A BRIEF INTRODUCTION to the genes and proteins of Sindbis virus. Refer to Strauss and Strauss, 1994 for a more detailed review.

Sindbis virus is an excellent gene expression vector.

Over 1800 Medline articles that mention Sindbis virus, 1966-2005.

Sindbis virus

Lifecycle and Genome

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Alphaviruses

Sindbis virus is the type species of the alphavirus genus in the Togaviridae family.

Members of the alphavirus genus include:

Old World viruses	Babanki, Barmah Forest, Bebaru, chikungunya, Getah, Karelian fever, Kyzylagach, Middleburg, Murweh, Ndumu, Ockelbo, o'nyong nyong, Ross River, Sagiyama, Semliki Forest, Sindbis, Whataroa
New World viruses	Aura, Buggy Creek, Cabassou, Everglades, Eastern equine encephalitis, Fort Morgan, Highlands J, Mayaro, Mucambo, Pixuna, Tonate, Una, Venezuelen equine encephalitis, Western equine encephalitis
Aquatic viruses	rainbow trout sleeping disease, salmon pancreas disease viruses

Most of the described alphaviruses are named after the place of first isolation
Some are quite pathogenic (see safety recommendations)
Sequence data are available for many of the viruses.

Sindbis virus

Sindbis virus (strain AR339) was first isolated in August 1952, from a pool of mosquitos (Culex pipiens and C. univittatus) trapped in the Sindbis health district, 30 km north of Cairo, Egypt (Hurlbut 1953; Taylor and Hurlbut 1953; Frothingham 1955; Taylor et al. 1955).

The HR (heat resistant) strain was derived from AR339 (Burge and Pfefferkorn, 1966). Essentially all ts mutants were derived from HR (Burge and Pfefferkorn, 1966; Strauss, et al., 1976).

The HRsp (small plaque) and lp (large plaque) strains were derived from the HR strain (Arias, et al., 1983).

The entire sequence (cDNA and protein coding, or GenBank format, or text only) of the SIN HRsp genome was determined by Strauss, et al., 1984, partly based on the slightly different SIN HRlp 26S mRNA sequence determined by Rice and Strauss, 1981. (The sequences of several other alphaviruses have also been determined).

By convention, positions along the genomic RNA are indicated as nucleotide distances from the 5' end (Strauss, et al., 1984). The nomenclature of the genes are according to the same reference. The nonstructural protein genes (and proteins) are called nsP1 to nsP4, and the structural protein genes (and proteins) are the capsid (C), and envelope proteins 1 and 2 (E1, E2).

	49S genomic RNA 11703 nt + poly-A				26S subgenomic mRNA 4106 nt + poly-A
Composition (excluding poly-A)	U 2908 20.8%	C 3049 26.1%	A 3308 28.3%	G 2908 24.8%	U 856 20.8%	C 1129 27.5%	A 1122 27.3%	G 999 24.3%

The idea of making an infectious cDNA clone of SIN was conceived in 1980. It took a while to get it to work (Rice, et al., 1987). The result was the creation of three types of cDNA clones that can be transcribed to produce infectious RNA:

• The sequence of the Toto1000 and 1100 series of plasmids is derived from HR, HRsp and HR(SS) (Rice, et al., 1987).
  It differs from the HRsp sequence at a number of positions (Lustig, et al., 1988; Polo, et al., 1988).
• The Toto100 series is derived from HR and HRsp.
• The Toto50 series is derived entirely from HRsp.

cDNA clones in the Toto1000 series have a Sst I site for runoff transcription of the genomic RNA.
Plasmids in the Toto1100 series are identical to the Toto1000 series, except for the Xho I site for runoff transcription.
Other full-length cDNA clones may have Mlu I and Not I runoff sites.

The Sindbis Virus lifecycle

Like other alphaviruses (except for the aquatic viruses), Sindbis virus is transmitted by vertebrate hosts to mosquitos. For the vertebrates, the alphaviruses are arthropod-borne pathogens; for mosquitos, they are food-borne pathogens.

Alphavirus virions consist of a nucleocapsid, wrapped inside a lipid bilayer, upon which the envelope proteins are displayed.

