Med. Microbiol. lmmunol. 164, 77-85 (1977)
Microbtq Y :and] C) by Springer-Verlag 1977
Arboviruses: Persistenceand Defectivenessin Sindbis Virus Infections* R. Walter Schlesinger Department of Microbiology, College of Medicine and Dentistry of New Jersey, Rutgers Medical School, Piscataway, New Jersey 08854, USA
The title originally assigned to me was 'Arboviruses'. For a 20 minute talk, that seemed like a formidable task. Therefore I proposed a more restricted topic, "Persistence and DI Particles in Sindbis Virus Infections". Once I settled down to put something on paper, it occurred to me that - surrounded as I would be by myxo- and retrovirologists I might as well take advantage of the educational opportunity to explain why the term 'arboviruses' has no place in a meeting on 'enveloped RNA viruses'. There are bona fide arboviruses which are not enveloped, mainly some 30 members of the family Reoviridae [1]. 'Arbovirus' denotes nothing other than a virus which is maintained in nature by an arthropod-vertebrate-arthropod ... transmission cycle. This implies that it is capable of replicating in both, arthropod vector and vertebrate host. The biological feature of continuing bidirectional crossing of phylogenetic barriers imposes on the arboviruses an unusual range of selective pressures. The identification of these pressures and of their phenotypic consequences is a uniquely challenging and fascinating problem. I shall summarize some.relevant experimental data later on. First, however, granted that by far the majority of known or suspected arboviruses are enveloped and contain RNA genomes and that two-thirds of them (about 220 of 340) are either toga- or bunyaviruses [1], even these two families leave us with at least three basically different kinds of viruses: a) The bunyaviruses and 'bunya-like' viruses, of which there are some 140 species, have a genome of ( - ) polarity consisting of three RNA segments (the virion carries RNA polymerase); b) the alphaviruses (about 20 species, with Sindbis as the prototype); c) theflaviviruses (about 57, for which type 2 dengue virus will be considered as the prototype). We shall discuss only certain aspects of the latter two. Although alpha- and flavivi~uses are classified together in the family Togaviridae, recent work has made it clear that they are substantially different. They resemble each other in a) the crude outline of general structure: both are enveloped, carry glycoprotein projections, and contain RNP cores which are more or less clearly defined *Dedicated to Professor Wemer Schiller on the occasion of ltis 6$th birthday
78
R.W. Schlesinger
as cubic in cross section. The alphavirions are larger (55 to 60 nm) than flavivirions (48.to 52 nm); b) both have linear ssRNA genomes of MW 4.3 x 106 dahons, sed. coeff. 42S; c) both kinds of genomes are infectious and therefore of presumed (+) polarity. Alphavirus genomes are polyadenylated and function as messengers in cellfree translation systems [3 ]. The differences are far more numerous. The most obvious and oldest one refers to the genus (group)-specific antigen(s) which led to their original separation into group A and B arboviruses [4]. A second distinction, in the historical sense, was the demonstrationby Sabin [24] that resistance of certain inbred mouse strains to flaviviruses is limited to these and does not extend to alphaviruses. The resistance is inherited as a dominant autosomal trait in accord with Mendelian segregation and expresses itself as depression of viral multiplication. The latter observation invites the speculation that some host factor controlling replication of flaviviruses is not operative in that of alphaviruses. The findings of Darnell and Koprowski [8] suggest that genetic resistance to a flavivirus (West Nile) may be associated with increased ability to produce defectiveinterfering (DI) virus particles. At the molecular and cellular level, fundamental differences include the following:
a) Structural Proteins. As illustrated in Table 1, alpbaviruses have a lysine-rich core protein of MW 33,000 daltons and 3 envelope glycoproteins assembled, in equimolar amounts, in the projections [10]. Flaviviruses have a core protein (VP-2), also lysine-rich, of only 13,500 dahons and 1 envelope glycoprotein (VP-3) of MW 59,000 daltons. A third protein (VP-1, MW 7 to 8,000 daltons) travels with the envelope or the core, depending on the detergent used to dissociate virions [36, 41]. b) Transcription. Sindbis or Semliki forest viruses produce 2 kinds of mRNA, one of genome size (42S), the other, predominant one (26S), representing one-third of the genome at the 3' end which codes for the structural proteins. The 26S RNA is thought to be generated by internal initiation in the region separating the genes for nonstructural from those for structural polypeptides (Fig.l) (Reviewed in [12]). The 26S and 42S species have been shown to be capped similarly at the 5' end (m7G[5 '] pppApUp...) and to have similar methylation patterns [9].
Table 1. Structural proteins of alpha- and flaviviruses
Location Core Envelope
?c
Alphaviruses (SFV) Designation MW
Haviviruses (Dengue-2) Designation MW
Ca GP E1 GP E2 GP E3
vP2a CAPVP~
13.5K 59K
VP1
7-8K
3 3K 52K 49K lO--20K b
aLysine-rich b45% CBH CDepending on detergent used for dissociation of vidon, separates with core or envelope
Arboviruses: Persistence and Defectiveness in Sindbis Virus Infections
tm7G(5/)ppp(5~)joPb'ci..'
..., c b a p(AP)9oAO,H
I
)
J
k X \
\
/./
."
/
5 ~;"
k x
t'
/
~.
.,
[
~
non- 26S recJion
ns-78
79
ns-86
?
