Research ELSEVIER Virus Research 38 (1995) 111-124
Nucleocapsid- and virus-like particles assemble in cells infected with recombinant baculoviruses or vaccinia viruses
expressing the M and the S segments of Hantaan virus
Michael Betenbaugh a, Manching Yu a, Kathleen Kuehl b, John White b, David Pennock c, Kristin Spik c, Connie Schmaljohn ~' *
a Department of Chemical Engineering, Johns Hopkins University, Baltimore, MD 21218, USA b Pathology Division, USArmy Medical Research Institute of Infectious Diseases, Fort Detrick,
Frederick, MD 21702-5011, USA c Virology Division, US Army Medical Research Institute of Infectious Diseases, Fort Detrick,
Frederick, MD 21702-5011, USA
Received 15 March 1995; revised 10 May 1995; accepted 11 May 1995
Abstract
The formation of Hantaan (HTN) virus nucleocapsid-like structures (NLS) or virus-like particles (VLP) from expressed gene products was investigated in two eukaryotic systems. Baculovirus expression of the HTN virus small segment (S), which encodes the viral nucleocapsid protein, resulted in assembly of NLS inside infected insect cells. The NLS and authentic ribonucleocapsids, prepared by detergent disruption of HTN virions, had similar sedimentation characteristics and morphologies, and were recognized by HTN virus N- specific antibodies. Co-expression of S and the medium segment (M), which encodes the two viral envelope glycoproteins (G1 and G2), did not efficiently generate VLP in the baculovirus-insect cell system, but VLP were observed in lysates and supernatants of cells infected with a recombinant vaccinia virus co-expressing HTN virus M and S. The VLP sedimented in sucrose to densities consistent with HTN virions, and some of them bore a striking resemblance to Hantaan virions when examined by immunoelectron microscopy.
112 M. Betenbaugh et al. / Virus Research 38 (1995) 111-124
I. Introduction
Nucleocapsid-like structures (NLS) or virus-like particles (VLP) have been found to assemble from the expressed gene products of a number of DNA and RNA viruses. For example, VLP of human papilloma virus (HPV), a double-strand DNA virus, were observed in insect cells infected with a recombinant baculovirus expressing the major capsid protein, L1 (Rose et al., 1993). Co-expression of L1 and the minor capsid protein L2 of HPV increased particle formation 4-fold (Kirnbauer et al., 1993). Empty-capsid VLP of a single-strand DNA virus, canine parvovirus, were also produced by baculovirus expression of viral structural pro- teins (Saliki et al., 1992).
Both baculovirus and vaccinia virus recombinants were used to make NLS of non-enveloped, plus strand RNA viruses, including picornaviruses (Ansardi et al., 1991; Brautigam et al., 1993) and caliciviruses (Laurent et al., 1994), double-strand RNA viruses, including rotaviruses (Redmond et al., 1993; Crawford et al., 1994; Dormitzer et al., 1994), reoviruses (Xu et al., 1993), and orbiviruses (Roy et al., 1992; Pearson and Roy, 1993; Hewat et al., 1994; Moss and Nuttall, 1994); and one enveloped negative-strand RNA virus, measles virus (Spehner et al., 1991; Fooks et al., 1993). The measles virus NLS were found to assemble in the absence of RNA, and were detected both in the cytoplasm and nuclei of recombinant virus-infected cells.
In addition to NLS, assembly of VLP by enveloped viruses has also been observed. A stably transfected eukaryotic cell line, expressing the capsid and E1 and E2 envelope proteins of rubella virus, produced enveloped VLP, but expressed E1 and E2 alone did not, suggesting that an interaction between the capsid and envelope proteins is required for assembly (Hobman et al., 1994). Similarly, VLP of alphaviruses were only produced with a Semliki Forest virus expression vector when the capsid and envelope proteins were co-expressed, although no RNA interaction was required (Suomalainen et al., 1992). Expression of the capsid (gag) gene of a number of retroviruses by baculovirus or vaccinia virus recombinants has been shown to generate VLP which can acquire envelopes by budding from cell surfaces. If gag and env genes were co-expressed, the VLP envelopes contained virus-specified surface glycoproteins (Karacostas et al., 1989; Haffar et al., 1990; Luo et al., 1990; Rasmussen et al., 1990; Morikawa et al., 1991; Smith et al., 1993; Hertig et al., 1994).
Because VLP would be a convenient source of viral antigen both for immuno- genicity and diagnostic studies and because such structures could provide an in vitro model for virus assembly, we sought to determine if expressed Hantaan (HTN) virus gene products could produce VLP. We used two eukaryotic cell expression systems, baculovirus and vaccinia virus, to express the small (S), and /o r medium (M) segments of HTN virus. Our studies indicate that NLS can assemble Srom exp~;essed N, and VLP can assemble when S and M are both expressed. Optimization of expression conditions might permit the further use of such VLP for development of vaccines or diagnostic reagents, or for studies on the morpho- genesis of HTN virus.
M. Betenbaugh et al. / Virus Research 38 (1995) 111-124 113
2. Materials and methods
2.1. Viruses, cell lines and culture media
Recombinant vaccinia viruses and baculoviruses, expressing the M or the S segments of HTN virus, and a recombinant vaccinia virus co-expressing the M and S segments were described previously (Schmaljohn et al., 1990, 1992). Bac- uloviruses were propagated in suspension or stationary cultures of the insect cell line SF-9, derived from Spodoptera frugiperda which were cultured in TNM-FH medium (Gibco-BRL) supplemented with 10% fetal bovine serum (FBS) and antibiotics (Schmaljohn et al., 1988). Vaccinia viruses were propagated in roller bottles of BHK-21 or Vero E6 cells cultured in Eagle's minimum essential medium (EMEM) with Earls' salts supplemented with 10% FBS, and antibiotics as de- scribed previously (Schmaljohn et al., 1990). The origin and growth characteristics in Vero E6 cells of authentic HTN virus, strain 76-118) have been described (Schmaljohn and Dalrymple, 1983).
2.2. Immune sera
The origin and characteristics of monoclonal antibodies to HTN virus have been described (Arikawa et al., 1989; Ruo et al., 1991). Polyclonal antibodies used were hyperimmune mouse ascitic fluid and rabbit immune sera prepared as described elsewhere (Schmaljohn et al., 1985; Liang et al., 1994). To prepare an immune rabbit serum specific for G1 and G2, a polyclonal serum was repeatedly adsorbed to cell lysates containing large amounts of baculovirus-expressed N as follows. SF-9 cells (9 X 108) infected with a baculovirus recombinant expressing HTN S, were harvested 3 days after infection, pelleted, and the cell pellets lysed with 5 ml of a buffer containing 1% TX-100, 10 mM Tris-HC1, pH 8.0, 1 mM EDTA, 0.5 M NaC1 and 0.25 mg/ml each of the proteinase inhibitors aprotinin and az-macroglobulin. Glutaraldehyde was added to a final concentration of 0.2% and the solution was incubated at room temperature for 1 h. Glycine was added to a final concentration of 0.2 M and the solution was stirred at room temperature for 1 h. The fixed cell lysates were pelleted, the pellet recovered, resuspended and washed two times in PBS, then divided into 4 equal aliquots and pelleted in a microcentrifuge. One milliliter of polyclonal anti-HTN rabbit serum was added to the pellet in one of the microtubes and mixed at 4°C overnight. The antigen was removed by centrifuga- tion and the procedure repeated 3 additional times. The specificity of the immune sera was confirmed by immune-precipitation of radiolabeled HTN virus proteins (not shown).
2.3. Polyacrylamide gel electrophoresis, and Western blots
Methods for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS- PAGE) were as described previously (Schmaljohn et al., 1983, 1987). Briefly, for Western blots, gradient fractions were diluted 1 : 4 in SDS and mercaptoethanol-
114 M. Betenbaugh et al. / Virus Research 38 (1995) 111-124
containing loading buffer and boiled for 2 min prior to electrophoresis. Gel fractionated proteins were transferred to Gene Screen Plus membranes (Dupont) electrophoretically and were reacted with polyclonal, hyperimmune mouse ascitic fluid to Hantaan virus, diluted 1 : 500 in 2.5% skimmed milk. After washing, the membranes were incubated with alkaline phosphatase conjugated goat anti-mouse antibody (Promega) diluted 1 : 4000, washed again, and reacted with NBT-BCIP color developer.