The envelope proteins mediate binding to host cell receptors, leading to the endocytosis of the virion. When the endocytic vesicle is acidified, the envelope proteins undergo conformational changes that result in the fusion of the lipid bilayer of the virion and that of the vesicle. The nucleocapsid, a complex of the capsid protein and the genomic RNA, is thus deposited into the cytoplasm of the host cell.

The Sindbis virus genome is a single-stranded RNA of 11703 nt (Strauss, et al., 1984), capped at the 5' terminus and poly-adenylated at the 3' terminus

The genomic 49S RNA is of plus sense, is infectious, and serves as mRNA in the infected cell. Translation of the genomic RNA produces the nonstructural proteins, nsP1, nsP2, nsP3 and nsP4.

The nonstructural proteins are synthesized as 2 polyproteins, P123 and P1234. The P123 polyprotein contains nsP1, nsP2 and nsP3. It terminates at an opal codon between the nsP3 and nsP4 coding sequences. The P1234 protein is made in lower abundances by read-through of the opal codon, and contains nsP1 through nsP4 (Lopez, et al., 1985).

The polyproteins are proteolytically processed, by the protease domain of nsP2 (Strauss, et al., 1992; Hardy and Strauss, 1989), to form various intermediates and the 'mature' nonstructural proteins (Hardy and Strauss, 1988; de Groot, et al., 1990; Shirako and Strauss, 1990; Lemm and Rice, 1993; Shirako and Strauss, 1994). Note that the polyproteins and the intermediates may have discrete roles in replication and transcription.

Early during infection, the nonstructural proteins, perhaps in association with host factors, use the genomic (+)-sense RNA as template to make a full-length, complementary (-) strand RNA. The (-) strand is template for synthesis of full-length genomic RNA. An internal promoter on the (-) strand is used for transcription of a subgenomic 26S mRNA. The 26S subgenomic mRNA is capped and polyadenylated, and is colinear wih the 3' terminal one-third of the genomic RNA.

The 26S subgenomic mRNA is translated to produce the structural polyprotein, that undergoes co-translational and post-translational cleavages to produce the C, E2 and E1 structural proteins.

The capsid protein encapsidates the genomic RNA to form nucleocapsids. These interact with the cytoplasmic domain of the cell-surface envelope proteins, resulting in the envelopment of the nucleocapsid inside a membrane bilayer containing the envelope proteins, and the budding of progeny virions out of the infected cell.

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Genome
	nonstructural proteins						structural proteins
49S 5' UT	nsP1	nsP2	nsP3	nsP4	Pro- moter	26S 5'UT	C	E3	E2	6K	E1	3'UT	poly-A

49S 5' Untranslated Sequence

Capped at the 5' terminus: m7GpppA....

Contains conserved 5' structure (Ou, et al., 1983). Mutagenesis: Niesters and Strauss, 1990. The structure is required for DI replication (Levis, et al., 1986). The complement (3' end of the minus-strand) is bound by host proteins, possiby for initiating plus-strnd synthesis (Pardigon, et al., 1993; Pardigon and Strauss, 1992; Pardigon and Strauss, 1996).
The wild type 5' end may be substituted by modified forms of cellular tRNA Asp and 5' end of 26S mRNA (Monroe and Schlesinger, 1983; Tsiang, et al., 1985).

Controls efficiency of translation of the genomic RNA (Berben-Bloemheuvel, et al., 1992).

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nsP1 (nonstructural protein 1)

nsP1/nsP2 cleavage is between GlyAla / AlaLeu, corresponding to position 1679/1680.

Sequence of alphavirus nsP1.

Homologous to amino half of TMV T1, BMV B1 and AMV A1 proteins (Ahlquist, et al., 1985).

51 nt conserved region extends from 155-205. Postulated double stem-loop structure. Mutagenesis: Niesters and Strauss, 1990.

