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Polysomal alterations in the larval fat body of Trichoplusia ni late in the course of a granulosis virus infection
JOURNAL
OF
ISVERTEBRATE
Polysomal
P.iTHOLOGY
Alterations Late
in the
18,
1‘i8-149
in the Course
(1971)
Larval
the
approval Experiment
Body
of a Granulosis
It is well est’ablished that virus diseases act to direct normal protein synthesis to form viral products. Currently, considerable attention is being given to the infectious process and the part played by polysomes in cellular response. Several aspects of the protein synthesizing mechanism of the cabbage looper, Tmkhoplusia ni, larval fat body are under investigation in our laboratory with emphasis on viral induced changes in protein synthesis at the translation level. Late 4th-instar T. ni larvae were infected per OS with a granulosis virus (GV) applied to a semisynthetic diet at a rate of 1.3 X lo5 capsular inclusion bodies/mm2 surface area. The virus, isolated and propagated from diseased larvae from our laboratory culture, appeared identical to that described by J. J. Hamm and J. D. Paschke (J. Insect Pathol. 5, 187-197, 1963) with the fat body being the primary target of infection. Adipose tissue, an active site for larval protein synthesis, was isolated by removal of the head and thorax, inverting the abdomen over a toothpick and immersing it in TKM buffer (0.03 M Tris, 0.025 M KCl, 0.005 M MgClz adjusted to pH 7.6 w-it’ll HCl). Fat bodies from 15 larvae were pooled, separated from adhering tissues, washed twice with TKM buffer, and then homogenized in a Pyrex ground glass homogenizer chilled to 4” c. The mitochondria-free supernatant was isolated by differential centrifugation for 10 min at 10,000 g and layered on discontinuous gradients. These consisted of 3 ml of 1.5 M sucrose layered with 4 ml of 0.5 M sucrose buffered with TKM (F. 0. Wettstein, T. Staehelin, and H. Noll, Nature 197, 4301 Published with Arkansas Agricultural
Fat
of Trichoplusia
Virus
ni
Infection’
435, 1963) in 13 ml tubes. Centrifugation on a Beckman L2-65B was for 4 hr at 40,000 rpm with a type 40 angle head rotor. The translucent pellet of free ribosomes was gently resuspended in cold TKI\I and subjected to sedimentation analysis. The free ribosome fraction was layered on 5-35 % (w/v) linear sucrose density gradients on a 50% sucrose cushion and centrifuged in an SW-27 rotor for 2 hr at 25,000 rpm. Sedimentation patterns of the free ribosome fraction from healthy and late
1
2
3 DEPTH
4 IN
5
6
7
CM
FIG. 1. Sucrose density gradient profiles of polyribosomes extracted of Trichoplusia ni 5th-instar larvae. D = GV infected.
sedimentation from fat body H = healthy,
diseased larval fat body are shown in Fig. 1. Analysis of free ribosomes with the analytical ultracentrifuge revealed the same spectrum of components (Fig. 2). Healthy larval fat body had components with average sedimentation coefficients of 8OS, 122S, 156S, 178S, and 210s. The 80 Z!Z 25 monoribosome is characteristic of eukaryotes in general and the polysomes are comparable to those described for mammals and other insect,s. In contrast to the sedimentation profile from healthy tissue, the profile from larval
of the Director, Station. 148
149
NOTES
FIG. 2. Sedimentation pattern of ribosomes from fat body of Trichopiusia healthy larvae, B = GV infected larvae. The photographs were taken G min was reached. Schlieren angle = 35”. Sedimentation is from left to right.