2.4. Purification o f NLS, VLP and H T N virions
For purification of NLS from insect cells, SF-9 cells, grown as monolayers in T150 plastic cell culture flasks (Falcon), were infected with a recombinant bac- ulovirus expressing the S segment of HTN virus (Schmaljohn et al., 1988) at a m.o.i, of 5. After 4 days, when approximately 50% of the cells demonstrated CPE, the cells were removed from the flasks by vigorous shaking, and pelleted by slow-speed centrifugation. The cell pellet was recovered, washed once in sterile PBS and centrifuged again. The pelleted cells were placed on ice and lysed by the addition of 2 ml of a buffer containing 4% Zwittergent 3-14 (Calbiochem), or 1% Triton X-100 (Pierce) in 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 0.5 M NaCI and 0.25 mg/ml each of the proteinase inhibitors aprotinin and az-macroglobulin (Gibco-BRL). Cell nuclei were removed by centrifugation, and the remaining detergent lysates were layered onto 15-40% cesium chloride gradients, or 10-60% sucrose gradients prepared in TNE buffer (100 mM NaCl, 10 mM Tris-HCl, pH 7.4, 1 mM EDTA), and centrifuged for 22 h at 30,000 rpm in a SW41Ti rotor (Beckman). Gradients were fractionated from the bottom into 0.50-ml aliquots, and densities were determined by calculations based on the refractive index of the gradient fractions as follows: density = 10.8601(refractive index) - 13.4974.
Recombinant vaccinia virus-infected Vero E6 cells or BHK cells (3 150-cm 2 flasks) were harvested when most cells were detached from the culture flasks (3-4 days postinfection). Cells remaining on the flask were scraped into the medium with a rubber policeman, and the culture fluid was centrifuged at 6000 g for 25 rain at 4°C. Particles in the supernatants were precipitated by adding polyethylene glycol 8000 (PEG, final concentration 8%) and NaCl (final concentration 0.5 M). After stirring at 4°C for 4 h, the particles were concentrated by centrifuging at 6000 g for 20 min. Resultant pellets were resuspended in 10 ml PBS and combined with cell pellets. The suspension was placed in a tight fitting, stainless steel dounce homogenizer on ice and cells disrupted with 10 strokes, after which the lysate was sonicated for 2 rain on ice at maximum output in a cup sonicator. The lysate was clarified by centrifugation at 500 g for 5 min and then placed into SW28 ultracentrifuge tubes and underlayed with 5 ml of 60% sucrose. After centrifuga- tion at 25,000 rpm for 1.5 h, the interface of the lysate and sucrose was collected and was diluted in PBS to 4 ml and layered onto a 10-60% sucrose gradient, prepared ,in TNE buffer. The gradients were centrifuged at 35,000 rpm in a SW41 rotor (Beckman) overnight. Gradient fractions (0.5 ml) were collected and densi- ties determined. Fractions with densities corresponding to those of authentic HTN virus were examined for NLS and VLP by immunoelectron microscopy.
M. Betenbaugh et al. / Virus Research 38 (1995) 111-124 115
To determine if VLP were being secreted into the supernatants, and if the particles contained NLS, recombinant-infected BHK cells (two roller bottles) were harvested 3 days postinfection, and putative particles were concentrated with PEG as above and then were resuspended in 2 ml of 10 mM Tris pH 8.8. Samples were layered onto 15-60% sucrose gradients, and centrifuged for 20 h at 38,000 rpm in a SW41Ti rotor (Beckman). Gradients were fractionated into 0.5-ml samples and the density of each fraction was determined. Fractions to be examined for the presence of NLS were treated with Triton X-100 (1% final concentration) and 0.5 M NaCI on ice for 10 min then layered on to 15-40% cesium chloride gradients and centrifuged for 22 h at 30,000 rpm in a SW41Ti rotor (Beckman).
Authentic Hantaan virus particles were purified as described previously, by using PEG precipitation and sucrose gradient sedimentation (Schmaljohn et al., 1983).
2.5. Enzyme-linked immunosorbent assay (ELISA)
Sandwich assays were performed in 96-well microtiter plates (Costar, high binding) coated with monoclonal antibodies (MAbs) against HTN virus N (BD01 and EC02), G1 (2D5 and 3D5), or G2 (HCO2). Characteristics of these MAbs were described previously (Arikawa et al., 1989; Ruo et al., 1991).
2.6. Sample preparation for immunoelectron microscopy
Gradient fractions determined by ELISA to have the highest concentration of N, or G1 and G2 were pooled, diluted in TNE buffer to 30 ml and pelleted through a 4-ml cushion of 15% sucrose for 2 h in an SW 28 rotor (Beckman) at 25 K rpm, 4°C. The pellet was resuspended in 20 /xl of PBS containing 0.2% glutaraldehyde. A 3 ~1 drop of each sample was applied to formvar and carbon coated, glow discharged, 200 mesh nickel grids, and washed with PBS buffer. Samples were blocked for 20 min in a PBS solution with 0.5% BSA and 4% normal goat serum in PBS buffer for 20 min. Samples were then incubated overnight at 4°C with a pool of HTN-specific MAbs at dilutions of 1 : 400 to 1 : 3200. The grids were washed with PBS containing 0.5% BSA, incubated for 2 h at 4°C with goat anti-mouse antibody conjugated to 5 or 10 nm gold particles, washed again with PBS, negatively stained, and then dried using filter paper. Grids were then examined using a Philips CM100 transmission electron microscope. A normal mouse serum was used as the negative control.
3. Results
3.1. Formation of NLS in insect cells
To determine if NLS were generated in SF-9 insect cells infected with the baculovirus-HTN S recombinant, we disrupted infected cells with a non-ionic
116 M. Betenbaugh et al. / Virus Research 38 (1995) 111-124
Fig. 1. Western blot and densities of cesium chloride gradient fractions containing recombinant baculovirus expressed Hantaan virus N. Insect cells, infected with a recombinant baculovirus expressing the S segment of Hantaan virus, were disrupted by treatment with 1% Triton X-100 and centrifuged in an isopycnic cesium chloride gradient. Densities of gradient fractions were calculated from refractive indices of fractions, and a portion of each fraction was subjected to polyacrylamide gel electrophoresis. Western blots were performed by using hyperimmune mouse ascitic fluid to authentic Hantaan virus.
detergent and centrifuged clarified cell lysates on cesium chloride or sucrose isopycnic gradients. Gradient fractions were assayed for the presence of viral N by Western blot, using N-specific MAbs, or hyperimmune mouse ascitic fluid to authentic HTN virus. Both in cesium chloride and sucrose gradients, nucleocapsid protein could be detected in all fractions, but there appeared to be a higher concentration of N at the bottom of the gradient, (Fig. 1, fraction 1) and in the center of the gradient (Fig. 1, fractions 5-10). We previously determined that the baculovirus-expressed N is partially insoluble after detergent treatment and could be pelleted even under slow-speed centrifugation conditions (Schmaljohn et al., 1988). Escherichia coli expressed hantaviral N proteins were found to be similarly insoluble (Z611er et al., 1993). Therefore, the protein detected in fraction 1 may be aggregates of insoluble N. The expected density of HTN ribonucleocapsids (RNP) is approximately 1.25-1.27 in CsC1, (Schmaljohn et al., 1983), which corresponds to fractions 5-7 of the gradient assayed and displayed in Fig. 1. Analysis of gradient fractions corresponding to these densities, in this and like experiments, by immu- noelectron microscopy (IEM) revealed the presence of structures similar in ap- pearance and size to authentic HTN RNPs (Fig. 2A), which were recognized by N-specific MAbs. The structures could be identified after sedimentation in either
M. Betenbaugh et al . / Virus Research 38 (1995) 111-124 117
Fig. 2. Comparison of authentic HTN virus ribonucleocapsids sedimented in CsCI gradients (A) to NLS recovered from insect cells infected with a recombinant baculovirus expressing the S segment of HTN virus after sedimentation once in sucrose gradients (B) or once (C) or twice (D) in cesium chloride gradients. All mierographs shown are at the same magnification and the calibration bar shown in A corresponds to 200 nm.