26S region
.~/
/
/
l!", '13 42S .RNA
C E~ Ez El u = I~:;~:J I;",~.,~'::~,':.m.'l ennH "z" g | . ~ ~:i,,-,~.~;;1 v V v
PROTEIN
Fig. 1. Model for organization of an alphavims genome based on studies of Sindbis and Semliki forest viruses. For details see Guild and Stollar (1977), from which this schema has been extracted In contrast, recent work in our laboratory [5] has shown that the only virus-specific RNA associated with polyribosomes of dengue-2 infected KB cells is of genome size (~42S). A similar preliminary report has appeared for Saint Louis encephalitis virus [22a]. Thus, the pattern of RNA transcription in flavivirus-infected cells appears to be closely similar to that in the picornavirus systems.
c) Translation and Processing. For alphaviruses, strong evidence is accumulating for the polycistronic function of the mRNA's [3, 6, 7, 34]. The 26S species is translated into a 130,000 d. precursor which is cleaved stepwise into the 4 structural polypeptides. The nonstructural proteins are probably derived from a "x,200,000 d. polyprotein precursor translated either from 42S or a more elusive, smaller mRNA representing the nonstructural genes [2a]. In the case of flaviviruses, the situation is much less clear. Although large (up to 98,000 d.) polypeptides have been found in cells infected with several members of the group [41], there is so far no evidence for posttranslational cleavage. On the contrary, a preliminary report claims the existence of multiple translational initiation sites on the 42S m R N A [22a]. Glycosylation of envelope proteins has been studied extensively for both kinds of viruses and will not be considered here. However, one noteworthy feature has been proposed for the processing of the non-glycosylated VP-1 in flavivirus-infected cells. It would involve terminal cleavage of a glycosylated precursor (NV-2) [29, 30, see below]. No similar process has been observed for the alphaviruses. d) Morpbogenesis and Release of Virus. In alphavirus-infected vertebrate cells, viral glycoproteins are transported to the plasma membrane where final cleavage occurs; the nucleo.capsid associates with the modified plasma membrane patches by proteinprotein interaction [10], signalling the release of progeny virions. In the morphogenesis of flaviviruses, the plasma membrane seems to play a minor (or more discrete) role. Most of the electron microscopic visualization of 'virion' assembly points to cytoplasmic membranes and vacuoles and to accumulation of crystalline aggregates of virus-like particles in the cytoplasm [22]. Release appears to be
80
R.W. Schlesinger
related to continuities of cytoplasmic membranes with the plasma membrane oia narrow canaliculi [2] in which chains of single virions are seen. However, the exceedingly low levels of cell-associated infectious virus are at variance with the massive number of virion-like particles visible in cell sections. This circumstance, coupled with the report that cell-associated virus carries mainly NV-2, thought to be a precursor of VP-1, rather than VP-1 [29, 30], suggests that intracellular accumulation reflects inefficient maturation and release rather than the manner in which extracellular infectious progeny (always of low titer compared with alphaviruses) is formed and released [26]. Poor coordination of the flavivirus assembly process is also indicated by the consistent observation that vertebrate cells release, in addition to rapidly sedimenting infectious, hemagglutinating particles (RHA), substantial amounts of a more slowly sedimenting virus-specific HA component (SHA, 70S) which lacks 42S viral RNA and the capsid protein VP-2 (Reviewed in [26]). Such coreless membrane fragments have not been reported as a regular accompaniment of alphavirus release. These examples, very briefly summarized, suggest that the fundamental differences between alpha- and flaviviruses may be as great as those between ortho- and paramyxoviruses which are now treated as distinct families. Therefore the time may not be far off when the former two genera will have to be similarly separated. Certainly virologists ought to stop referring to arboviruses or even togaviruses as a homogeneous taxonomic group except in terms of common questions relating directly to their unique biological-ecological features. That the latter questions offer many opportunities for stimulating discoveries is illustrated by recent studies by Dr. Victor Stollar and associates in our department [12, 13, 14, 18, 19, 23, 31, 32, 33, 37, 38, 39, 40]. These have dealt with comparative aspects of Sindbis virus (SV) replication in vertebrate (BHK21 or CEF) and mosquito (Aedes albopictus) cell cultures. Attention has been paid especially to the generation of defective viral genomes during undiluted serial passage in vertebrate cells, the apparent failure of mosquito cells to make them under the same conditions, and mechanism(s) involved in viral persistence in the latter cells. Some aspects of these studies will be reviewed briefly. Undiluted serial passage of SV in BHK21 or chick embryo fibroblasts (CEF) leads to repeated cycles of diminishing and increasing infectious yields [28, 20, 31, 12]. The role of DI particles in this process can be demonstrated by mixing experiments and by examination of intracellular viral RNA species. The latter approach has been especially fruitful. It has revealed that, as the undiluted passage series continues, intracellular double-stranded (ds) viral RNA species of progressively reduced S value and singlestranded (ss) species of increasing migration rate in polyacrylamide gels are generated [31, 12]. Electron microscopic length measurement of the ds species predominating at various passage levels has shown that the reduction in length proceeds in integral steps. The same is true for ss species, as determined by electrophoretic migration rates in polyacrylamide gels (Table 2). Furthermore, progeny virion populations from the corresponding undiluted passages in CEF contain ssRNA molecules of analogous size [13, 141 . The latter finding was unexpected because it had not been possible to separate the DI from standard virus particles [12], or the respective nucleocapsids from each other [11], by various gradient centrifugation techniques. It had also been found that
Arboviruses: Persistence and Defectiveness in Sindbis Virus Infections
81
