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J. gen. Virol. (I973), I9, I45-I5O

Printed in Great Britain 145

Nuclear Polyhedrosis Virus: a Possible Example of de novo Intranuclear Membrane Morphogenesis

(Accepted 19 December I972 )

Membranes are intimately involved in the life-cycle of nuclear polyhedrosis viruses (NPV), and the related (Bellett, I969) granulosis viruses (GV). Generally, two different types of membrane are seen, which we categorize as envelopes and 'transport' membranes. The former represent those membranes which closely surround mature, occluded, nucleocapsids and which are also involved in the initial membrane fusion event thought necessary for virus uptake by midgut epithelial cells (Harrap, 197o; Summers, I97I; Kawanishi et aL I972). The acquisition of envelopes is also a necessary requisite for occlusion (Summers & Arnott, 1969), a process that usually occurs only in tissues other than the gut. 'Transport' mem- branes, on the other hand, are involved in the movement of non-occluded nucleocapsids from the site of primary (gut) infection to other tissues, such as fat body, in which the progeny of a second round of virus replication become occluded; unlike envelopes, these membranes are often vesicular in profile (Harrap, I97o; Summers, I97I ).

The origin of such diverse membranes is of considerable interest. Preliminary observa- tions suggest that in at least some cases, NPV particles acquire 'transport' membranes by budding through the lateral and basal plasma membranes of midgut cells (C. Y. Kawanishi, unpublished observations); this also seems likely in the case of granulosis viruses (Summers, 1970. Much less is known concerning the source of virus envelopes. The source of GV envelopes is uncertain since breakdown of nuclear membranes occurs rapidly in those tissues in which occlusion takes place (Arnott & Smith, I968). However, the suggestion has been made (Summers & Arnott, 1969) that those of NPV particles might be derived from the inner nuclear membrane. This idea is attractive by virtue of the fact that some other nuclear viruses, such as herpes-type viruses (Darlington & Moss, I969; Leestma et al. i969) and rhabdoviruses (Kitajima, Lauritis & Swift, I969; Chiu et aL 197o ), acquire envelopes derived by budding through the inner nuclear membrane. However, in the case of NPV, no convincing electron micrographs in support of such a process have as yet appeared in the literature.

In the present report, the involvement of membrane in NPV infections is examined using an unusual virus/host system in which no 'transport' membranes appear to be produced; NPV infections of the fly Rhynchosciara angelae (Diptera: Sciaridae) are restricted to gut epithelial cells (midgut and caeca), with no transport of progeny virus to the haemocoele occuring (D. B. Stoltz, unpublished observations). Occlusion, then, takes place in the primary and, in this case, only site of infection. In a recent preliminary report, Da Cunha et al. (1972) describe some aspects of the cytopathology of this disease, and point out that polyhedra develop on the inner nuclear membrane. With the exception of an NPV in another dipterous species, Tipulapaludosa (Smith, I955), this situation is in marked contrast to that obtaining for all other known nuclear polyhedroses, in which polyhedra develop in the interior rather than at the periphery of the nucleus. Direct involvement of the inner nuclear membrane in the formation of polyhedra led us to examine the question of whether it might also function as a source of membrane for virus envelopes. Laboratory rearing procedures for this insect have been previously described (Lara, Tamaki & Pavan, I965).

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I46 Short communications

Infections were induced by painting infective material on to egg masses. Electron micro- graphs were taken with a Hitachi HU I ~E electron microscope.

In addition to the development of polyhedra on the inner nuclear membrane, our observa- tions have now revealed another interesting facet of this disease: namely, the appearance of discrete intranuclear accumulations of short, open-ended, membrane profiles (Figs. I, 2). These membranes represent the source of virus envelopes (Figs. 3, 4)- Preliminary serial sectioning reveals no points of contact between the intranuclear membranes and those of the nuclear envelope. The latter in fact show no morphological signs of either cytopathologi- cal damage or participation in the production of the intranuclear profiles: membranes of the nuclear envelopes of both infected and non-infected cells are identical in morphology and both appear to be intact, with no evident blebbing or infolding. Intranuclear mem- brane was not detected in uninfected cells. The membranes of the nuclear envelope are distinctly different in morphology from the intranuclear profiles. In particular, the latter are always obviously trilaminar, unlike the nuclear membranes. A marked difference in width of the two types of membranes is also evident (Fig. z). These morphological observa- tions, taken together, have prompted us to advance the hypothesis that the envelopes of Rhynchosciara angelae NPV particles may originate by a process of de novo membrane morphogenesis.

Rigorous proof of this hypothesis could be achieved by complete serial sectioning of an entire infected nucleus. Unfortunately, in the case of a caecal cell nucleus, this would be prohibitively difficult: such nuclei may be as large as 3oo/zm in diameter, requiring at least 5ooo sections for complete analysis by that method. One of us (D.B.S.) is presently engaged in a comprehensive comparative study of NPV morphogenesis in hopes of finding analogous systems which might be more amenable to analysis by serial sectioning.

Whether, in addition to assembly, de novo synthesis of membrane components is involved cannot be decided here. In any case, elements of the nuclear envelope do not appear to function in any direct sense as a template, or as a source for microscopically detectable membrane precursor. This, of course, does not preclude the presence of cellular, as well as virus, components such as phospholipid and protein in these membranes. We suggest only that, to our knowledge, this is the first clear example of what appears to be intranuclear, de novo membrane morphogenesis. Experiments designed to further characterize the process by means of high resolution autoradiography are in progress.