Methyltransferase domain (Rozanov, et al., 1992; Wang, et al., 1996):

4 conserved motifs:
• I = invariant His
• II= AspXXArg
• III=?
• IV=invariant Tyr

Genetic and biochemical evidence for RNA methyltransferase and guanylyltransferase activities:

• SV-MPA (mycophenolic acid resistance) (Scheidel, et al., 1987)
  • C 120 to A, Gln 21 to Lys
  • G 127 to A, Ser 23 to Asn
  • G 963 to A, Val 302 to Met changes
  • A 963 alone or A 120 plus A 127 give partial phenotype (Scheidel and Stollar, 1991).
  • SV-MPA virus does not grow well in secondary CEF derived from aged primary CEF (Rosenblum, et al., 1994). Virus selected to grow well in these cells have lost A 120, still retain A 127 and A 963, and are still MPA resistant.
• LM21 (Scheidel, et al., 1989; Mi, et al., 1989; Mi and Stollar, 1990; Mi and Stollar, 1991).
  • altered methyltransferase activity
  • able to grow in mosquito cells in the presence of low levels of methionine.
  • Mutations are
    • 319 G to U, Arg 87 to Leu
    • 321 A to U, Ser 88 to Cys
  • Individual changes give partial phenotype

Cloned SFV nsP1 has methyltransferase and guanylyltransferase activities (Laakkonen, et al., 1994; Ahola and Kaariainen, 1995; Ahola, et al., 1997)).

Cloned and virus-expressed nsP1 is ester-linked to palmitate (Laakkonen, et al., 1996), and is associated with membranes: plasma membrane, endocytic vesicles, and large, lysosomal vescicles (Peranen, et al., 1995). Palmitoylation is at cysteine residues 418 to 420 of SFV nsP1, and at cys 420 of SIN nsP1 (Ahola et al., 2000.

Genetic (ts-11, complementation group B, [1101 G to A, nsP1 Ala 348 to Thr], (Hahn, et al., 1989) evidence for involvement in (-) strand synthesis: ts-11 suppresses/epistatic over the ts24R (maps in nsP4) phenotype (re-activation of (-) strand synthesis) (Sawicki, et al., 1981; Wang, et al., 1991).

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nsP2 (nonstructural protein 2)

nsP1/nsP2 cleavage is between GlyAla / AlaLeu, corresponding to position 1679 / 1680.
nsP2/nsP3 cleavage between GlyAla / AlaPro, corresponding to position 4100 / 4101.

Sequence of alphavirus nsP2.

Amino half of nsP2 is homologous to carboxyl half of TMV T1, BMV B1 and AMV A1 proteins (Ahlquist, et al., 1985).

It has NTP-binding/helicase motifs (Hodgman, 1988; Company, et al., 1991).

It does have ATPase and GTPase activities (Rikkonen, et al., 1994). Helicase activity has not been detected.

• Helicase motif I (NTP-binding A site): V(184)IGTPGSGKSAIIK. See Hodgman, 1988.
• Helicase motif II (NTP-binding B site): V(248)LYVDEAFACH. Also DEAD/DEAH box.
• Helicase motif III (DNA polymerase-like): V(275)VLCGDPMQCG
• Helicase motif IV: Y(311)ISRRCTQPV.
• Helicase motif V: V(379)MTAAASQGLTRKGVYAV.
• Helicase motif VI: H(412)VNVLLTRT

View the structure of a DNA helicase (SF1 super-family) (481 kbytes) to see the position of the helicase motifs, and residues conserved among the DNA helicase and the alphavirus nsP2 proteins; or the structure of the HCV NS3 helicase (SF2 super-family) (338 kbytes).
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Carboxyl half has protease activity (Strauss, et al., 1992; Hardy and Strauss, 1989), that cleaves in cis and trans to process the nonstructural polyproteins P123 and P1234 (Hardy and Strauss, 1988; de Groot, et al., 1990; Shirako and Strauss, 1990; Lemm and Rice, 1993; Shirako and Strauss, 1994).

• Deletion of the amino half up to EcoRV 2750 does not impair protease activity. Deletion up to PvuII 3103 gives decreased protease activity.
• Cys-481 and His-558 active site residues of nsP2 protease (Strauss, et al., 1992).
• Unlike other papain-like cysteine proteinases, nsP2 has an essential Trp-559 instead of the usual Ala or Gly immediately after the His-558

Genetic evidence for involvement in 26S mRNA synthesis (Scheele and Pfefferkorn, 1969; Waite, 1973; Keranen and Kaariainen, 1979; Sawicki and Sawicki, 1985; Hahn, et al., 1989; Suopanki, et al., 1998).