Table 2. Estimated molecular weights of intracellular viral RNA species generated during undiluted serial passage of sindbis virus in chick embryo fibroblasts (CP) passage No. _c CP 6 CP 15 CP 24
ds RNA
Species 22S 18S 15S 12S
MWa 8.7 x 4.4 x 2.8 x 2.0 x
ss RNA
106 106 106 106
Species MWb 42S 4.3 x 106 33S 2.2 x 106 24S 1.4 x 106 18-22S 0.75--1.0 x 106
Genome Equivalent (%) 100 50 33 20-25
aBased on electron microscopic length measurements. bBased on migration rate in polyacrylamide gel electrophoresis. CStandard virus passage. For experimental details, see [131 and [14]
extraction of DI particles produced in BHK21 cells under less drastic denaturing conditions yielded only 42S RNA [31]. The findings in CEF therefore suggest a) that the DI particles contain multiple copies of the short genome fragments linked together in some way, b) that these multiples add up to one entire genome equivalent, c) that integrally divided genome residues are preferentially packaged in nucleocapsids due to assembly constraints, and hence, d) that a presumably random process of deletions is de-randomized by accumulation of defective fragments o f these selected sizes in succeeding virus passages. This interpretation is strengthened by structural studies of the defective RNA's by hybridization [13] and oligonucleotide analysis [21]. Briefly, both methods have revealed that the deletions are all internal, that the defective genomes retain about 1000 nucleotides o f the 3' end (which is believed to include the initiation site for transcription) and decreasing proportions of the 5' end of the complete genome (Fig. 2), and that they are of (+) polarity [13]. Thus, all defective genomes which can be replicated in the presence of helper virus contain the two ends whose nucleotide sequences display inverted homology. One can envisage the linking together of multiple strands by a 3' to 5' end base-pairing mechanism. This has, however, not yet been shown. Serial passage at high input multiplicities is an experimental artifact designed to amplify the production of defectives. It should be noted, however, that virions released even in the first passage (at m.o.i = 1 PFU/cell) of plaque-purified standard SV contain, in addition to standard 42S, clearly detectable, though small, peaks of RNA in the 33 and 25S regions [14], i.e., 1/2 and 1/3 genome equivalents. This observation is in line with the view that generation of defectives is an inescapable byproduct of viral replication and that it may contribute significantly to the self-limiting character of virus infections (reviewed in [ 1 5 - 1 7 ] ) . It is all the more striking that serial undiluted passage of SV in A. albopictus cell cultures has failed to provide convincing evidence for production of DI virions in terms of significantly reduced yields or of evolving RNA patterns [18]. Equally striking is the fact that infection o f A . albopictus cells with BHK21- or CEF-derived SV preparations containing high proportions of DI particles produce neither reduced yields of infectious virus nor the defective RNA species found in vertebrate cells [18]. The reasons for this apparent failure to produce or 'recognize' DI particles are not known.
82
R.W. Sehlesinger
8000 Np
5'I I
I I
! ~
5360 Np
DELETED
3040Np
DELETED
1880 Np
DELETED
1470 Np
4600 Np
13' SVs-ro 42S 1000Nfo
D!
33S
I
DI
24S
I
DI 22S
l
DI 20S
I
DI 18S
DELETED ' i
I
1150Np
(o)
l
DELETED ,
(o)
Fig. 2. Schematic presentation of defective Sindbis genomes evolvingduring undiluted serial passages in chick embryo fibroblast cultures. For details see Guild and Stollar (1977), from which this schema has been extracted
Infection of mosquito cell cultures with standard SV leads to initial yields comparable with those produced in vertebrate cells. Generally, the cells remain morphologically normal and continue to divide at the normal rate [35]. Cultures remain persistently infected, producing relatively small amounts of virus, all of which, eventually, becomes temperature-sensitive (ts) and shows small plaque morphology [23, 38, 33, 19]. Persistently infected cultures can be segregated into virus (+) and virus ( - ) clones [19]. Prolonged treatment of cultures with antiviral immune serum results in curing [19]. Both results indicate that persistent infection depends on continuous cell-tocell transfer of virus v/a the medium. Despite the lack of significant accumulation of DI virus particles in persistently infected A. albopictus cells, they do, after several weeks in culture, produce a prominent amount of 12S dsRNA which may indicate that defective genomes are made. It remains to be determined whether this material is virus-specific and plays a role in keeping the amounts of infectious ts, sp virus at such low levels. Elsewhere [25, 27] we have speculated on the possible implications of the differential behavior of SV in vertebrate and mosquito cells for the natural life cycle of this arbovirus. Cold selection of ts variants would clearly be favored in life-long persistence of such viruses in arthropod vectors, especially in those hibernating in cold climates; the massive production of DI particles by persistently infected mosquitoes would greatly reduce the effectiveness of transmission to vertebrates, for which a certain threshold titer is required. On the other hand, as already noted, the production of defective genomes and virus particles in vertebrate hosts could contribute, along with the immune response and interferon induction, to the self-limitation of a potentially destructive infection. Time does not permit further elaboration of these points, but they illustrate the potential contributions that detailed study at the molecular and comparative cellular level can make to an understanding of the epidemiological life cycle of arboviruses. It should be stressed again, however, that the fundamental differences between bunya-,
Arboviruses: Persistence and Defectiveness in Sindbis Virus Infections
83
alpha-, flavi- and other kinds of arboviruses will undoubtedly be found to impose totally disparate patterns on each of these families.
Acknowledgements: The work in the author's laboratory was supported in part by Grant AI11290 from the National Institute of Allergy and Infectious Diseases, by the United States-Japan Cooperative Medical Science Program through Public Health Service Grant AI-05920, and by the National Institutes of Health Training Grant CA-05234 from the National Cancer Institute References
1. 2.
2a.
3.
4. 5.
6, 7. 8.
9.
10. 11.
12.
