight at 4” in 150 ~1 of PBS. Prior to velocity gradient sedimentation (see legend to Fig. 1) the concentrated virus was sonicated for 15 sec. Inte$erence assay. Passaged virus stocks or purified DI particles were assayed for the ability to interfere with VSV,.,, replication in the following manner. A series of fivefold dilutions of the test sample was made in cold PBS containing 0.1% BSA. Sufficient VSV,,, was added to each dilution so that when 0.2 ml of each mixture was added to cell monolayers in 60 mm plates (3.6 x 10fi cell/plate) the final m.o.i. was 10 PFUicell. After adsorption for 45 min the monolayers were rinsed three times with cold PBS and fed with 4 ml of E medium. Cultures were incubated for 24 hr at 28” (A. albopictus) or 34” (BHK) after which samples of media were assayed by plaque assay on BHK cells. In each experiment cultures were also infected with VSV,,, alone (m.o.i. 10 PFUicell) to determine the control titers in the absence of interference. Analysis sf‘ intracellular and particleassociated viral RNA made during in&tion with passagedvirus. Duplicate cultures in 100mm plates were infected with VSVsT,

DEFECTIVE

INTERFERING

(10 PFUlcell) alone or together with a 1:lO dilution of the passaged virus stock to be tested. One monolayer was used for the analysis of intracellular RNA as described (Gillies and Stollar, 1980), and the second for particle-associated RNA. The second culture was labeled from 2 to 24 hr after infection with [3H]uridine (10 pCi/ml) in the presence of actinomycin D (0.1 kg/ ml) at 28” (A. albopictus) or 34” (BHK). Medium was then layered directly on lo40% (w/w) sucrose-D20 gradients (Shenk and Stollar, 1973) and centrifuged at 26,000 rpm for 24 hr (4”) in an SW27.1 rotor. Viruscontaining fractions (detected by cont;inuous absorbance monitoring) were pooled, diluted eightfold in PBS, and pelleted as described. RNA was phenol-extracted from virus pellets as from whole cells (Gillies and Stollar, 1980). Agarose gel electrophoresis of RNA. Electrophoresis of single-stranded intracellular RNA was performed in agaroseformaldehyde gels under denaturing conditions (Gillies and Stollar, 1980). RNA soluble in 2.0 M LiCl (double-stranded) was analyzed by agarose gel electrophoresis under nondenaturing conditions (McDonnel et al., 1977). Ethidium bromide (0.5 pglml) was included in all gels for visualization of marker RNA or DNA. Electrophoresis was at 25 V for 14 hr with buffer recirculation. Gels were prepared for fluorography by dehydration with two rinses in absolute methanol (45 min each) followed by impregnation with 2,5-diphenyloxazole (10% w/w in acetone) for 16 hr. Gels were dried without heat in a Hoefer gel dryer and exposed to prefogged Kodak XR-5 X-ray film at -70”. RESULTS

Serial Undiluted Passage of VSV in BHK and A. albopictus Cells

Serial undiluted passage of cloned stocks of VSV at high multiplicity generally results by the third or fourth passage in the generation of defective interfering particles (Huang, 1973) and rapidly declining titers of infectious virus. Table 1 shows that titers in an undiluted passage series in BHK cells remained unchanged in the first two

PARTICLES

OF VSV

499

TABLE INTERFERING

1

ACTIVITY OF UNDILUTED STOCKS OF VSV”

PASSAGE

Fraction control yield in interference assay Virus passaged

in

Passage number

Titer (PFUlml)

A. albo (cm

BHK21

A. albopictus cells

AP 1 2 3 4 5

2.0 2.5 3.0 3.4 6.2

x x x x x

10” 10’ 106 10’ 10’