Biochemical evidence for:

• association of nsP2 with the nuclear matrix (Peranen, et al., 1990);
• import into the nucleus /nucloelus (Rikkonen, et al., 1992; Rikkonen, et al., 1994);
  • Infection by a nsP2 mutant that does not localize to the nucleolus has lower levels of inhibition of host DNA synthesis, and the virus is attenutated compared to wildtype infections (Rikkonen, 1996)
• association with ribosomes (Ranki, et al., 1979).

Mutations (Sawicki and Sawicki, 1985; Hahn, et al., 1989):

• ts-21 (A)
  • 2590 G to A, Cys 304 to Tyr.
• Ala 382 (nt 2824) to Val is lethal (Rice, et al., 1987)
• ts-118 (F)
  • 2953 U to C, Val 425 to Ala
  • One of 2 mutations (the other is at 6046 in nsP4).
  • 2953 U to C by itself gives minute plaques but not reduction in plaque numbers.
• Leu 438 (nt 2992) to Pro is lethal (Rice, et al., 1987)
• ts-18 (G)
  • 3204 U to C, Phe 509 to Leu.
• ts-17 (A) and ts-133 (A)
  • ts-17
    • 3228 G to A, Ala 517 to Thr
  • ts-133 (A) (Sawicki and Sawicki, 1993):
    • A 2605 to U. Still present in revertant
    • A 3579 to G. Wildtype A in revertant. Not needed to confer mutant phenotype.
    • C 3779 to G. Changed to U (and wildtype Asn) in revertant. Probably causal mutation.
    • C 3879 to G. Still present in revertant
  • Both ts-17 and ts-133 (Sawicki and Sawicki, 1993)
    • reactivate (-) strand synthesis
    • have defective proteolytic processing of nonstructural polyprotein
    • have defective transcription of subgenomic mRNA
• ts-7 (G)
  • 3243 G to A, Asp 552 to Asn
  • One of 2 mutations (other is at 5035 in nsP3).
  • This one alone is RNA±, perhaps because the Asn deaminates to give Asp.
• ts-24 (A)
  • 3885 G to A, Gly 736 to Ser.
• SIN-1 (Dryga, et al., 1997)
  • 3855 C to T, Pro 726 to Ser
  • Mutation that allows establishment of persistent infection in BHK-21 cells.
  • RNA synthesis is 60% of wildtype.
  • Other mutations in the SIN-1 virus are required for the decreased RNA synthesis (10% wt) of SIN-1.

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nsP3 (nonstructural protein 3)

Sequence of alphavirus nsP3.

nsP3 is a phosphoprotein, phsphorylated on serines and threonines (Peranen, et al., 1988; Peranen, 1991; Lastarza, et al., 1994). This explains its anomalously low mobility in SDS gels. It is phosphorylated even when it is expressed by itself, in the absence of other viral proteins (Peranen, 1991) (the specific residues phosphorylated is not known in either case). Much of it is localized to the mitochodrial pellet (P15) fraction and vescicular structures (Peranen, et al., 1988; Peranen, 1991). How this is accomplished, and the functional significance of the phosphrylation/localization are not known.

nsP3 is the only nonstructural protein with no homology with other virus families. Its amino-terminus is homologous to a region of the rubella virus nonstructural proteins.

nsP3 usually terminates at an opal termination codon (5748-5750). Low frequency (10%) read-through produces P1234. The precise cleavage site has not been published, but is supposed to be at Tyr 5769.
Thus, in theory, there are 2 forms of nsP3. The nsP3 produced by cleavage between NS34 has a 6-residue carboxyl extension compared to nsP3 produced by terminatation at the opal codon.

The carboxyl domain (beyond nt 5079, residue 326) is extremely variable among alphaviruses (See sequence comaprison of alphavirus nsP3). Much of it is dispensable for growth in vertebrate cells, while mutations in the amino-domain are deleterious (Lastarza, et al., 1994; LaStarza, et al., 1994).