Berge, T.O.: International Catalogue of Arboviruses, 2nd Edition, U.S. Dept. of HEW, Publ. No. (CDC) 75--8301, 789 pp. (1975) Boulton, R.W., Westaway, E.G.: Replication of flavivirus Kunjin: proteins, glycoproteins, and maturation associated with cell membranes. Virology 69, 4 1 6 - 4 3 0 (1976) Bracha, M., Leone, A., Schlesinger, M.J.: Formation of a Sindbis virus nonstructural protein and its relation to 42S mRNA function. J. Virol. 2 0 , 6 1 2 620 (1976) Cancedda, R., Villa-Komaroff, L., Lodish, H.F., Schlesinger, M.: Initiation sites for translation of Sindbis virus 42S and 26S messenger RNAs. Cell 6, 215-222 (1975) Casals, J., Brown, L.V.: Hemagglutination with arthropod-borne viruses. J. Exp. Med. 99, 429-449 (1954) Cleaves, G.R., Schlesinger, R.W.: Characterization of polysomal RNAs from dengue virus infected KB cells. Abstracts Ann. Mtg. Am. Soc. Microbiol., p. 287 (1977) Clegg, J.C.S.: Sequential translation of capsid and membrane protein genes of alphaviruses. Nature 254, 454-455 (1975) Clegg, J.C.S., Kennedy, S.I.T.: Initiation of synthesis of the structural proteins of Semliki Forest virus. J. Mol. Biol. 97,401--411 (1975) Darnell, M.B., Koprowski, H.: Genetically determined resistance to infection with group B arboviruses. II. Increased production of interfering particles in cell cultures from resistant mice. J. Infect. Dis. 129, 453-463 (1974) Dubin, D.T., Stollar, V., HsuChen, C., Timko, K., Guild, G.M.: Sindbis virus messenger RNA: The 5'-termini and methylated residues of 26 and 42S RNA. Virology 77,457--470 (1977) Garoff, H., Simons, K., Renkonen, O.: Isolation and characterization of the membrane proteins of Semliki forest virus. Virology 6 1 , 4 9 3 - 5 0 4 (1974) Guild, G.M.: Defective interfering particles of Sindbis virus - Generation and characterization of defective RNA species. Thesis submitted to the Graduate School of Rutgers University in partial fulfillment for the degree of Ph.D. (1976) Guild, G.M., Stollar, V.: Defective interfering particles of Sindbis virus. III. Intracellular viral RNA species in chick embryo cell cultures. Virology 67, 2 4 - 4 1 (1975)
84
13.
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Guild, G.M., Stollar, V.: Defective interfering particles of Sindbis virus. V. Sequence relationships between SVSTD42S RNA and intracellular defective viral RNAs. Virology 77,175-188 (1977) 14. Guild, G.M., Flores, L., Stollar, V.: Defective interfering particles of Sindbis virus. IV. Virion RNA species and molecular weight determination of defective double-stranded RNAs. Virology 77,158--174 (1977) 15. Huang, A.S.: Defective interfering viruses. Ann. Rev. Microbiol. 2 7 , 1 0 1 - 1 1 7 (1973) 16. Huang, A.S., Baltimore, D.: Defective viral particles and viral disease processes. Nature 226, 325-327 (1970) 17. Huang, A.S., Baltimore, D.: Defective-interfering animal viruses. Comprehensive Virology (H. FrankebConrat and R.R. Wagner, eds.) (in press) 18. Igarashi, A., Stollar, V.: Failure of defective interfering particles of Sindbis virus produced in BHK or chick cells to affect viral replication in A. albopictus cells. J. Virol. 19, 398-408 (1976) 19. Igarashi, A., Koo, R., Stollar, V.: Evolution and properties of Aeries albopictus cell cultures persistently infected with Sindbis virus. Virology (in press) 20. Inglot, A.D., Albin, M., Chudzio, T.: Persistent infection of mouse cells with Sindbis virus: Role of virulence of strains, auto-interfering particles and interferon. J. Gen. Virol. 20, 105-110 (1973) 21. Kennedy, S.I.T., Bruton, C.J., Weiss, B., Schlesinger, S.: Defective interfering particles of Sindbis virus: Nature of the defective virion RNA. J. Virol. 19, 1034-1043 (1976) 22. Matsumura, T., Stollar, V., Schlesinger, R.W.: Studies on the nature of dengue viruses. V. Structure and development of dengue virus in Veto cells. Virology 46, 344-355 (1971) 22a. Naeve, C.W., Trent, D.W.: Identification of Saint Louis encephalitis virus mRNA. Abstracts Ann. Mtg. Am. Soc. Microbiol., p. 287 (1977) 23. Peleg, J., Stollar, V.: Homologous interference in Aedes aegypti cell cultures infected with Sindbis virus. Arch. Ges. Virusforsch. 4 5 , 3 0 9 - 3 1 8 (1974) 24. Sabin, A.B.: Genetic factors affecting susceptibility and resistance to virus disease of the nervous system. Proc. Assn. for Research in Nervous and Mental Disease, Vol. XXXIII, pp. 5 7 - 6 6 (1954) 25. Schlesinger, R.W.: Sindbis virus replication in vertebrate and mosquito cells: an interpretation. Medical Biology 53,295--301 (1975) 26. Schlesinger, R.W.: Dengue-viruses. Virology Monographs, Vol. 16. Wien, New York: Springer (in press) 27. Schlesinger, R.W.: The significance and nature of defective interfering viruses. K.F. Meyer Symp., Bull. Swiss Academy of Medical Sciences 1977 (in press) 28. Schlesinger, S., Schlesinger, M.J., Burge, B.W.: Defective virus particles from Sindbis virus. Virology 4 5 , 6 1 5 - 6 1 7 (1972) 29. Shapiro, D., Brandt, W.E., Russell, P.K.: Change involving a viral membrane glycoprotein during morphogenesis of group B arboviruses. Virology 50, 906-911 (1972) 30. Shapiro, D., Kos, K., Russell, P.K.: Protein synthesis in Japanese encephalitis virus-infected cells. Virology 56, 95-109 (1973)
Arboviruses: Persistence and Defectiveness in Sindbis Virus Infections
31.