0.667 0.007 0.680 0.911 0.800

0.614 0.214 0.669 0.882 0.920

BHK21

BP 1 2 3 4 5

4.1 1.2 1.0 3.1 2.5

x 109

0.138

x

109

0.118

x 108 x 106 x 109

0.005 0.662 1.000

0.429 0.130 0.006 0.409 0.804

cells

* A stock of VSV was prepared by three consecutive plaque purifications in BHK cells followed by a low multiplicity infection (0.01 PFUlcell), also of BHK cells. Undiluted passages were initiated by infecting monolayers of BHK and A. albopictus (C7) cells in lO@mm petri dishes (containing approximately 1.0 x lo7 cells) with 0.5 ml of the VW stock to give a final multiplicity of 1000 PFUicell. After 18 hr of incubation in E medium at 34 and 28” for BHK and A. albo. (CT) cells, respectively, medium was removed and cell debris pelleted by low-speed centrifugation. Consecutive passages were continued using 0.5 ml amounts of undiluted medium. Each passaged stock was assayed for infectivity on BHK cells, and for interfering activity against standard VW in both cell types (see Materials and Methods). Stocks passaged in Aedes albopictus cells are designated as AP, and those passaged in BHK cells as BP.

passages but declined by the third passage (VSV&. In A. albopictus cells, each of the first three passages reduced the yield of infectious virus by about 90%, suggesting that VSV DI particles were generated quite efficiently in A. albopictus cells, probably even during the first high multiplicity infection. To confirm the interfering activity, each passaged stock was mixed with a fixed amount of standard VSV to bring the final multiplicity to 10 PFU/cell and used to infect cultures of BHK and A. albopictus cells. Yields of infectious virus at 24 hr after infection were determined and expressed as the fraction of control yields from cells infected with standard virus alone (Table 1). Maximum interfering activity was found with the second passage virus from A.

500

GILLIES

AND

STOLLAR

cells (VSV,,,) and with the third were produced in A. albopictus cells as well passage virus from BHK cells (VSV,,,). as in BHK cells. To show more conclusively When the ability of the various passaged that DI particles were responsible for the stocks to interfere in the homologous and interfering activity of VSVAPZ, [“Hluridineheterologous cell type was compared, a labeled virus produced from VSV,,,-infected significant difference between VSV,, and A. albopictus cells was subjected to sucrose VSV,, was observed. Whereas each stock velocity gradient centrifugation; each graof VSV,, interfered to approximately the dient fraction was assayed for radioactivity, same extent in both cell types, the A. infectivity in BHK cells, and interfering ability in A. albopictus cells. albopictus passage stock with the most The gradient shown in Fig. 1 contained interfering ability (VSV& inhibited VSV two major peaks of radioactivity. The faster replication in A. albopictus cells 30 times more efficiently than in BHK cells. Since sedimenting peak contained virtually all of the infectivity while most of the interfering VSVAp, and VSV,,, contained the greatest interfering ability, and presumably the activity cosedimented with the smaller, component. A signifihighest concentration of DI particles, fur- slower sedimenting ther comparative studies were carried out cant amount of interfering activity was also found in the region of the gradient interwith these two virus stocks. mediate between the standard virus and the In the A. albopictus cell passage there major DI species, suggesting that more was not always a good correlation between the interfering activity of a given stock than one size of DI particle was produced (as measured by the interference assay) by the A. albopictus cells. That the slower and the level of infectious virus in the sedimenting component was made up of T subsequent passage. Since (1) in a co- particles was confirmed by electron microscopy (not shown). infection with standard and DI particles the type of progeny produced is deterEffect of an Undiluted Passage in the Hetmined not only by the absolute amounts erologous Cell Type on, the Interfering of each type of particle in the inoculum Ability of VSV DI Particles but also by the ratio of standard to DI particles and (2) in the interference assay Figure 2 shows that, in agreement with but not in the passage series the m.o.i. of Table 1, VSV,,, interfered significantly infectious virus is kept constant, then the with infectious virus production only in interference assay should provide a better A. albopictus cells, whereas VSV,,,, interguide to the concentration of interfering fered to approximately the same extent in particles in passaged stocks than simply the both cell types. When VSV,,, was passaged levels of PFU/ml in an undiluted passage once in BHK cells, a virus stock (VSV,,,,,,,,) series, during which the m.o.i. can vary up was produced that interfered equally well to 104-fold (Table 1). In view of the m.o.i. in both cell types and closely resembled the effect (Gillies and Stollar, 1980) on the rate VSV,,, stock. Passage of VSV,,, in A. of virus release (at m.o.i.‘s
DEFECTIVE