Mutations:

• ts-4 (A) (Wang, et al., 1994)
  • 4903 C to U, Ala-268 to Val
  • defect in (-) strand synthesis
• ts-7 (G)
  • 5035 G to A, nsP3 Phe 312 to Ser
  • another mutation at 3243 in nsP2
  • Either 5035 G to A or 3243 G to A is RNA-.
• insertion and deletion mutations in the conserved and variable domains (Lastarza, et al., 1994):
  • Those in the variable domain have no apparent effect on the virus lifecycle.
  • CR3.36 and CR3.39, that map near residues 58 and 226, respectively.
    • temperature-sensitive, defective in synthesis of minus-strand RNAs at the nonpermissive temperature.
    • CR3.39 complements ts mutants from complementation groups A, B, F, and G.
  • CR3.34
    • non-ts for plaque formation or RNA synthesis
    • forms smaller plaques
    • defective in subgenomic RNA synthesis at all temperatures examined.

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nsP4 (nonstructural protein 4)

Sequence of alphavirus nsP4.

Homologous to TMV T2, BMV B2 and AMV A2 proteins (Ahlquist, et al., 1985)

Produced by readthrough of opal termination codon. Amino terminal is reputed to be Y, 7th residue from the opal codon, giving a 610 residue protein. Some alphaviruses (e.g. Semliki Forest virus) do not have a termination codon after nsP3, and nsP4 is produced as part of a single polyprotein.
Readthrough efficiency depends on the C immediately downstream of the opal termination codon (Li and Rice, 1993; Li and Rice, 1989).

Mutations (Hahn, et al., 1989):

• ts-118 (F): 6046 A to G, nsP4 Gln 93 to Arg. One of 2 mutations (the other is at 2953 in nsP2). This one alone has no apparent phenotype, but is ts in combination with mutation at 2953.
• ts-6 (F): 6226 G to A, nsP4 Gly 159 to Glu.
• ts-24R1: 6339 C to A, nsP4 Gln 195 to Lys. Fails to shut off (-) strand synthesis (Sawicki and Sawicki, 1986; Sawicki, et al., 1990).
• ts-110 (F): 6739 G to A, nsP4 Gly 330 to Glu.

Probably the polymerase;

• ts-6, that ceases all RNA synthesis at the nonpermissive temperature in vivo (Hahn, et al., 1989) and in vitro (Barton, et al., 1988);
• homology to putative polymerases of other viruses, e.g., the presence of the conserved GDD nucleotide-binding motif at residues 470-473.

Carboxyl-end is shorter among some New World alphaviruses (Western, Eastern and Venezuelan equine encephalitis viruses), but not Aura.
3' end of nsP4 coding sequences overlaps with the promoter (Levis, et al., 1990).

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26S mRNA Promoter

The sequence in the junction region between the nonstructural and structural protein genes is conserved (Ou, et al., 1982).

The conserved region is congruent with the minimal promoter (shortest sequence that can still direct transcription of a mRNA), that extends from position 7579 to 7602; -19 to +5 relative to 26S mRNA start site at 7598 (Levis, et al., 1990).

The minimal promoter overlaps with the carboxyl terminus of nsP4.

The conservation is due to selection for promoter function and for encoding nsP4 (Hertz and Huang, 1995).

Promoter utilization is host-dependent (Durbin, et al., 1991; Hertz and Huang, 1995).

Sindbis virus is able to recognize and use the junction sequences of other alphaviruses for transcription of subgenomic mRNAs (Hertz and Huang, 1992).

The minimal promoter is about 3-6 fold less active than a region that includes, e.g., -98 to +14. Thus there are sequences external to the minimal promoter that enhances its activity (Raju and Huang, 1991).

The region from -40 to +14 appears to have full promoter strength.

Mutations:

• CR4.1 (Grakoui, et al., 1989)
  • GTC insertion between 7590 and 7591 (or, equivalently, between 7593 and 7594)
  • very low levels of 26S mRNA synthesis.

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26S mRNA 5' Untranslated Sequence

Overlaps 5 nt with minimal promoter.

Capped at the 5' terminus.

Controls efficiency of translation of the subgenomic RNA (Berben-Bloemheuvel, et al., 1992).

Excellent Kozak consensus through capsid AUG, although +4 is A instead of consensus G.

Toto1002 has TCTAGA (Xba I site) inserted between 7611 and 7612.

Derivatives may serve as 5' ends of DI's (Tsiang, et al., 1988).

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Structural Polyprotein

Proteolytically processed to produce the capsid (C), E3 peptide, envelope proteins 2 (E2), 6K peptide, and envelope protein 1 (E1).