32. 33.
34. 35.
36. 37. 38.
39.
40.
41.
85
Shenk, T.E., Stollar, V.: Defective-interfering particles of Sindbis virus. I. Isolation and some chemical and biological properties. Virology 53,162-173 (1973a) Shenk, T.E., Stollar, V.: Defective-interfering particles of Sindibs virus. II. Homologous interference. Virology 5 5 , 5 3 0 - 5 3 4 (1973b) Shenk, T.E., Koshelnyk, K.A., Stollar, V.: Temperature-sensitive virus from Aedes albopictus cells chronically infected with Sindbis virus. J. Virol. 13, 439-447 (1974) Simmons, D.T., Strauss, J.H.: Translation of Sindbis virus 26S RNA and 49S RNA in lysates of rabbit reficulocytes. J. Mol. Biol. 86, 397--409 (1972) Stevens, T.M.: Arbovirus replication in mosquito cell lines (Singh) grown in monolayer or suspension culture. Proc. Soc. Exp. Biot. Ivied. 134, 356-361 (1970) Stollar, V.: Studies on the nature of dengue viruses. IV. The structural proteins of type 2 dengue virus. Virology 39, 426-438 (1969) Stollar, V., Shenk, T.: Homologous viral interference in Aedes albopictus cultures chronically infected with Sindbis virus. J. ViroL 11J, 592-595 (1973) Stollar, V., Peleg, J., Shenk, T.: Temperature sensitivity of a Sindbis mutant isolated from persistently infected Aedes aegypti cell culture. Intervirology 2, 337--344 (1974) Stollar, V., Shenk, T.E., Koo, R., Igarashi, A., Schlesinger, R.W.: Observations on Aedes albopictus cell cultures persistently infected with Sindbis virus. Ann. N.Y. Acad. Sci. 266, 214--231 (1975) Stollar, V., Shenk, T.E., Koo, R., lgarashi, A., Schlesinger, R.W.: Established mosquito cell lines and the study of togaviruses. In: Invertebrate Tissue Culture. Applications in Medicine, Biology and Agriculture (E. Kurstak and K. Maramorosch, eds.) pp. 49-67. New York: Academic Press 1976 Westaway, E.G.: Proteins specified by group B togaviruses in mammalian cells during productive infections. Virology 5 1 , 4 5 4 - 4 6 5 (1973)
Microbtq Y :and] C) by Springer-Verlag 1977
Arboviruses: Persistenceand Defectivenessin Sindbis Virus Infections* R. Walter Schlesinger Department of Microbiology, College of Medicine and Dentistry of New Jersey, Rutgers Medical School, Piscataway, New Jersey 08854, USA
The title originally assigned to me was 'Arboviruses'. For a 20 minute talk, that seemed like a formidable task. Therefore I proposed a more restricted topic, "Persistence and DI Particles in Sindbis Virus Infections". Once I settled down to put something on paper, it occurred to me that - surrounded as I would be by myxo- and retrovirologists I might as well take advantage of the educational opportunity to explain why the term 'arboviruses' has no place in a meeting on 'enveloped RNA viruses'. There are bona fide arboviruses which are not enveloped, mainly some 30 members of the family Reoviridae [1]. 'Arbovirus' denotes nothing other than a virus which is maintained in nature by an arthropod-vertebrate-arthropod ... transmission cycle. This implies that it is capable of replicating in both, arthropod vector and vertebrate host. The biological feature of continuing bidirectional crossing of phylogenetic barriers imposes on the arboviruses an unusual range of selective pressures. The identification of these pressures and of their phenotypic consequences is a uniquely challenging and fascinating problem. I shall summarize some.relevant experimental data later on. First, however, granted that by far the majority of known or suspected arboviruses are enveloped and contain RNA genomes and that two-thirds of them (about 220 of 340) are either toga- or bunyaviruses [1], even these two families leave us with at least three basically different kinds of viruses: a) The bunyaviruses and 'bunya-like' viruses, of which there are some 140 species, have a genome of ( - ) polarity consisting of three RNA segments (the virion carries RNA polymerase); b) the alphaviruses (about 20 species, with Sindbis as the prototype); c) theflaviviruses (about 57, for which type 2 dengue virus will be considered as the prototype). We shall discuss only certain aspects of the latter two. Although alpha- and flavivi~uses are classified together in the family Togaviridae, recent work has made it clear that they are substantially different. They resemble each other in a) the crude outline of general structure: both are enveloped, carry glycoprotein projections, and contain RNP cores which are more or less clearly defined *Dedicated to Professor Wemer Schiller on the occasion of ltis 6$th birthday
78
R.W. Schlesinger
as cubic in cross section. The alphavirions are larger (55 to 60 nm) than flavivirions (48.to 52 nm); b) both have linear ssRNA genomes of MW 4.3 x 106 dahons, sed. coeff. 42S; c) both kinds of genomes are infectious and therefore of presumed (+) polarity. Alphavirus genomes are polyadenylated and function as messengers in cellfree translation systems [3 ]. The differences are far more numerous. The most obvious and oldest one refers to the genus (group)-specific antigen(s) which led to their original separation into group A and B arboviruses [4]. A second distinction, in the historical sense, was the demonstrationby Sabin [24] that resistance of certain inbred mouse strains to flaviviruses is limited to these and does not extend to alphaviruses. The resistance is inherited as a dominant autosomal trait in accord with Mendelian segregation and expresses itself as depression of viral multiplication. The latter observation invites the speculation that some host factor controlling replication of flaviviruses is not operative in that of alphaviruses. The findings of Darnell and Koprowski [8] suggest that genetic resistance to a flavivirus (West Nile) may be associated with increased ability to produce defectiveinterfering (DI) virus particles. At the molecular and cellular level, fundamental differences include the following:
a) Structural Proteins. As illustrated in Table 1, alpbaviruses have a lysine-rich core protein of MW 33,000 daltons and 3 envelope glycoproteins assembled, in equimolar amounts, in the projections [10]. Flaviviruses have a core protein (VP-2), also lysine-rich, of only 13,500 dahons and 1 envelope glycoprotein (VP-3) of MW 59,000 daltons. A third protein (VP-1, MW 7 to 8,000 daltons) travels with the envelope or the core, depending on the detergent used to dissociate virions [36, 41]. b) Transcription. Sindbis or Semliki forest viruses produce 2 kinds of mRNA, one of genome size (42S), the other, predominant one (26S), representing one-third of the genome at the 3' end which codes for the structural proteins. The 26S RNA is thought to be generated by internal initiation in the region separating the genes for nonstructural from those for structural polypeptides (Fig.l) (Reviewed in [12]). The 26S and 42S species have been shown to be capped similarly at the 5' end (m7G[5 '] pppApUp...) and to have similar methylation patterns [9].