INTERFERING

DI particles passaged in A. albopictus cells interfered to a much greater extent in the homologous cell type (Fig. 3A) while DI particles passaged in BHK cells interfered to the same extent in both cell types (Fig. 3B). This experiment also confirms that the interfering activity of passaged virus stocks is due to DI particles and not to soluble antiviral factors produced by the cells. As an additional control, interference assays were repeated using as helper virus standard WV grown in A. albopictus cells; identical results were obtained (not shown). This shows that the cell-specific interference seen with the VSV,,, stock is not related to the use of heterologous standard

7

“S”SP3

\

\

loo

200

30(

200

xx)

lDD2co3xl

Volume DI Added I pl I

FIG. 3. Interfering ability of DI particles purified from the VSV,., and VSV,,,,,,,, stocks. DI particles were purified by velocity gradient centrifugation (see Materials and Methods and legend to Fig. 1) of VSV,,, (see legend to Fig. 2). Gradient (Table 1) or VSV,,,,,,, fractions containing DI particles (determined by continuous absorbance monitoring) were pooled and diluted to equivalent optical density. Interfering activity was then quantitated as described in the legend for Fig. 2. (A) Interfering ability of VSV,,,, as a function of the relative number of DI particles added, determined in A. albopictus (0 __ 0) and BHK cells (0 __ 0). (8) Interfering ability of VSV,,,,,,, in A. nlbopictus (0 0) and BHK cells (0 0). The multiplicity of infection for VSV,,, was 10 PFUlcell in each case.

RNA I00

501

OF VSV

virus but rather to some property particles themselves.

“S”APZBPI

VOLUME

PARTICLES

i

of the DI

Synthesis in VSV-Infected A. albopictus and BHK Cells under Conditions of Interference

PASSAGE0 STOCK ADOED~pl~

FIG. 2. Interfering activity of VSV,,, and VSV,,, after passage in the heterologous cell type. VSV,,,, (Table 1) was passaged without dilution in BHK cells to give the VSV,.,,,., stock, and VSV,r3 (Table 1) in A. albopictus cells to give the VSVBP3,,,,, stock. The interfering ability of each stock was then tested in both BHK and A. albopictus cells. Fivefold dilutions of the passaged stocks were prepared and mixed with an equal volume of VSV,,, to give a final multiplicity of 10 PFUicell. After 24 hr incubation at 28” (A. albopictus cells) or 34” (BHK cells), media were assayed for infectious virus on BHK cells. The control yields, set at 1.0, were obtained from duplicate cultures infected with VSV,,, alone, and in different experiments ranged from 1.0 to 2.0 x 10’” PFU/ml for BHK and from 8.0 x 10” to 1.5 X 10y PFU/ml for A. albopictus cells. The values on the abscissa are equivalent to the volume of the undiluted stock added for each infection. Yields from A. albopictus cells (0 0); yields from BHK cells (0 __ 0).

In order to determine whether the host cell-determined specificity of interference (as determined by infectious for VSV,, virus yields) was the result of interference at the level of viral RNA synthesis, the effect of VSV,,, and VSV,,, on VSV,,, RNA synthesis was examined in both BHK and A. albopictus cells. Duplicate cell cultures were infected with VSV,,, (10 PFU/cell) alone, or together with VSVAPP or VSV,,,. One monolayer from each type of infection was used for analysis of intracellular RNA while the second was used for the analysis of extracellular virus. Phenol-extracted intracellular RNA was fractionated with LiCl into single-stranded (LiCI precipitable) and double-stranded (LiCl soluble) RNA fractions. The RNA packaged into progeny virus particles was

502

GII,LIES VSVST

42s

+AP2

AND

+ BP3

-

1.46

NS,M

-

A

B

C

D

E

F

FIG. 4. Analysis of the LiCl-precipitable VSVspecific RNA synthesized in BHK cells under conditions of interference. BHK cell monolayers were infected with VSV,.,.,, at a multiplicity of 10 PFUicell either alone or together with a 1:lO dilution of VSV,,., or VSV,,,, and labeled at 34” with [“Hluridine (25 &ii ml) in the presence of actinomycin D (4 kg/ml) from 3 to 6 hr after infection. RNA was extracted and fractionated with LiCl as described in Materials and Methods. The LiCl-insoluble fraction was suspended in 200 ~1 of 2~ denaturing electrophoresis sample buffer (4.4 M formaldehyde, 0.04 M phosphate pH 7.0, 0.2% SDS). To label extracellular particles l:‘H]uridine was added to infected cultures from 2 to 24 hr after infection, and RNA was obtained by phenol extraction of purified particles. Samples (20 ~1) were mixed with an equal volume of 99% deionized formamide and electrophoresed as described in Materials and Methods. (A) Intracellular RNA from cells infected with VW’,.,,,; (B) RNA from extracellular particles released from cells infected with VSV,,,,; (C) intracellular RNA from cells infected with VSV,,.,, and VSV,,,, diluted 1:lO; (D) RNA from extracellular particles released from cells infected with VSV,,,, and VSV,,,? 1:lO; (E) intracellular RNA from cells infected with VSV,.,,, and a 1:lO dilution of VSV,,,:,; (F) RNA from extracellular particles released from cells infected with VSV,,, and a I:10 dilution of VSV,,,,,. Molecular weight estimates (X lOP), determined as described in the text are indicated. The values obtained for the RNA species seen in VSV,,,-infected cells (average of three determinations) were: 42 S, 4.0 x 10”; L, 2.4 x 10”; G, 0.64 x 10”; N, 0.54 x 10G; M and NS, 0.34 x 10”.

STOLLAR

obtained after equilibrium centrifugation (sucrose-D,O) of the culture medium and extraction of the RNA from the viruscontaining fractions. Since both VSVsTu and DI particles have the same buoyant density (Wagner et al., 1969), the relative proportion of standard and DI particles can be determined from the ratio of the standard and truncated RNAs extracted from the total particle population. The RNAs obtained by this method were analyzed by electrophoresis in denaturing agarose gels; intracellular single-stranded (LiCl insoluble) viral RNA was run in a parallel channel. Molecular weights of the DI RNAs were estimated from their relative mobilities using Sindbis virus 42 S and 26 S RNA (Simmons and Strauss, 1974) and bacteriophage T7 early mRNA (Studier, 1973) as markers. The effects of VSV,,, and VSV,,, on standard virus RNA synthesis are shown in Figs. 4 and 5. As expected from the yields of infectious virus, VSV,,, greatly reduced the amount of 42 S virion RNA released from both BHK (Fig. 4, channel F) and A. albopictus cells (Fig. 5, channel G) when compared to controls infected with standard virus alone (channel B, in Figs. 4 and 5). Intracellular single-stranded (ss)RNA was also drastically reduced in both cell types following infection with VSV,,, (Fig. 4, channel E, and Fig. 5, channel F). The predominant ssRNA synthesized in each case corn&rated with the major DI RNA species extracted from particles released by BHK cells (Fig. 4, channel F) and had a molecular weight of 0.89 x 10’. This species along with a second, minor RNA (molecular weight 1.46 x 10”) represent the genomes of the DI particles contained in this virus stock. In contrast to VSVnr3, VSV,i,2 had little effect on standard virus RNA synthesis in BHK cells (Fig. 4, channel C) or on the amount of virion 42 S RNA released from BHK cells (Fig. 4, channel D). Two new (presumably DI) RNAs were detected, however, and had different molecular weights (0.76 and 0.95 x 106) from those seen after infection with VSV,,,,. More importantly, at least one of these species was synthesized in relative abundance, despite the lack of significant interference