It is of interest to consider whether de novo morphogenesis of virus envelopes, as suggested here for Rhynchosciara angelae NPV, might also obtain in the case of other nucleopoly- hedrosis virus infections. Unfortunately, the question of possible sources of envelopes for this type of virus has rarely been taken up in the literature, and in any case there has usually been no distinction made by previous workers between envelopes and transport membranes. A survey of published micrographs does, however, occasionally reveal the presence of what can reasonably be interpreted as intranuclear membrane profiles and virus envelopment apparently not involving membranes derived from or connected to the nuclear envelope (some Figs. in Adams et al. I968; Kislev, Harpaz & Zelcer I969; Tanada, Hukuhara & Chang, I969). In addition, a recent ultrastructural investigation of nucleopolyhedrosis in a mosquito, Aedes sollicitans, (D. B. Stoltz, T. Fukuda and H. C. Chapman, unpublished work) has revealed the presence of intranuclear membrane foci entirely analogous to those seen in Rhynchosciara angelae. The available evidence is thus at least suggestive of possible de novo intranuclear morphogenesis of NPV envelopes in general. Since, however, no other detailed studies aimed at elucidating this point have as yet been conducted, the above generalization must be regarded as highly speculative. Alternative sources for NPV envelope

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I 4 8 Short communications

Figs. ~, 2. Serial sections through an intranuclear focus of short, open-ended membrane segments. Note that the inner nuclear membrane (ira) shows no connexions with the intranuclear profiles. The latter are more obviously trilaminar and appear wider than the nuclear membranes (compare circled areas and respective insets in Fig. 2). In both micrographs, the intimate association between the polyhedron and the inner nuclear membrane is evident. S, virogenic stroma; P, polyhedron.

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15 ° Short communications

m e m b r a n e , s u c h as t h e i n n e r n u c l e a r m e m b r a n e , c a n b y n o m e a n s as ye t b e d i s c o u n t e d in

t he case o f all n u c l e o p o l y h e d r o s e s .

T h i s s t u d y w as s u p p o r t e d b y N I H g r a n t G M 15769-05 to D r P a v a n .

Cell Research Institute University of Texas Austin, Texas 78712, U.S.A.

Departmento of Zoology University o f Texas Austin, Texas 7 8 7 t 2 , U.S.A.

Departmento de Biologia A . B . DA CUNHA

C. P . 11230

Universidade de Sao Paulo Sao Paulo, Brazil

REFERENCES

ADAMS, J. R., WALLIS, R. L., WaLCOX, T. A. & fAUST, R. M. (I968). A previously undescribed polyhedrosis of the zebra caterpillar, Ceramica picta. Journal of Invertebrate Pathology xx, 45-58.

ARNOTT, H. J. & SMITH, K. M. (I968). An ultrastructural study of the development of a granulosis virus in the cells of the moth Plodia interpunctella (Hbn.). Journal of Ultrastructure Research zI, 251-268.

BELLETT, A. J. D. (I969)- Relationships among the polyhedrosis and granulosis viruses of insects. Virology 37, II7-I23.

CHIU, R. J . , LIU, H. Y., MACLEOD, R. & BLACK, L. M. (I97o). Potato yellow dwarf virus in leafhopper cell culture. Virology 4o, 387-396.

DA CUNHA, A . B . , PAVAN, C., BIESELE, J. J. , RIESS, R. W. & SIMOES, L. C. G. (I972). Studies in genetics VII. University of Texas' Publications 7213, I I7-I43.

DARLINGTON, It. W. & MOSS, L. H. nI. (I969). The envelope of herpesvirus. Progress in Medical Virology H, 16-45I.

HARRAP, I<. A. 097O). Cell infection by a nuclear polyhedrosis virus. Virology 42, 3I 1-318. KAWANISHI, C. Y., SUMMERS, M. D., STOLTZ, D. B. & ARNOTT, H. J. (I972). Entry of an insect virus in vivo by

fusion of virion envelope and microvillar membrane. Journal of Invertebrate Pathology 2o, IO4-IO8. KISLEV, N., HARPAZ, I. & ZELCER, A. (I969). Electron-microscopic studies on hemocytes of the Egyptian cotton-

worm, Spodoptera littoralis (Boisduval) infected with a nuclear-polyhedrosis virus, as compared to noninfected hemocytes. II. Virus-infected hemocytes. Journal of Invertebrate Pathology x4, 245-257.

KITAJIMA, E. W. , LAURITIS, J. A. & SWIFT, H. (1969). Morphology and intracellular localization of a bacilliform latent virus in sweet clover. Journal of Uhrastructure Research 29, 14I-I5O.

LARA, F. J. S., TAMAKI, H. & PAVAN, C. (1965). Laboratory culture of Rhynchosciara angelae. American Naturalist 99, 189-I 9 I.

LEESTMA, J. E., BORNSTEIN, M. B., SHEPPARD, a . D. & FELDMAN, L. A. (I969). Ultrastructural aspects of herpes simplex virus infection in organized cultures of mammalian nervous tissue. Laboratory Investigation 2o, 7o-78.

SMITH, g. M. (I955). Intranuclear changes in the polyhedrosis of Tipula paludosa (Meig.). Parasitology 45, 482-487.

SUMMERS, M. D. (I97I). Electron microscopic observations on granulosis virus entry, uncoating and replica- tion processes during infection of the midgut cells of Trichoplusia hi. Journal of Ultrastructure Research 35, 606-625.

SUMMERS, M. D. & ARNOTT, n. J. (I969). Ultrastructural studies on inclusion formation and virus occlusion in nuclear polyhedrosis and granulosis virus-infected cells of Trichoplusia ni (Hfibner). Journal of Ultra- structure Research 28, 462-480.

TANADA, Y., HUKUHARA, T. & CHANG, G. Y. (I969). A strain of nuclear-polyhedrosis virus causing extensive cellular hypertrophy. Journal of Invertebrate Pathology x3, 394-4o9.

D . B. STOLTZ

C. PAVAN

(Received I2 September I972)