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Characterization of the top component of the “T” strain of mosquito iridescent virus
VIROLOGY
76, 426-428
Characterization
(1977)
of the Top Component of the “T” Strain of Mosquito Iridescent Virus’ G. W. WAGNER
Department
of Entomology,
Purdue
J. D. PASCHKE
AND
University,
West Lafayette,
Indiana
47907
AND
D. B. HOGG Department
of Entomology,
University Accepted
of California, August
Berkeley,
California
94720
23,1976
The molecular weight of the top component of TMIV was found to be 1.94 x log. The particles contain between 5.0 and 5.7% DNA, 4.3% lipid, and 87.1% protein. The antigenicity and electrophoretic mobility of top component differ from those of TMIV, although the number and molecular weight of top component proteins as determined in polyacrylamide gels appear identical to those of the virus. Top component particles are neither infectious nor do they affect the infectivity of virus suspensions.
Upon purification of crude preparations of mosquito iridescent virus (MIV), two components of different sedimentation rates are found (1,2). The faster sedimenting virus component has been well characterized (141, but little analysis has been carried out on the slower sedimenting component, termed “top component” (I). This paper describes some characteristics of the top component (TC) of the “T” strain of MIV in comparison with the virus. The top component of TMIV was purified following the method previously described for the virus (2). After collection of the top component band from sucrose gradients, the suspensions were centrifuged for 60 min at 30,000 rpm and the pellets were resuspended in 0.1 M sodium phosphate buffer, pH 7.0. Pellets of TC were easily distinguished by their lavender color compared with the blue-green pellets of TMIV. Figure 1 shows a comparison of the density gradient centrifugation patterns of 1 Purdue University Station Journal Paper
Agricultural No. 6308.
Experiment
purified TC as monitored in an ISCO Model 620 density gradient fractionator modified to read the absorbancy at 254 and 280 nm simultaneously. TC sedimented more slowly than the virus and had a relatively greater absorption at 280 nm than the virus. The 260/280 ratio of the TC was 1.37 compared with 1.82 for TMIV. The sedimentation coefficient (sZow) of TC as measured in a Spinco Model E analytical centrifuge was 2141 S compared with 3318 S for TMIV, and the molecular weight (M) of the TC calculated from the Svedberg equation M =
where R and T are the gas constant and absolute temperature, respectively, p is the density of water, D is the diffusion coefficient (1.00 x 1Om8cm2/sec), and d is the partial specific volume (0.7303 ml/g), was 1.94 x log compared with 2.10 x log for TMIV (2). The diffusion coefficient and partial specific volume of TC were determined as before (2). 426
Copyright All rights
0 1977 by Academic Press, Inc. of reproduction in any form reserved.
RT smw D (1 - tip)’
SHORT
A
C
RELATIVE FIG. 1. Tracings of the 254 c-----j absorbance distribution tions of (A) top component of (C) mixture of top component gation was for 40 min at 15,000 in gradients of lo-40 mg of containing 0.1 M phosphate mentation is to the right.
427
COMMUNICATIONS
DEPTH nm (-1 and 280 nm of purified preparaTMIV, (B) TMIV, and and TMIV. Centrifurpm in an SW41 rotor sucrose per milliliter buffer, pH 7.0. Sedi-
Using the methods previously described (2), the percentage lipid in TC was found to be 4.3% or 8.34 x lo7 daltons which is about the same quantity of lipid (8.19 x lo7 daltons) found in TMIV (2). The protein compositions of TMIV and TC are very similar (Fig. 2), as well as the quantity of protein in each particle, 1.67 x log and 1.69 x lo9 daltons, respectively. TMIV contains 13% DNA (2). Thus, if the molecular weight difference between the two components is due only to the total absence of DNA from the TC, its molecu-
lar weight should be 1.83 x 10s. The difference between the measured molecular weight of the TC and DNA-less TMIV is 1.10 x 10H daltons. This value suggests TC should have about 5.7% DNA, and when determined by the method of Burton (51, TC was found to be 5.06 + 0.14%. The bouyant density of TC determined in 33% cesium chloride gradients prepared in SW 39 rotor tubes spun for 48 hr at 35,000 rpm was 1.2810 compared to 1.3121 g/ml for TMIV. TC appeared similar to TMIV in the electron microscope (Fig. 3) although some particles of TC appeared deformed and collapsed. TC is antigenically related to the virus but is not identical. Ring interface precipitin tests showed a titer of 64 when TC was reacted with virus antiserum of a homologous titer of 256, and when virus was reacted with TC antiserum, a 64 titer was observed compared with a 128 homologous titer. In quantitative tube precipitin tests, TC titered to 0.03 mg of protein compared with 0.06 mg of protein for the virus when reacted with a l/8 dilution of TC antiserum. Using a l/16 dilution of virus antiserum, TMIV titered to 0.02 mg of protein compared with 0.06 mg of protein for TC.
c A
DISTANCE MOVED* FIG. 2. Optical density profiles on 10% polyacrylamide gels of (A) TMIV and (B) TMIV top component. Electrophoresis was for 2.5 h at 8 mA/gel. Migration is to the right. Method according to Wagner et al. (2).
428
with
SHORT
FIG. 3. Electron micrographs 1% PTA, pH 7.0 x 50,000. TABLE
ELECTROPHORETIC
MOBILITIES
COMMUNICATIONS
of purified
preparations
1 OF TMIV
AND
TMIV
TOP COMPONENT PH
CL
Buffer”
Mobility (cm* volt-’ set-* 105) TMIV
3.0 4.0 5.0 6.0 7.0 8.0
0.1 0.1 0.1 0.1 0.1 0.1
Glycine-HCl Sodium acetate Sodium acetate Sodium phosphate Sodium phosphate Sodium Verona1
1.00 -1.79 -3.38 -4.06 -4.35 -4.77
X
Top component *b -1.30 -3.48 -4.55 -5.00 -5.35
n According to Miller and Golder (6). * Top component was unstable below pH 4.0.
The electrophoretic mobilities of TC and TMIV are different (Table 1). The isoelectric point of TMIV was pH 3.4; however, the isoelectric point of TC could not be determined precisely since the particles became unstable before the isoelectric point was reached. From the above analyses, we conclude that TC contains between 5.0 and 5.7% DNA, which is about 38% of the DNA of TMIV. However, TC is not infectious nor does it affect the infectivity of purified TMIV (data not shown). The smaller
of (A) TMIV
and (B) TMIV
top component
stained
amount of DNA in TC is most likely responsible for the other characteristics which distinguish it from TMIV, i.e., structural collapse, lesser stability at low pH, lower buoyant density, and 260/280nm ratio, and a possible change in the orientation of the protein subunits which would account for the different antigenicity and electrophoretic mobility. ACKNOWLEDGMENTS The authors acknowledge the technical assistance of Ms. Ramona Lambert, Ms. Done11 Litzenberg, and Ma. Sandra Spitznagle. This investigation was supported in part by U. S. Public Health Service Research Grant No. AI 09972. REFERENCES 1. MATTA, J. F., J. Znuertbr. Pathol. 16, 157-164 (1970). 2. WAGNER, G. W., PASCHKE, J. D., CAMPBELL, W. R., and WEBB, S. R., Virology 52,72-80 (1973). 3. WAGNER, G. W., PASCHKE, J. D., CAMPBELL, W. R., and WEBB, S. R. Zntervirology 3, 97-105 (1974). 4. WAGNER, G. W., WEBB, S. R., PASCHKE, J. D., and CAMPBELL, W. R. Virology 64, 430-437 (1975). 5. BURTON, K., Biochem. J. 62, 315-323 (1956). 6. MILLER, G. G., and GOLDER, R. H., Arch. Biothem. 29, 420-423 (1950).
76, 426-428
Characterization
(1977)
of the Top Component of the “T” Strain of Mosquito Iridescent Virus’ G. W. WAGNER
Department
of Entomology,
Purdue
J. D. PASCHKE
AND
University,
West Lafayette,
Indiana
47907
AND
D. B. HOGG Department
of Entomology,
University Accepted
of California, August
Berkeley,
California
94720
23,1976
The molecular weight of the top component of TMIV was found to be 1.94 x log. The particles contain between 5.0 and 5.7% DNA, 4.3% lipid, and 87.1% protein. The antigenicity and electrophoretic mobility of top component differ from those of TMIV, although the number and molecular weight of top component proteins as determined in polyacrylamide gels appear identical to those of the virus. Top component particles are neither infectious nor do they affect the infectivity of virus suspensions.
Upon purification of crude preparations of mosquito iridescent virus (MIV), two components of different sedimentation rates are found (1,2). The faster sedimenting virus component has been well characterized (141, but little analysis has been carried out on the slower sedimenting component, termed “top component” (I). This paper describes some characteristics of the top component (TC) of the “T” strain of MIV in comparison with the virus. The top component of TMIV was purified following the method previously described for the virus (2). After collection of the top component band from sucrose gradients, the suspensions were centrifuged for 60 min at 30,000 rpm and the pellets were resuspended in 0.1 M sodium phosphate buffer, pH 7.0. Pellets of TC were easily distinguished by their lavender color compared with the blue-green pellets of TMIV. Figure 1 shows a comparison of the density gradient centrifugation patterns of 1 Purdue University Station Journal Paper
Agricultural No. 6308.
Experiment
purified TC as monitored in an ISCO Model 620 density gradient fractionator modified to read the absorbancy at 254 and 280 nm simultaneously. TC sedimented more slowly than the virus and had a relatively greater absorption at 280 nm than the virus. The 260/280 ratio of the TC was 1.37 compared with 1.82 for TMIV. The sedimentation coefficient (sZow) of TC as measured in a Spinco Model E analytical centrifuge was 2141 S compared with 3318 S for TMIV, and the molecular weight (M) of the TC calculated from the Svedberg equation M =
where R and T are the gas constant and absolute temperature, respectively, p is the density of water, D is the diffusion coefficient (1.00 x 1Om8cm2/sec), and d is the partial specific volume (0.7303 ml/g), was 1.94 x log compared with 2.10 x log for TMIV (2). The diffusion coefficient and partial specific volume of TC were determined as before (2). 426
Copyright All rights
0 1977 by Academic Press, Inc. of reproduction in any form reserved.
RT smw D (1 - tip)’
SHORT
A
C
RELATIVE FIG. 1. Tracings of the 254 c-----j absorbance distribution tions of (A) top component of (C) mixture of top component gation was for 40 min at 15,000 in gradients of lo-40 mg of containing 0.1 M phosphate mentation is to the right.
427
COMMUNICATIONS
DEPTH nm (-1 and 280 nm of purified preparaTMIV, (B) TMIV, and and TMIV. Centrifurpm in an SW41 rotor sucrose per milliliter buffer, pH 7.0. Sedi-
Using the methods previously described (2), the percentage lipid in TC was found to be 4.3% or 8.34 x lo7 daltons which is about the same quantity of lipid (8.19 x lo7 daltons) found in TMIV (2). The protein compositions of TMIV and TC are very similar (Fig. 2), as well as the quantity of protein in each particle, 1.67 x log and 1.69 x lo9 daltons, respectively. TMIV contains 13% DNA (2). Thus, if the molecular weight difference between the two components is due only to the total absence of DNA from the TC, its molecu-
lar weight should be 1.83 x 10s. The difference between the measured molecular weight of the TC and DNA-less TMIV is 1.10 x 10H daltons. This value suggests TC should have about 5.7% DNA, and when determined by the method of Burton (51, TC was found to be 5.06 + 0.14%. The bouyant density of TC determined in 33% cesium chloride gradients prepared in SW 39 rotor tubes spun for 48 hr at 35,000 rpm was 1.2810 compared to 1.3121 g/ml for TMIV. TC appeared similar to TMIV in the electron microscope (Fig. 3) although some particles of TC appeared deformed and collapsed. TC is antigenically related to the virus but is not identical. Ring interface precipitin tests showed a titer of 64 when TC was reacted with virus antiserum of a homologous titer of 256, and when virus was reacted with TC antiserum, a 64 titer was observed compared with a 128 homologous titer. In quantitative tube precipitin tests, TC titered to 0.03 mg of protein compared with 0.06 mg of protein for the virus when reacted with a l/8 dilution of TC antiserum. Using a l/16 dilution of virus antiserum, TMIV titered to 0.02 mg of protein compared with 0.06 mg of protein for TC.
c A
DISTANCE MOVED* FIG. 2. Optical density profiles on 10% polyacrylamide gels of (A) TMIV and (B) TMIV top component. Electrophoresis was for 2.5 h at 8 mA/gel. Migration is to the right. Method according to Wagner et al. (2).
428
with
SHORT
FIG. 3. Electron micrographs 1% PTA, pH 7.0 x 50,000. TABLE
ELECTROPHORETIC
MOBILITIES
COMMUNICATIONS
of purified
preparations
1 OF TMIV
AND
TMIV
TOP COMPONENT PH
CL
Buffer”
Mobility (cm* volt-’ set-* 105) TMIV
3.0 4.0 5.0 6.0 7.0 8.0
0.1 0.1 0.1 0.1 0.1 0.1
Glycine-HCl Sodium acetate Sodium acetate Sodium phosphate Sodium phosphate Sodium Verona1
1.00 -1.79 -3.38 -4.06 -4.35 -4.77
X
Top component *b -1.30 -3.48 -4.55 -5.00 -5.35
n According to Miller and Golder (6). * Top component was unstable below pH 4.0.
The electrophoretic mobilities of TC and TMIV are different (Table 1). The isoelectric point of TMIV was pH 3.4; however, the isoelectric point of TC could not be determined precisely since the particles became unstable before the isoelectric point was reached. From the above analyses, we conclude that TC contains between 5.0 and 5.7% DNA, which is about 38% of the DNA of TMIV. However, TC is not infectious nor does it affect the infectivity of purified TMIV (data not shown). The smaller
of (A) TMIV
and (B) TMIV
top component
stained
amount of DNA in TC is most likely responsible for the other characteristics which distinguish it from TMIV, i.e., structural collapse, lesser stability at low pH, lower buoyant density, and 260/280nm ratio, and a possible change in the orientation of the protein subunits which would account for the different antigenicity and electrophoretic mobility. ACKNOWLEDGMENTS The authors acknowledge the technical assistance of Ms. Ramona Lambert, Ms. Done11 Litzenberg, and Ma. Sandra Spitznagle. This investigation was supported in part by U. S. Public Health Service Research Grant No. AI 09972. REFERENCES 1. MATTA, J. F., J. Znuertbr. Pathol. 16, 157-164 (1970). 2. WAGNER, G. W., PASCHKE, J. D., CAMPBELL, W. R., and WEBB, S. R., Virology 52,72-80 (1973). 3. WAGNER, G. W., PASCHKE, J. D., CAMPBELL, W. R., and WEBB, S. R. Zntervirology 3, 97-105 (1974). 4. WAGNER, G. W., WEBB, S. R., PASCHKE, J. D., and CAMPBELL, W. R. Virology 64, 430-437 (1975). 5. BURTON, K., Biochem. J. 62, 315-323 (1956). 6. MILLER, G. G., and GOLDER, R. H., Arch. Biothem. 29, 420-423 (1950).