Table 1. Structural proteins of alpha- and flaviviruses
Location Core Envelope
?c
Alphaviruses (SFV) Designation MW
Haviviruses (Dengue-2) Designation MW
Ca GP E1 GP E2 GP E3
vP2a CAPVP~
13.5K 59K
VP1
7-8K
3 3K 52K 49K lO--20K b
aLysine-rich b45% CBH CDepending on detergent used for dissociation of vidon, separates with core or envelope
Arboviruses: Persistence and Defectiveness in Sindbis Virus Infections
tm7G(5/)ppp(5~)joPb'ci..'
..., c b a p(AP)9oAO,H
I
)
J
k X \
\
/./
."
/
5 ~;"
k x
t'
/
~.
.,
[
~
non- 26S recJion
ns-78
79
ns-86
?
26S region
.~/
/
/
l!", '13 42S .RNA
C E~ Ez El u = I~:;~:J I;",~.,~'::~,':.m.'l ennH "z" g | . ~ ~:i,,-,~.~;;1 v V v
PROTEIN
Fig. 1. Model for organization of an alphavims genome based on studies of Sindbis and Semliki forest viruses. For details see Guild and Stollar (1977), from which this schema has been extracted In contrast, recent work in our laboratory [5] has shown that the only virus-specific RNA associated with polyribosomes of dengue-2 infected KB cells is of genome size (~42S). A similar preliminary report has appeared for Saint Louis encephalitis virus [22a]. Thus, the pattern of RNA transcription in flavivirus-infected cells appears to be closely similar to that in the picornavirus systems.
c) Translation and Processing. For alphaviruses, strong evidence is accumulating for the polycistronic function of the mRNA's [3, 6, 7, 34]. The 26S species is translated into a 130,000 d. precursor which is cleaved stepwise into the 4 structural polypeptides. The nonstructural proteins are probably derived from a "x,200,000 d. polyprotein precursor translated either from 42S or a more elusive, smaller mRNA representing the nonstructural genes [2a]. In the case of flaviviruses, the situation is much less clear. Although large (up to 98,000 d.) polypeptides have been found in cells infected with several members of the group [41], there is so far no evidence for posttranslational cleavage. On the contrary, a preliminary report claims the existence of multiple translational initiation sites on the 42S m R N A [22a]. Glycosylation of envelope proteins has been studied extensively for both kinds of viruses and will not be considered here. However, one noteworthy feature has been proposed for the processing of the non-glycosylated VP-1 in flavivirus-infected cells. It would involve terminal cleavage of a glycosylated precursor (NV-2) [29, 30, see below]. No similar process has been observed for the alphaviruses. d) Morpbogenesis and Release of Virus. In alphavirus-infected vertebrate cells, viral glycoproteins are transported to the plasma membrane where final cleavage occurs; the nucleo.capsid associates with the modified plasma membrane patches by proteinprotein interaction [10], signalling the release of progeny virions. In the morphogenesis of flaviviruses, the plasma membrane seems to play a minor (or more discrete) role. Most of the electron microscopic visualization of 'virion' assembly points to cytoplasmic membranes and vacuoles and to accumulation of crystalline aggregates of virus-like particles in the cytoplasm [22]. Release appears to be
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related to continuities of cytoplasmic membranes with the plasma membrane oia narrow canaliculi [2] in which chains of single virions are seen. However, the exceedingly low levels of cell-associated infectious virus are at variance with the massive number of virion-like particles visible in cell sections. This circumstance, coupled with the report that cell-associated virus carries mainly NV-2, thought to be a precursor of VP-1, rather than VP-1 [29, 30], suggests that intracellular accumulation reflects inefficient maturation and release rather than the manner in which extracellular infectious progeny (always of low titer compared with alphaviruses) is formed and released [26]. Poor coordination of the flavivirus assembly process is also indicated by the consistent observation that vertebrate cells release, in addition to rapidly sedimenting infectious, hemagglutinating particles (RHA), substantial amounts of a more slowly sedimenting virus-specific HA component (SHA, 70S) which lacks 42S viral RNA and the capsid protein VP-2 (Reviewed in [26]). Such coreless membrane fragments have not been reported as a regular accompaniment of alphavirus release. These examples, very briefly summarized, suggest that the fundamental differences between alpha- and flaviviruses may be as great as those between ortho- and paramyxoviruses which are now treated as distinct families. Therefore the time may not be far off when the former two genera will have to be similarly separated. Certainly virologists ought to stop referring to arboviruses or even togaviruses as a homogeneous taxonomic group except in terms of common questions relating directly to their unique biological-ecological features. That the latter questions offer many opportunities for stimulating discoveries is illustrated by recent studies by Dr. Victor Stollar and associates in our department [12, 13, 14, 18, 19, 23, 31, 32, 33, 37, 38, 39, 40]. These have dealt with comparative aspects