fat body late in the course of a GV infection exhibited a large increase in the concentration of monoribosomes at the expense of heavier polysomal components. This breakdown, c&aggregation, of host cell polysomes implies that inhibition of protein synthesis is induced late in infection by a reduction or elimination of large polysomes. These preliminary results support the logical hypothesis that insect viral infections induce macromolecular alterations similar to those of mammalian viral infections responsible for inhibition of protein synthesis by disaggregation of polysomes. Attempts to releasebound ribosomesfrom rough ER in the postmitochondrial supernatant using conventional sodium deoxycholate treatment were unsuccessful. Failure
ni 5th.instar after a speed
larvae. A = of 40,000 rpm
to increase the concentration of polysomes isolated with the free ribosome fraction is in agreement with studies of other insect systems (S. Litvak and 11. Agosin, Biochem. 7, 1560-1567, 1968; C. M. Tsiapalis, Y. Hayashi, IV. Chefurka, Nuture 214, 358361, 1967). The authors thank Dr. H. A. Scott for his interest and advice and Dr. J. D. Paschke, Purdue University, for helpful criticism of the manuscript. S. R. WEBB S. Y. YOUNG Virology-Biocontrol Laboratory Department of Entomology Univers ‘ty of Arkansas Fayetteville, Arkansas YRYOi ReceivedDecember21, 1970
OF
ISVERTEBRATE
Polysomal
P.iTHOLOGY
Alterations Late
in the
18,
1‘i8-149
in the Course
(1971)
Larval
the
approval Experiment
Body
of a Granulosis
It is well est’ablished that virus diseases act to direct normal protein synthesis to form viral products. Currently, considerable attention is being given to the infectious process and the part played by polysomes in cellular response. Several aspects of the protein synthesizing mechanism of the cabbage looper, Tmkhoplusia ni, larval fat body are under investigation in our laboratory with emphasis on viral induced changes in protein synthesis at the translation level. Late 4th-instar T. ni larvae were infected per OS with a granulosis virus (GV) applied to a semisynthetic diet at a rate of 1.3 X lo5 capsular inclusion bodies/mm2 surface area. The virus, isolated and propagated from diseased larvae from our laboratory culture, appeared identical to that described by J. J. Hamm and J. D. Paschke (J. Insect Pathol. 5, 187-197, 1963) with the fat body being the primary target of infection. Adipose tissue, an active site for larval protein synthesis, was isolated by removal of the head and thorax, inverting the abdomen over a toothpick and immersing it in TKM buffer (0.03 M Tris, 0.025 M KCl, 0.005 M MgClz adjusted to pH 7.6 w-it’ll HCl). Fat bodies from 15 larvae were pooled, separated from adhering tissues, washed twice with TKM buffer, and then homogenized in a Pyrex ground glass homogenizer chilled to 4” c. The mitochondria-free supernatant was isolated by differential centrifugation for 10 min at 10,000 g and layered on discontinuous gradients. These consisted of 3 ml of 1.5 M sucrose layered with 4 ml of 0.5 M sucrose buffered with TKM (F. 0. Wettstein, T. Staehelin, and H. Noll, Nature 197, 4301 Published with Arkansas Agricultural
Fat
of Trichoplusia
Virus
ni
Infection’
435, 1963) in 13 ml tubes. Centrifugation on a Beckman L2-65B was for 4 hr at 40,000 rpm with a type 40 angle head rotor. The translucent pellet of free ribosomes was gently resuspended in cold TKI\I and subjected to sedimentation analysis. The free ribosome fraction was layered on 5-35 % (w/v) linear sucrose density gradients on a 50% sucrose cushion and centrifuged in an SW-27 rotor for 2 hr at 25,000 rpm. Sedimentation patterns of the free ribosome fraction from healthy and late
1
2
3 DEPTH
4 IN
5
6
7
CM
FIG. 1. Sucrose density gradient profiles of polyribosomes extracted of Trichoplusia ni 5th-instar larvae. D = GV infected.
sedimentation from fat body H = healthy,
diseased larval fat body are shown in Fig. 1. Analysis of free ribosomes with the analytical ultracentrifuge revealed the same spectrum of components (Fig. 2). Healthy larval fat body had components with average sedimentation coefficients of 8OS, 122S, 156S, 178S, and 210s. The 80 Z!Z 25 monoribosome is characteristic of eukaryotes in general and the polysomes are comparable to those described for mammals and other insect,s. In contrast to the sedimentation profile from healthy tissue, the profile from larval
of the Director, Station. 148
149
NOTES
FIG. 2. Sedimentation pattern of ribosomes from fat body of Trichopiusia healthy larvae, B = GV infected larvae. The photographs were taken G min was reached. Schlieren angle = 35”. Sedimentation is from left to right.
fat body late in the course of a GV infection exhibited a large increase in the concentration of monoribosomes at the expense of heavier polysomal components. This breakdown, c&aggregation, of host cell polysomes implies that inhibition of protein synthesis is induced late in infection by a reduction or elimination of large polysomes. These preliminary results support the logical hypothesis that insect viral infections induce macromolecular alterations similar to those of mammalian viral infections responsible for inhibition of protein synthesis by disaggregation of polysomes. Attempts to releasebound ribosomesfrom rough ER in the postmitochondrial supernatant using conventional sodium deoxycholate treatment were unsuccessful. Failure
ni 5th.instar after a speed
larvae. A = of 40,000 rpm
to increase the concentration of polysomes isolated with the free ribosome fraction is in agreement with studies of other insect systems (S. Litvak and 11. Agosin, Biochem. 7, 1560-1567, 1968; C. M. Tsiapalis, Y. Hayashi, IV. Chefurka, Nuture 214, 358361, 1967). The authors thank Dr. H. A. Scott for his interest and advice and Dr. J. D. Paschke, Purdue University, for helpful criticism of the manuscript. S. R. WEBB S. Y. YOUNG Virology-Biocontrol Laboratory Department of Entomology Univers ‘ty of Arkansas Fayetteville, Arkansas YRYOi ReceivedDecember21, 1970