Sequence comparison of alphavirus structural polyproteins

C (Capsid Protein)

Crystal structure of the carboxyl proteolytic fragment (Choi, et al., 1996; Choi, et al., 1991; Tong, et al., 1992; Tong, et al., 1993)

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CryoEM structure (Paredes, et al., 1993; Paredes, et al., 1998; Paredes, et al., 1992)

Encapsidates genomic RNA to form nucleocapsids (Wengler, et al., 1982; Wengler, et al., 1984; Weiss, et al., 1989; Geigenmuller-Gnirke, et al., 1993; Forsell, et al., 1996), by binding of the amino-terminal domain to a packaging signal in the nsP1 region (Geigenmuller-Gnirke, et al., 1991; Weiss, et al., 1994; Forsell, et al., 1996).

The carboxyl domain is a serine protease that cleaves capsid off from E3 and the rest of the structural polyprotein.

• Cleavage is between GluTrp / SerAla, corresponding to between positions 8438 / 8439.

The carboxyl protease domain functions in the absence of the amino domain (Forsell, et al., 1995).

Associated with large subunit of ribosome (Wengler, 1984; Wengler and Wurkner, 1992), that may be involved in the uncoating of the nucleocapsid early during infection (Wengler, 1987; Wengler, 1991; Wengler and Gros, 1996).

Mutations:

• ts-2, ts-5 (C): 8298 C to G, capsid Pro 218 to Ser.

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E3 peptide

N-terminal domain is uncleaved leader peptide for E2. E3+E2 = pE2 (Schlesinger and Schlesinger, 1972; Welch and Sefton, 1979).

E3/E2 cleavage between LysArg / SerVal, corresponding to between positions 8630 / 8631 (Mayne, et al., 1984).

During viron assembly, E3 cleavage is a required step (Lobigs, et al., 1990; Jain, et al., 1991; Heidner, et al., 1994).

Cleaved E3 is not found in the mature Sindbis virus virion (Mayne, et al., 1984; Paredes, et al., 1998), but it is retained as part of the virion of Semliki Forest virus (Luukkonen, et al., 1977).

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E2 envelope protein

Envelope glycoprotein that forms heterodimer with E1, 3 copies of the heterodimer in each spike.

E2 Asn 196 and Asn 318 are glycosylated.

E2 carries the major neutralizing epitopes.

E2 residues ca. 365 to ca. 390 = trans-membrane domain.

Residues ca. 391 onwards constitute its cytoplasmic domain, that interacts with the nucleocapsid during virion assembly (Kail, et al., 1991).

The transmembrane/intracellular domain of E2, like that of E1, is acylated with long-chain fatty acids, primarily palmitic acid (Schmidt, et al., 1979; Schmidt and Schlesinger, 1980; Schmidt, 1982; Schmidt, et al., 1988; Schmidt, et al., 1995).

E2 / 6K cleavage between AsnAla / GluThr, corresponding to position 9900 / 9901.

Mutations:

• SB-RL: 8972 C to A, E2 Ser 112 to Arg. (Gidwitz, et al., 1988; Olmsted, et al., 1986; Pence, et al., 1990; Polo, et al., 1988; Russell, et al., 1989)
  • Mutant has rapid penetration of BHK
  • increased reactivity with R6 and R13 neutralizing monoclonal antibodies
  • decreased neurovirulence for neonatal mice.
• ts-103: E2 Ala 344 to Val.
• ts-103 maps in E2.

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6K peptide

Leader for E1 (Welch and Sefton, 1979; Welch and Sefton, 1980).

6K/E1 cleavage between AspAla / TyrGlu, corresponding to position 10064 / 10065.

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E1 Envelope protein

Envelope glycoprotein that forms heteroimer with E2. Each spike on the virion surface contains 3 molecules of the heterodimers.

E1 Asn 139 and Asn 245 are glycosylated.

The transmembrane/intracellular domain of E1, like that of E2, is acylated with long-chain fatty acids, primarily palmitic acid (Schmidt, et al., 1979; Schmidt and Schlesinger, 1980; Schmidt, 1982; Schmidt, et al., 1988; Schmidt, et al., 1995).

Low pH treatment results in the rapid dissociation of the E1-E2 heterodimer in the spike, formation of E1 trimers, binding to target membranes, and fusion (Wahlberg, et al., 1989; