Neuroscience and Biobehavioral Reviews, Vol. 22, No. 6, pp. 721–723, 1998 䉷 1998 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0149-7634/98 $32.00 + .00
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A Review of Alphavirus Replication in Neurons DIANE E. GRIFFIN Department of Molecular Microbiology and Immunology, Johns Hopkins University, School of Hygiene and Public Health, Room E5132, 615 North Wolfe Street, Baltimore, MD 21205, USA
GRIFFIN, E.E. A review of alphavirus replication in neurons. NEUROSCI BIOBEHAV REV 22(6) 721–723, 1998.—Alphaviruses are important causes of mosquito-borne viral encephalitis. The prototype alphavirus, Sindbis virus, causes encephalomyelitis in mice. The primary target cell for nervous system infection is the neuron. Thus, Sindbis virus infection of mice provides a model system for studying virus–neuron interactions. The outcome of infection is dependent on the maturity of the targeted neurons and on the strain of Sindbis virus used for infection. Most Sindbis virus strains can induce programmed cell death or apoptosis in cultured lines of mammalian cells and in immature postmitotic neurons both in vitro and in vivo. As neurons mature they become increasingly resistant to Sindbis virus-induced apoptosis presumably due to increased expression with differentiation of cellular antiapoptotic proteins. Therefore, in the absence of an effective immune response, these relatively avirulent strains of Sindbis virus establish persistent nonfatal infection in mature neurons. More virulent strains of Sindbis virus can overcome this intrinsic resistance of mature neurons to apoptosis and cause neuronal death. Amino acid changes in the virion glycoproteins are the main determinants of neurovirulence and knowledge of the effects of specific changes allows the investigator to design Sindbis viruses of specified neurovirulence for animals of different ages. 䉷 1998 Elsevier Science Ltd. All rights reserved Neurovirulence
Apoptosis
Sindbis virus
Virus replication in neurons
Age-dependent virulence
INTRODUCTION
AGE-DEPENDENT VIRULENCE
ALPHAVIRUSES ARE small, relatively simple, enveloped, message-sense RNA viruses that are geographically restricted in distribution. In nature, these viruses are transmitted to mammals and birds by mosquitoes. Several members of this virus family cause encephalomyelitis in their vertebrate hosts (4). Sindbis virus (SV) is the prototype alphavirus, infects neurons and causes encephalomyelitis in mice, and has been extensively used as a model system for understanding virus infection of the nervous system, specifically acute viral encephalomyelitis. The SV genome has 11 700 nucleotides that encode two large polyproteins. The capped and polyadenylated genomic RNA serves as mRNA for the four nonstructural proteins that comprise the replicase complex. A subgenomic RNA is transcribed from the negative strand copy of the genome and serves as mRNA for the three major and two minor structural proteins. The three major structural proteins are the capsid (C) protein that surrounds the genomic RNA and two transmembrane glycoproteins, E1 and E2, that form a heterodimer that trimerizes to form spikes on the surface of the virion (24). The outcome of SV infection is dependent on the strain of the virus used for infection and the age of the animal at the time of infection. Several sequenced and recombinant strains of SV that vary in biological properties have been studied (2,6,13,17,19–21,26,28). These investigations have led to an expanding knowledge of the molecular basis of SV neurovirulence in mice and an ability to control the outcome of infection in a particular host.
The original strain of SV (AR339) was isolated in 1953 by inoculating a pool of culicene mosquitoes collected in Sindbis, Egypt into newborn mice. In these early studies it was recognized that the newly isolated virus caused fatal disease in these young mice, but was avirulent for older mice (25). Subsequent studies have shown that the target tissues for SV infection in mice are muscle, brown fat and the central nervous system (CNS) (9). In the CNS, the target cells are ependymal cells and neurons (7,8). Motor neurons in the brain and spinal cord are particularly susceptible to infection (Fig. 1) and an important clinical manifestation of infection is paralysis (7). Recent studies have shown that SV damages the cells it infects by inducing apoptosis or programmed cell death (10). Fatal encephalomyelitis correlates with apoptotic neuronal cell death in vivo (11). As neurons mature they become increasingly resistant to induction of apoptosis by a variety of insults, including SV infection. Studies of cultured dorsal root ganglion cells matured in vitro in the presence of nerve growth factor (10) and of mice infected at different ages (11) have shown that the induction of apoptosis and outcome of infection are correlated. For any one strain of virus, susceptibility to apoptosis is determined by the maturity of the neuron infected. The change in susceptibility to the induction of programmed cell death with neuronal maturation is presumably due to increased expression of cellular inhibitors of apoptosis and provides a plausible explanation for agedependent virulence of SV, and perhaps other, neurotropic viruses. 721
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FIG. 2. Diagram of the adaptation of the AR339 strain of Sindbis virus by passage through tissue culture cells or mouse brain. Virulence for newborn (1-day-old) mice is decreased by prolonged passage in tissue culture while neurovirulence is increased by passage through mouse brain. FIG. 1. In situ hybridization of the spinal cord of a mouse infected with Sindbis virus. The target cells for virus replication in the nervous system are neurons, with motor neurons being particularly susceptible.
VIRUS STRAIN AS A DETERMINANT OF VIRULENCE
A large number of strains of SV that differ in virulence for mice have been isolated. These strains come from independent isolates from a variety of sources: naturally infected mosquitos (12,20,25), viruses carried in different ways in vitro and in vivo (2,5,12,20,23) and construction of recombinant viruses using a cDNA clone that can be transcribed into infectious RNA allowing the recovery of genetically defined viruses (13,14,17,19,21,26,28). We have studied avirulent strains of AR339 derived by passage in tissue culture and virulent strains derived by intracerebral passage in mice. Comparisons of the biologic properties of these strains have provided insight into the determinants of virulence. Neurons are target cells for all strains of SV, but the levels of replication in neurons, the numbers of neurons infected and the abilities of the viruses to induce neuronal apoptosis differ between strains. For instance, the tissue cultureadapted, avirulent, heat resistant, small plaque (HRSP) strain does not reproducibly cause fatal encephalitis even in newborn mice (23), while the mouse brain-passaged neurovirulent NSV strain causes fatal disease in 3–4 week old mice (5,7,8) (Fig. 2). When the CNS infection is not fatal, immunologically normal mice clear virus within 7–8 days and recover uneventfully. Differences in neurovirulence correlate with the efficiency and level of SV replication in mature neurons and in cultured cells overexpressing inhibitors of apoptosis such as bcl-2 (8,11,30). The most neurovirulent strains (e.g., NSV) replicate to higher titer in the brain and spinal cord of 3–4 week old mice and spread to more neurons than less virulent strains (8). These strains also induce apoptosis in AT3 rat prostatic carcinoma cells overexpressing bcl-2 (AT3-bcl-2 cells) (30). The least neurovirulent strains (e.g., HRSP) replicate to only a limited extent in brain and spinal cord, even those of newborn mice (23), and establish a persistent infection in AT3-bcl-2 cells (10).
E2, are important determinants of virulence (2,13,17). These glycoproteins heterodimerize and undergo significant conformational changes during the virus life cycle. Single amino acid changes, particularly in E2, can have profound effects on the ability of the virus to infect neurons, spread within the CNS and cause fatal encephalomyelitis (26,28). E2 is the major attachment protein and is presumed to initiate interaction with the neuron or other target cell. The cellular receptor(s) for SV have not been clearly defined and it is unknown whether neurons do or do not have a special receptor that accounts for their unique in vivo vulnerability to SV infection (29). Amino acid changes in E2 that affect virulence often affect an early step in virus replication (Table 1) suggesting that the efficiency of interaction with a specific neuronal receptor or coreceptor may be an important determinant of neurovirulence. An amino acid change at E2-172 in HRSP (arginine in HRSP, glycine in AR339 and NSV) decreases the ability of this virus to bind to neural cells, a change that probably contributes to the decreased neurovirulence of this strain of SV (26). Other changes in E2 affect virulence in less well-defined ways, but data suggest that efficient entry of the virus into the neuron is a critical step that is sensitive to amino acid changes in E2. A change from serine to arginine at E2-114 decreases virulence. Paradoxically, this change is associated with more rapid viral penetration of BHK cells (2,15,16). Presumably, penetration of neural cells is simultaneously decreased, although that has not yet been evaluated for these viruses. The amino acid change leading to the most profound increase in neurovirulence is a substitution of histidine for glutamine at E2-55 (28). This amino acid change alters viral entry and affects early steps of replication more profoundly in neural cells than in nonneural cells (3,27). These observations suggest that cellular proteins or lipids participate actively in entry and that these, as yet unidentified, molecules differ in neural and nonneural cells leading TABLE 1 AMINO ACID CHANGES IN THE E2 GLYCOPROTEIN THAT AFFECT VIRULENCE AND THE APPARENT STEP OF VIRUS REPLICATION THAT IS ALTERED E2 residue
MOLECULAR BASIS OF SV NEUROVIRULENCE
Sequence analyses and construction of recombinant viruses have shown that the virus glycoproteins, E1 and
55 114 172
↑ Virulence
↓ Virulence
Replication step
His Ser Gly
Gln Arg Arg
Entry Entry Receptor binding
ALPHAVIRUS REPLICATION IN NEURONS
723
to selective vulnerability of cells to infection. In addition, amino acid substitutions at E2-1, E2-62, E2-96 and E2-159 also alter virulence, but the steps in virus replication that are affected by these changes have not yet been determined (6,21,22). The E1 glycoprotein contains an internal hydrophobic sequence that is postulated to be the region that is exposed during the acid-induced conformational change of the E1E2 heterodimer in the endosome. This change then initiates fusion of the viral lipid envelope with the endosomal membrane of the cell to allow release of the genome-containing nucleocapsid into the cytoplasm (24). E1 glycoproteins from strains of SV differing in virulence have revealed only four amino acid substitutions. These substitutions are located at E1-72, E1-75, E1-237 and E1-313. Construction
and testing of recombinant viruses have implicated at least E1-75 and E1-237 as determinants of virulence (13,18). The mechanism of this change in virulence is not known, but does not appear to be directly associated with alterations in the optimal pH of fusion, at least as measured in BHK cells (1). SUMMARY
Sindbis virus is a neuronotropic virus for which there is an increasingly detailed knowledge of the molecular determinants of neurovirulence. Construction of recombinant viruses using a cDNA clone from which infectious RNA can be transcribed allows the investigator to design viruses of defined virulence.