of Sindbis virus (SV) replication in vertebrate (BHK21 or CEF) and mosquito (Aedes albopictus) cell cultures. Attention has been paid especially to the generation of defective viral genomes during undiluted serial passage in vertebrate cells, the apparent failure of mosquito cells to make them under the same conditions, and mechanism(s) involved in viral persistence in the latter cells. Some aspects of these studies will be reviewed briefly. Undiluted serial passage of SV in BHK21 or chick embryo fibroblasts (CEF) leads to repeated cycles of diminishing and increasing infectious yields [28, 20, 31, 12]. The role of DI particles in this process can be demonstrated by mixing experiments and by examination of intracellular viral RNA species. The latter approach has been especially fruitful. It has revealed that, as the undiluted passage series continues, intracellular double-stranded (ds) viral RNA species of progressively reduced S value and singlestranded (ss) species of increasing migration rate in polyacrylamide gels are generated [31, 12]. Electron microscopic length measurement of the ds species predominating at various passage levels has shown that the reduction in length proceeds in integral steps. The same is true for ss species, as determined by electrophoretic migration rates in polyacrylamide gels (Table 2). Furthermore, progeny virion populations from the corresponding undiluted passages in CEF contain ssRNA molecules of analogous size [13, 141 . The latter finding was unexpected because it had not been possible to separate the DI from standard virus particles [12], or the respective nucleocapsids from each other [11], by various gradient centrifugation techniques. It had also been found that
Arboviruses: Persistence and Defectiveness in Sindbis Virus Infections
81
Table 2. Estimated molecular weights of intracellular viral RNA species generated during undiluted serial passage of sindbis virus in chick embryo fibroblasts (CP) passage No. _c CP 6 CP 15 CP 24
ds RNA
Species 22S 18S 15S 12S
MWa 8.7 x 4.4 x 2.8 x 2.0 x
ss RNA
106 106 106 106
Species MWb 42S 4.3 x 106 33S 2.2 x 106 24S 1.4 x 106 18-22S 0.75--1.0 x 106
Genome Equivalent (%) 100 50 33 20-25
aBased on electron microscopic length measurements. bBased on migration rate in polyacrylamide gel electrophoresis. CStandard virus passage. For experimental details, see [131 and [14]
extraction of DI particles produced in BHK21 cells under less drastic denaturing conditions yielded only 42S RNA [31]. The findings in CEF therefore suggest a) that the DI particles contain multiple copies of the short genome fragments linked together in some way, b) that these multiples add up to one entire genome equivalent, c) that integrally divided genome residues are preferentially packaged in nucleocapsids due to assembly constraints, and hence, d) that a presumably random process of deletions is de-randomized by accumulation of defective fragments o f these selected sizes in succeeding virus passages. This interpretation is strengthened by structural studies of the defective RNA's by hybridization [13] and oligonucleotide analysis [21]. Briefly, both methods have revealed that the deletions are all internal, that the defective genomes retain about 1000 nucleotides o f the 3' end (which is believed to include the initiation site for transcription) and decreasing proportions of the 5' end of the complete genome (Fig. 2), and that they are of (+) polarity [13]. Thus, all defective genomes which can be replicated in the presence of helper virus contain the two ends whose nucleotide sequences display inverted homology. One can envisage the linking together of multiple strands by a 3' to 5' end base-pairing mechanism. This has, however, not yet been shown. Serial passage at high input multiplicities is an experimental artifact designed to amplify the production of defectives. It should be noted, however, that virions released even in the first passage (at m.o.i = 1 PFU/cell) of plaque-purified standard SV contain, in addition to standard 42S, clearly detectable, though small, peaks of RNA in the 33 and 25S regions [14], i.e., 1/2 and 1/3 genome equivalents. This observation is in line with the view that generation of defectives is an inescapable byproduct of viral replication and that it may contribute significantly to the self-limiting character of virus infections (reviewed in [ 1 5 - 1 7 ] ) . It is all the more striking that serial undiluted passage of SV in A. albopictus cell cultures has failed to provide convincing evidence for production of DI virions in terms of significantly reduced yields or of evolving RNA patterns [18]. Equally striking is the fact that infection o f A . albopictus cells with BHK21- or CEF-derived SV preparations containing high proportions of DI particles produce neither reduced yields of infectious virus nor the defective RNA species found in vertebrate cells [18]. The reasons for this apparent failure to produce or 'recognize' DI particles are not known.
82
R.W. Sehlesinger
8000 Np
5'I I
I I
! ~
5360 Np
DELETED
3040Np
DELETED
1880 Np
DELETED
1470 Np
4600 Np
13' SVs-ro 42S 1000Nfo
D!