sucrose (Fig. 2B) or cesium chloride gradients (Fig. 2C). Less dense gradient fractions (e.g., fractions with densities less than 1.2 in CsCI), examined by IEM, contained only scattered gold particles with no distinct structures apparent (not shown). Because our Western blot results indicated that N protein was found in most fractions, we attempted to further purify the NLS before subsequent analysis. Gradient fractions shown to contaia the structures were pooled and centrifuged in CsCI gradients as before. Most fractions were once again found to contain N when examined by Western blot (not shown). IEM revealed NLS at the correct density for hantavirus RNPs once again, but these were less abundant and shorter than observed after only one centrifugation, suggesting that they were unstable (Fig. 2D). Although NLS were readily recovered from the baculovirus-insect cell system, few VLP could be detected in SF-9 cells infected with baculovirus recombinants independently expressing the M or the S segments of HTN virus, or with a recombinant virus co-expressing both the M and S segments (not shown). Interest-
118 M. Betenbaugh et al. / Virus Research 38 (1995) 111-124
ingly, we did find NLS that could be labeled with both N-specific and with GI- and G2-specific antibodies indicating that association between the expressed envelope proteins and nucleocapsid proteins had occurred (not shown).
3.2. VLP in mammalian cells infected with a recombinant vaccinia virus co-expressing the M and the S segments of H T N virus
Because VLP were not commonly observed in recombinant baculovirus-infected insect cells, we sought to determine if a mammalian cell system might prove more permissive for particle formation. Toward this goal, we infected Vero E6 or BHK cells with recombinant vaccinia viruses co-expressing the M and the S segments of HTN virus (Schmaljohn et al., 1992). Cell lysates and PEG concentrated super- natants were combined and concentrated by step gradient centrifugation followed by isopycnic sucrose gradient sedimentation. Examination of gradient fractions with densities corresponding to those of authentic HTN virions (1.16-1.17) or viral
A .. F =
Fig 3. VLP from recombinant vaccinia virus-infected cells immunolabeled with a pool of monoclonal antibodies to HTN virus G1 and G2 (A), with polyclonal rabbit antibodies to HTN virus G1 and G2 prepared by repeatedly adsorbing a polyclonal rabbit sera with N to deplete antibodies specific for N. The specificity of the resultant serum for G1 and G2 was demonstra ted by its inability to immune-pre- cipitate radiolabeled N after adsorption (not shown) (B); polyclonal mouse antibodies to total HTN virus (C); or with a monoclonal antibody to HTN N (D). Authentic HTN virus double-labeled with rabbit antibodies to G1 and G2 (10 nm gold particles) and with a monoclonal antibody to N (5 nm gold particles) (E); or with a monoclonal antibody to HTN N (F). Magnification of all micrographs is the same and the calibration bar shown in A corresponds to 100 nm.
M. Betenbaugh et al. / Virus Research 38 (1995) 111-124 119
~i~ ii~ii~il;ii;il ¸̧II ~ ; i~ "~:: ~ :
Fig. 4. The surface structure of the VLPs, including a 'fringe' on some of the particles (A and C), bore a striking resemblance to HTN virions (B) prepared under similar conditions. NLS (D) appeared indistinguishable from authentic HTN nucleocapsids (E). Micrographs A, B, and C are at the same magnification and the calibration bar shown in A corresponds to 100 nm. Micrographs D and E are at the same magnification and the calibration bar shown in D corresponds to 200 nm. Immunolabeling of A and C is with MAbs to HTN virus G1 and G2, and labeling of D and E is with MAbs to HTN virus N.
R N P s (1 .18-1.19) by I E M revea led the p re sence of bo th H T N VLP and NLS. The VLP could be i m m u n o l a b e l e d with monoc lona l or po lyc lona l an t ibod ies to G1 and G2 (Fig. 3 A - C ) , bu t d id not labe l extensively with M A b s to N (Fig. 3D). H T N vir ions s imilar ly were not l abe l ed with N-speci f ic an t ibodies , even when they were sub jec ted to f r e e z e - t h a w condi t ions which resu l t ed in pa r t i a l d e g r a d a t i o n of the par t ic les into p l e o m o r p h i c forms of the vir ion (Fig. 3E, F). The surface s t ruc ture of the VLP, inc luding a ' f r inge ' on some of the par t ic les , bo re a s t r iking r e semblance to H T N vir ions p r e p a r e d u n d e r s imilar condi t ions (Fig. 4 A - C ) . NLS were indis t in- gu ishable f rom au then t i c H T N nuc leocaps ids by I E M (Fig. 4D, E). VLP were not r ecove red f rom ceils in fec ted with a r e c o m b i n a n t vaccinia virus express ing only M, a l though m e m b r a n o u s a p p e a r i n g s t ructures , which could be i m m u n o l a b e l e d with M A b s to G1 and G2, were obse rved (not shown).
3.3. VLP in cell supernatants
To d e t e r m i n e if V L P were sec re t ed into cell supe rna t an t s , P E G - c o n c e n t r a t e d s u p e r n a t a n t s were f r ac t i ona t ed on sucrose g rad ien t s and assayed by c a p tu r e E L I S A for the p re sence of viral ant igens . A n an t igen p e a k at a dens i ty of abou t
Capture ELISA o f Sucrose Gradient-Fractionated R e c o r n b i n a n t - l n f e c t e d Cell S u p e r n a t a n t s
A 1.3
1.25
1.2
0.7
0.6
0.5 O
0.4 121 O 0.3
0.2
0.1
0 , iI ii i l,i, Ii ,ill ,, I, iI I, I
2 4 6 8 10 12
Fraction Number
>,,
1.15 ~- a
1.1
1.05
B 1.2
1 14
0.8 o cO 04
0 0.6 0
0.4
0.2
, , i , , , , , , , ,
0 2 4 6 8 Fraction Number
120 M. Betenbaugh et al. / Virus Research 38 (1995) 111-124
I0 12
1.3
1.25
1.2 > ,
1.15 a
1.1
1.05
1 4
Fig. 5. Capture ELISA of sucrose gradient fractions from recombinant vaccinia virus-infected BHK cell supernatants before (A), or after (B) treatment with Triton X-100. Densities of the gradient fractions were determined from refractive indices. ELISA plates were coated with N-, G1- or G2-specific MAbs to capture HTN virus-specific antigen. The antigen was detected by sandwich hybridization using polyclonal rabbit-anti-HTN virus serum, peroxidase-conjugated goat-anti-rabbit IgG and ABTS sub- strate. (×) N, ([]) GI, (~) G2, (0) density.
1.17, was readily detected with MAbs to N, G1 or G2 (Fig. 5 A). IEM examination of these fractions revealed clumps of indistinct VLP that could be labeled with Gl - and G2-specific MAbs (Fig. 6A). NLS were not observed in the cell supernatant samples. To ascertain if NLS were present within VLP, the VLP-containing gradient fractions were treated with Triton X-100 and sedimented to density in sucrose gradients. Capture ELISA of the resultant fractions revealed a small peak of N at 1.18-1.19, the expected densities of NLS, while most of the G1 and G2 antigens were found in less dense fractions, although there did appear to be residual envelope proteins (especially G2) present in the N-containing fractions (Fig. 5B). Examination of the N-containing fractions by IEM revealed only a few
M. Betenbaugh et al. / I/irus Research 38 (1995) 111-124 121
Fig. 6. IEM of VLP recovered from cell supernatants, sedimented to density in sucrose gradients, and immunolabeled with G1- and G2-specific MAbs (A). VLP-containing gradient fractions were treated with Triton X-100 and samples centrifuged in isopycnic sucrose gradients. NLS observed in gradient fractions corresponding in density to that of authentic HTN RNPs could be immunolabeled with N-specific MAbs (B). Calibration bars correspond to 100 nm.
NLS (Fig. 6B). Although we cannot rule out the possibility that the NLS co-sedi- mented with the VLP, these results suggest that at least some of the VLP may contain nucleocapsids.
4. Discussion
The results presented in this study demonstrate that NLS can assemble from baculovirus or vaccinia virus-expressed N, and that VLP can assemble from vaccinia virus-expressed N, G1 and G2. The NLS that we recovered from bac- ulovirus or vaccinia virus-infected cells were found to sediment at the same buoyant density as authentic RNP. Although N proteins were found at other densities, NLS were less frequent or absent. This finding is different from results with measles virus NLS, which sedimented to a lower buoyant density than authentic RNP, a finding interpreted to indicate the absence of nucleic acid in the NLS (Fooks et al., 1993). In contrast, our results suggest that nucleic acid was present in the H T N NLS. At tempts to verify the presence of and to identify the type of nucleic acid associated with the NLS were inconclusive because of high background levels of nucleic acid (primarily DNA) in all of the gradient fraction- ated samples (not shown). Results of other studies, using E. coli-expressed H T N N, suggested that N non-specifically bound to nucleic acid, with a preference for double-stranded R N A over single-stranded R N A or DNA, a finding consistent with ours (Gf t t et al., 1993). In experiments not described in this paper, we were able to demonstrate co-sedimentation of NLS and H T N virus M RNA, which was transcribed f rom c D N A and transfected into insect cells infected with the bac- ulovirus-HTN S recombinant. Thus, it is possible that there would be a preference for H T N virus-specific R N A if it was available. It would be interesting to determine if such R N A might enhance assembly of NLS.
122 M. Betenbaugh et al. / Virus Research 38 (1995) 111-124
In addition to NLS, VLP were readily identified in recombinant vaccinia virus-infected cells. The Vero E6 cell line was chosen for these studies because it is permissive for authentic HTN virus replication. BHK cells were also used because our recombinant vaccinia virus replicates to slightly higher titers in them than in Vero E6 cells. The sedimentation characteristics and morphologies of these VLP recovered from cell lysates, were very similar to authentic H T N virions. The VLP were recognized by G1- and G2-specific MAbs, suggesting that the presentation of the viral envelope proteins is similar to that of authentic H T N virus. We were not able to determine the efficiency of secretion of these VLP, because the recombi- nant vaccinia viruses quickly caused extensive CPE in the host cells, thus making it impossible to discriminate particles which were truly secreted from those that were released after cell death. To address this question, it would be necessary to study infected monolayers of cells at various times after infection, and to determine if VLP accumulate in the Golgi and if they are transported to cell surfaces in Golgi vesicles as are authentic HTN viruses.
Treatment of VLP, recovered either from cell supernatants or cell lysates, with non-ionic detergent changed the sedimentation characteristics of the structures. This finding is consistent with our conjecture that NLS are present within at least some of the VLP. Only a few NLS were observed in gradient fractions containing detergent-disrupted VLP; however, as we found in other experiments described in this report, it was difficult to maintain NLS structural integrity through two gradient sedimentations. Thus, we feel that it is probable that the VLP do contain NLS. Support for this conclusion also comes from our inability to purify VLP from cells infected with a recombinant vaccinia virus expressing H T N M (not shown), although membranous structures similar to those found in the baculovirus experi- ments could be identified.
Our discovery of VLP formation in the recombinant vaccinia virus system may have relevance to the immunogenicity of these products. It is logical to assume that VLP could assemble in individuals immunized with recombinants expressing N, G1 and G2. If so, such particles might elicit enhanced immune responses, by allowing presentation of the viral antigens in a more native context than could be achieved if particles were not formed. Support for this hypothesis comes from other studies in which animals vaccinated with non-replicating poxviruses expressing both M and S displayed higher immunogenicity to challenge than was observed in animals immunized with recombinants expressing only M or S (Schmaljohn et al., 1994). Finally, use of VLP as a non-infectious subunit vaccine might be possible, if conditions for optimal expression and secretion of the structures could be deter- mined.
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