DEFECTIVE

INTERFERING

PARTICLES

OF VSV M

+ BP3

NS,M

503

-

A

6

c

D

E

F

G

H

FIG. 5. Analysis of the LiCl-precipitable VW-specific RNA synthesized in A. albopictus (C7) cells under conditions of interference. A. albopictus (Ci’) monolayers were infected exactly as described for Fig. 4. For analysis of intracellular RNA, labeling was from 6 to 12 hr after infection and from 2 to 24 hr for analysis of extracellular particle RNA. RNA was phenol-extracted and analyzed as in Fig. 4. A mock-infected control culture was included for C7 cells due to the presence of a low level of actinomycin D-resistant RNA synthesis in these cells (Mento and Stollar, 1978; Eaton and Randlett, 1978). (A) Intracellular RNA from cells infected with VSV,,,; (B) RNA from extracellular particles released from cells infected with VSV,,,; (C) intracellular RNA from cells infected with VSV,,, and a 1:lO dilution of VSV,,,,, labeled from 6 to 12 hr after infection; (D) same as C except that labeling was from 18 to 24 hr after infection; (E) RNA from extracellular particles released from cells infected with VSV,,, and a I.10 dilution of VSV,,,; (F) intracellular RNA from cells infected with VSV STD and a 1: 10 dilution of VSV,,,; (G) RNA from extracellular particles released from cells infected with VSV,,, and a 1:lO dilution of VSV,,+ (H) intracellular RNA from mock-infected cells. Molecular weight estimates (X lo-“) are indicated.

with 42 S synthesis normally associated with DI particle replication. effect VSVAP2 had a quite different on standard virus RNA synthesis in A. albopictus cells. Figure 5 shows that coinfection with VSV,,, and VSVsTD greatly reduced the synthesis of virus-associated (channel E) and intracellular 42 S RNA at both early (channel C) and late times (channel D). Unlike what was observed after VSV,,, infection of either cell type, RNA species corn&rating with the mRNAs for the G and N proteins were detected under conditions of VSV,,,-mediated interference in A. albopictus cells (channel C). The identity of these species as mRNA has not been confirmed; however, it does seem surprising that these messengers would

continue to be synthesized while the L, NS, and M messengers would not. On the other hand, it seems unlikely that these species are DI RNAs since they are not observed in the extracellular virus population. Also, in contrast to VSVBP,-infected cells, there was no correspondence between the extracellular particle DI RNA (Fig. 4, channel D) and those species of ssRNA found in VSV,p,-infected cells. This finding led us to examine the intracellular LiClsoluble or double-stranded RNA produced during standard infections and under conditions of interference in both cell types (Fig. 6). BHK cells infected with VSV,,, contained only one species of dsRNA with an apparent molecular weight of 8.30 x 10fi (channel A), approximately twice that of the

504

GILLIES

AND

BHK SA0 T 023

s P

P

STOLLAR

A. olbo. C7 A A0

T

P

PP

D

2

23M

A

-

13.70

-

474

8.30 -

= 3:s -

302

-

2 13

2.69 -

1.74 1.63 I.36 -

‘i% = 000 0.74 -

?+

0.60 -

ABCDEFGHL FIG. 6. Analysis of the LiCl-soluble VSV-specific RNA synthesized in A. albopictus (C7) and BHK cells under conditions of interference. Double-stranded RNA was purified from A. albopictus and BHK cells by phenol extraction and LiCl fractionation as described in Materials and Methods. The LiCl soluble fractions of the RNA samples described in Figs. 4 and 5 were reprecipitated with 2.5 volumes of 95% ethanol and suspended in electrophoresis buffer (10 mJ4 Tris pH 7.4, 5 m&f sodium acetate. 0.5 mM EDTA, 0.2% SDS). Electrophoresis was carried out on a 0.7% agarose gel containing ethidium bromide (0.5 pg/ml) along with an EcoRI digest of lambda DNA. After photographing the DNA markers, the gel was fluorographed, dried, and exposed to prefogged X-ray film. (A) RNA from BHK cells infected with VSV,,,; (B) RNA from BHK cells infected with VSV,.,-,, and a 1:lO dilution of vsv,,,; (C) RNA from BHK cells infected with VSV,,.,, and a 1:lO dilution of VSV,,.,,; (D) RNA from A. nlhopict~s cells infected with VSV,.r,; (E) RNA from A. oZbo[)irt~s cells infected with VSV,,, and a 1:lO dilution of VSV,,,, labeled from 6 to 12 hr after infection; (F) RNA from A. albopictz~s cells infected with VSV,,,,, and a 1:lO dilution of VSV,,,, labeled from 18 to 24 hi after infection; (G) RNA from A. albopiclus cells infected with VSV,,,, and a 1:lO dilution of VSV,,,,,; (H) RNA from mock-infected A. alhopict~s cells; (I) E’coRI restriction endonuclease fragments of lambda DNA. Molecular weights (X 10~ “) of restriction fragments are from Thomas and Davis (1975).

42 S genome. An RNA of similar size was seen in A. albopictus cells upon longer exposure of the gel (not shown). This species represents the annealed positive and negative strands of the genome length RNA. Double-stranded DI RNAs were seen in cells coinfected with VSV,,, and VSV,,,, (channels C and G); their estimated molecular weights were 1.63 and 2.69 x 10fi, or slightly less than twice the molecular weight of the major DI RNAs seen in these infections (0.89 and 1.46 x 106). The estimate of molecular weights of the doublestranded RNA may be low since DNA was

used as marker. The presence of DI dsRNA in VSV,,,,-infected BHK cells was also correlated with a reduction in the amount of dsRNA related to the standard virus genome. The double-stranded RNA fraction from BHK and A. albopictus cells coinfected with VSV,,, and VSV,, (Figure 6, channels B and E) consisted largely of two species of DI RNA that were much more abundant than the DI dsRNAs seen in VSVB,,-infected cells. These molecules represent the only detectable RNA synthesized in A. albopictus cells from 18 to 24 hr after coinfection

DEFECTIVE

INTERFERING

with VSV,,, and VSV,,, (channel F). The molecular weights of the major species, as determined by their migration relative to DNA restriction fragments (0.74 and 0.89 x lo”), were not double those of the DI RNAs seen in released particles (0.76 and 0.95 x lo6 see Fig. 4, channel D), as would be expected for annealed (+) and (-) strands. Instead, their molecular weights in neutral agarose closely resembled those obtained for the DI particle-associated RNA examined under fully denaturing conditions. These findings strongly suggest that the major DI particles in VSV,,, (and those produced by cells infected with VSV,,,) contain RNAs with extensive internal complementary regions which, upon phenol extraction, are able to self-anneal. Such RNAs would be soluble in 2 M LiCl. Similar VSV DI RNAs have already been described (Lazzarini et al., 1975; Perrault, 1976). In contrast to what was observed in BHK cells infected with VSV,,, and VSVBp3, coinfection with VSVAP2 and VSV,,,, caused little if any reduction in the amount of the 8.3 x 10’ MW dsRNA, despite abundant DI RNA synthesis. Analysis

gf Double-Stranded

DI RNA

In order to determine the extent of selfcomplementarity in the RNA from VSV,,, DI particles, LiCl-soluble DI RNA was prepared from A. albopictus cells infected with regions of VSVA,, and single-stranded the molecules were digested with RNase. The remaining RNase-resistant (doublestranded) RNA was then analyzed by electrophoresis under denaturing and nondenaturing conditions. Self-complementary molecules would be resistant to RNase throughout the base-paired region but would be susceptible in the region that covalently links the complementary strands (Perrault, 1976). By comparing RNasetreated molecules to the untreated control under conditions of denaturing and nondenaturing electrophoresis the degree of base pairing can be calculated from the reduction in molecular weight. Electrophoretic analysis of RNasetreated DI RNA under nondenaturing conditions is shown in Fig. 7. DI RNA

PARTICLES

OF VW

505

0.7

4.4

2.8

2.0

-

FIG. 7. Analysis of RNase-treated ds DI RNA by neutral agarose gel electrophoresis. RNA from VSV,,,Z-infected A. albopictus cells (conditions of infection were as described in Fig. 4) was labeled from 3 to 8 hr after infection with [“Hluridine (25 &ii ml) and the LiCl-soluble fraction split into two equal portions. One-half of the sample was treated with RNase A (12 wgiml) in 0.24 Jf NaCl at 37” for 15 min and then phenol-extracted, while the remaining portion served as the untreated control. Each RNA was then precipitated with ethanol, resuspended in electrophoresis buffer, and analyzed as in Fig. 6, except that a 0.8% agarose gel was used. Marker dsRNA was photographed and the gel dried and fluorographed as described. (A) RNA extracted from particles produced by BHK cells following infection with VWkr,; (B) LiCl-soluble RNA untreated; (C) LiCl-soluble RNA treated with RNase A; (D) Sindbis virus dsRNAs: replicative forms of the standard genome and of three DI genomes. Estimated molecular weights (Guild et cr2., 1977) are indicated.

extracted from the progeny particles of BHK cells infected with VSV,,, (channel A) comigrated with both the untreated (channel B) and the RNase-treated DI RNA (channel C) extracted from A. albopictus cells coinfected with VSVsTD and VSV,, demonstrating that (1) intracellular as well as DI particle-associated RNA self-anneal to form extensively base paired molecules and (2) RNase treatment had no detectable effect on the mobility of these doublestranded RNAs. When the same samples were analyzed

506

GILLIES A

6

AND

C

-42s

-

1.67 1.46

-

a95 0.76 0.62

-

0.44 0.36

FIG. 8. Analysis of RNase-treated ds DI RNA by denaturing agarose gel electrophoresis. The RNasetreated, untreated, and particle-associated DI RNAs described in the legend to Fig. 7 were denatured and analyzed on a 1% agarose gel containing 2.2 &f formaldehyde as described in Materials and Methods, and in the legend to Fig. 4. Molecular weights (X lo- “) are indicated and were determined as described in the text. (A) RNA extracted from particles produced by BHK cells following infection with WV,,,,; (B) LiCl-soluble RNA untreated: (C) LiCl-soluble RNA treated with RNase A.

on denaturing gels after reaction with formaldehyde, DI particle RNA (Fig. 8, channel A) again comigrated with the untreated intracellular DI RNA, whereas the RNase-treated sample (channel C) contained two RNA species with estimated molecular weights of 0.36 and 0.44 x 10” or slightly less than half of the original single-strand molecular weights (0.76 and 0.95 x 106). These small RNA species presumably arise by the digestion of the RNase-susceptible sequences that covalently link the self-complementary regions of the molecules, and the subsequent denaturation of the resulting linear duplexes. From the molecular weights of the untreated and RNase-treated RNAs, it was determined that each species contains from 90 to 95% self-complementary sequences. DISCUSSION

The results that as with

presented in this report show most other cell types, serial

STOLLAR

undiluted passage of VSV in A. ulbopictxs cells led rapidly to the appearance of interfering activity. That this interfering activity was due to DI particles is shown by (1) the finding in sucrose velocity gradients of slowly sedimenting particles which were not infectious but which interfered with standard virus production in a dose dependent manner and (2) the observation by electron microscopy of truncated particles. Of special interest is our finding that although these DI particles could be replicated in both BHK cells and A. albopictm cells they manifested much less interfering activity in the former than in the latter. When viral RNA synthesis was examined in both cell types under conditions of interference, it was found that in a given infection the decrease in the amount of 42 S genome-length RNA synthesis correlated well with the degree of interference as determined by infectious virus yields. For example, in BHK cells infected with VSV.,,,Z there was relatively little interference with the replication of standard virus and these cells contained approximately the same amount of 42 S intracellular RNA and genome length dsRNA and released about as much particle 42 S RNA as cells infected with VSV,.,,, alone (Figs. 4 and 6). On the other hand A. albopictzcs cells, coinfected with VSV,,, and VSV,.,I,, contained greatly reduced levels of 42 S RNA and, consistent with the lower yields of infectious virus, very little standard genome RNA was released in virus particles. These results indicate that in BHK cells, but not in A. albopictus cells, VSV,,,> DI particles may function as defective noninterfering particles. Adachi and Lazzarini (1978) have reported that three different DI particles of VSV Indiana were abundantly replicated using the Hazelhurst strain of VSV New Jersey as helper, although no significant interference with helper virus replication occurred. When the same DIs were tested with VSV (Indiana) as helper, DI replication occurred but now at the expense of infectious virus production. Thus, the replication of DI particles can be associated in one case with a marked interference with the production of standard virus (as in A. albopictus cells

DEFECTIVE

INTERFERING

infected with VSV,, DI particles) or with only a slight reduction in the yield of standard virus (as in BHK cells infected with VSV,, DI particles). Schnitzlein and Reichmann (1977) used pseudotype VSV DI particles containing Indiana serotype RNA and New Jersey serotype proteins to examine the interaction between standard (helper) virus and heterologous DI particles. They observed that DI particles containing Indiana DI RNA, but encapsidated and enveloped with New Jersey viral proteins, interfered with standard VSV New Jersey much better than did DI particles composed of both RNA and protein of the Indiana serotype. They further observed that when the pseudotype DI particles were passaged once in cells coinfected with VSV (Indiana) as helper, this enhanced interference ability was completely abolished. This suggests very strongly that the viral proteins contained in DI particles must influence the specificity of interference. Since both standard virus and DI RNA are replicated as a nucleocapsid- enzyme complex (Huang and Palma, 1974), and interference occurs at the level of replication (Huang, 1973) it is likely that one or more of the nucleocapsid proteins (N, NS, or L) is crucial in determining interference specificity. That the same type of specificity is responsible for the defective but noninterfering nature of VSV,, DI particles in BHK cells is suggested by the following observations. (1) A single passage of VSV,,, DI particles in BHK cells resulted in the production of DI particles with equal interfering ability in both cell types. (2) RNA extracted from DI particles produced in VSV ,1,,2-infected BHK cells was identical to that found in A. a,lbopictus cells infected with VSV,,, and different from the DI RNA seen in VSV,,,, infections. (3) Although high yields of VSV,A,. DI particles were produced by BHK cells there was little interference with the production of standard virus, just as was observed when heterologous VSV DI particles (Indiana) were replicated using VSV (NJ) as helper (Adachi and Lazzarini, 1978). In contrast to the pseudotype DI particles, the proteins synthesized in A.

PARTICLES

OF VSV

507

or BHK cells and incorporated into DI particles were difficult to differentiate since all of the polypeptides were of the VSV (Ind.) serotype and differed presumably only in the nature or extent of hostspecified, posttranslational modification. The glycoprotein of VSV,. (,,,,,, migrated faster in SDS-polyacrylamide gels than that of VSV,,, (Gillies and Stollar, 1980) presumably as a result of differences in hostspecified glycosylation. We found, however, that such differences had no effect on the adsorption properties of VSV,,, (and presumably of DI particles) grown in A. albopictus cells (Gillies and Stollar, 1980). In addition to the G protein, the NS and M proteins are known to be modified (phosphorylated) and to interact with the nucleocapsld in the expression of replicative and transcriptive functions (Clinton et al., 1978a, b). Analysis of these proteins and the nucleocapsid (N) protein by partial proteolysis (Cleveland et al., 1977) has so far not revealed differences between vsv .,. n~~jJland VSVnfIK (Gillies and Stollar, unpublished results). Modifications that do not alter electrophoretic mobility in SDSpolyacrylamide gels, however, would not be detected in this system. The production of DI particles with cellspecific interfering ability might also be explained in terms of RNA modification such as methylation. Recent studies (Moyer and Gatchell, 1979) have shown that VSV DI RNA is not methylated in BHK cells; such modifications could, however, be sequence-dependent or cell-specific. By determining the modification(s) effected by passage of VSV DI particles in A. albopictus and BHK cells it might be possible to clarify the factors which determine whether DI RNA replication will or will not interfere with standard virus replication. albopictus

ACKNOWLEDGMENTS This investigation was supported by Grant AI-11290 from the National Institute of Allergy and Infectious Diseases, by the United States-Japan Medical Science Program through Public Health Service Grant AI-05920, and by the Institutional National Research Service Award CA-09069 from the National Cancer Institute.

GILLIES

508

AND STOLLAR

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