33S
I
DI
24S
I
DI 22S
l
DI 20S
I
DI 18S
DELETED ' i
I
1150Np
(o)
l
DELETED ,
(o)
Fig. 2. Schematic presentation of defective Sindbis genomes evolvingduring undiluted serial passages in chick embryo fibroblast cultures. For details see Guild and Stollar (1977), from which this schema has been extracted
Infection of mosquito cell cultures with standard SV leads to initial yields comparable with those produced in vertebrate cells. Generally, the cells remain morphologically normal and continue to divide at the normal rate [35]. Cultures remain persistently infected, producing relatively small amounts of virus, all of which, eventually, becomes temperature-sensitive (ts) and shows small plaque morphology [23, 38, 33, 19]. Persistently infected cultures can be segregated into virus (+) and virus ( - ) clones [19]. Prolonged treatment of cultures with antiviral immune serum results in curing [19]. Both results indicate that persistent infection depends on continuous cell-tocell transfer of virus v/a the medium. Despite the lack of significant accumulation of DI virus particles in persistently infected A. albopictus cells, they do, after several weeks in culture, produce a prominent amount of 12S dsRNA which may indicate that defective genomes are made. It remains to be determined whether this material is virus-specific and plays a role in keeping the amounts of infectious ts, sp virus at such low levels. Elsewhere [25, 27] we have speculated on the possible implications of the differential behavior of SV in vertebrate and mosquito cells for the natural life cycle of this arbovirus. Cold selection of ts variants would clearly be favored in life-long persistence of such viruses in arthropod vectors, especially in those hibernating in cold climates; the massive production of DI particles by persistently infected mosquitoes would greatly reduce the effectiveness of transmission to vertebrates, for which a certain threshold titer is required. On the other hand, as already noted, the production of defective genomes and virus particles in vertebrate hosts could contribute, along with the immune response and interferon induction, to the self-limitation of a potentially destructive infection. Time does not permit further elaboration of these points, but they illustrate the potential contributions that detailed study at the molecular and comparative cellular level can make to an understanding of the epidemiological life cycle of arboviruses. It should be stressed again, however, that the fundamental differences between bunya-,
Arboviruses: Persistence and Defectiveness in Sindbis Virus Infections
83
alpha-, flavi- and other kinds of arboviruses will undoubtedly be found to impose totally disparate patterns on each of these families.
Acknowledgements: The work in the author's laboratory was supported in part by Grant AI11290 from the National Institute of Allergy and Infectious Diseases, by the United States-Japan Cooperative Medical Science Program through Public Health Service Grant AI-05920, and by the National Institutes of Health Training Grant CA-05234 from the National Cancer Institute References
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2a.
3.
4. 5.
6, 7. 8.
9.
10. 11.
12.
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Guild, G.M., Stollar, V.: Defective interfering particles of Sindbis virus. V. Sequence relationships between SVSTD42S RNA and intracellular defective viral RNAs. Virology 77,175-188 (1977) 14. Guild, G.M., Flores, L., Stollar, V.: Defective interfering particles of Sindbis virus. IV. Virion RNA species and molecular weight determination of defective double-stranded RNAs. Virology 77,158--174 (1977) 15. Huang, A.S.: Defective interfering viruses. Ann. Rev. Microbiol. 2 7 , 1 0 1 - 1 1 7 (1973) 16. Huang, A.S., Baltimore, D.: Defective viral particles and viral disease processes. Nature 226, 325-327 (1970) 17. Huang, A.S., Baltimore, D.: Defective-interfering animal viruses. Comprehensive Virology (H. FrankebConrat and R.R. Wagner, eds.) (in press) 18. Igarashi, A., Stollar, V.: Failure of defective interfering particles of Sindbis virus produced in BHK or chick cells to affect viral replication in A. albopictus cells. J. Virol. 19, 398-408 (1976) 19. Igarashi, A., Koo, R., Stollar, V.: Evolution and properties of Aeries albopictus cell cultures persistently infected with Sindbis virus. Virology (in press) 20. Inglot, A.D., Albin, M., Chudzio, T.: Persistent infection of mouse cells with Sindbis virus: Role of virulence of strains, auto-interfering particles and interferon. J. Gen. Virol. 20, 105-110 (1973) 21. Kennedy, S.I.T., Bruton, C.J., Weiss, B., Schlesinger, S.: Defective interfering particles of Sindbis virus: Nature of the defective virion RNA. J. Virol. 19, 1034-1043 (1976) 22. Matsumura, T., Stollar, V., Schlesinger, R.W.: Studies on the nature of dengue viruses. V. Structure and development of dengue virus in Veto cells. Virology 46, 344-355 (1971) 22a. Naeve, C.W., Trent, D.W.: Identification of Saint Louis encephalitis virus mRNA. Abstracts Ann. Mtg. Am. Soc. Microbiol., p. 287 (1977) 23. Peleg, J., Stollar, V.: Homologous interference in Aedes aegypti cell cultures infected with Sindbis virus. Arch. Ges. Virusforsch. 4 5 , 3 0 9 - 3 1 8 (1974) 24. Sabin, A.B.: Genetic factors affecting susceptibility and resistance to virus disease of the nervous system. Proc. Assn. for Research in Nervous and Mental Disease, Vol. XXXIII, pp. 5 7 - 6 6 (1954) 25. Schlesinger, R.W.: Sindbis virus replication in vertebrate and mosquito cells: an interpretation. Medical Biology 53,295--301 (1975) 26. Schlesinger, R.W.: Dengue-viruses. Virology Monographs, Vol. 16. Wien, New York: Springer (in press) 27. Schlesinger, R.W.: The significance and nature of defective interfering viruses. K.F. Meyer Symp., Bull. Swiss Academy of Medical Sciences 1977 (in press) 28. Schlesinger, S., Schlesinger, M.J., Burge, B.W.: Defective virus particles from Sindbis virus. Virology 4 5 , 6 1 5 - 6 1 7 (1972) 29. Shapiro, D., Brandt, W.E., Russell, P.K.: Change involving a viral membrane glycoprotein during morphogenesis of group B arboviruses. Virology 50, 906-911 (1972) 30. Shapiro, D., Kos, K., Russell, P.K.: Protein synthesis in Japanese encephalitis virus-infected cells. Virology 56, 95-109 (1973)
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