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15. Olmsted, R.A., Baric, R.S., Sawyer, B.A. and Johnston, R.E., Sindbis virus mutants selected for rapid growth in cell culture display attenuated virulence in animals. Science, 1984, 225, 424–426. 16. Olmsted, R.A., Meyer, W.J. and Johnston, R.E., Characterization of Sindbis virus epitopes important for penetration in cell culture and pathogenesis in animals. Virology, 1986, 148, 245–254. 17. Polo, J.M., Davis, N.L., Rice, C.M., Huang, H.V. and Johnston, R.E., Molecular analysis of Sindbis virus pathogenesis in neonatal mice by using virus recombinants constructed in vitro. J. Virol., 1988, 62, 2124–2133. 18. Polo, J.M. and Johnston, R.E., Attenuating mutations in glycoproteins E1 and E2 of Sindbis virus produced a highly attenuated strain when combined in vitro. J. Virol., 1990, 64, 4438–4444. 19. Polo, J.M. and Johnston, R.E., Mutational analysis of a virulence locus in the E2 glycoprotein gene of Sindbis virus. J. Virol., 1991, 65, 6358– 6361. 20. Russell, D.L., Dalrymple, J.M. and Johnston, R.E., Sindbis virus mutations which coordinately affect glycoprotein processing, penetration, and virulence in mice. J. Virol., 1989, 63, 1619–1629. 21. Schoepp, R.J. and Johnston, R.E., Directed mutagenesis of a Sindbis virus pathogenesis site. Virology, 1993, 193, 149–159. 22. Schoepp, R.J. and Johnston, R.E., Sindbis virus pathogenesis: phenotypic reversion of an attenuated strain to virulence by second-site intragenic suppressor mutations. J. Gen. Virol., 1993, 74, 1691–1695. 23. Sherman, L.A. and Griffin, D.E., Pathogenesis of encephalitis induced in newborn mice by virulent and avirulent strains of Sindbis virus. J. Virol., 1990, 64, 2041–2046. 24. Strauss, J.H. and Strauss, E.G., The alphaviruses: gene expression, replication and evolution. Microbiol. Rev., 1994, 58, 491–562. 25. Taylor, R.M., Hurlbut, H.S., Work, T.H., Kingsbury, J.R. and Frothingham, T.E., Sindbis virus: a newly recognized arthropodtransmitted virus. Am. J. Trop. Med. Hyg., 1955, 4, 844–846. 26. Tucker, P.C. and Griffin, D.E., The mechanism of altered Sindbis virus neurovirulence associated with a single amino acid change in the E2 glycoprotein. J. Virol., 1991, 65, 1551–1557. 27. Tucker, P.C., Lee, S.H., Bui, N., Martinie, D. and Griffin, D.E., Amino acid changes in the Sindbis virus E2 glycoprotein that increase neurovirulence improve entry into neuroblastoma cells. J. Virol., 1997, 71, 6106–6112. 28. Tucker, P.C., Strauss, E.G., Kuhn, R.J., Strauss, J.H. and Griffin, D.E., Viral determinants of age-dependent virulence of Sindbis virus in mice. J. Virol., 1993, 67, 4605–4610. 29. Ubol, S. and Griffin, D.E., Identification of a putative alphavirus receptor on mouse neural cells. J. Virol., 1991, 65, 6913–6921. 30. Ubol, S., Tucker, P.C., Griffin, D.E. and Hardwick, J.M., Neurovirulent strains of alphavirus induce apoptosis in bcl-2-expressing cells: role of a single amino acid change in the E2 glycoprotein. Proc. Natl. Acad. Sci. USA, 1994, 91, 5202–5206.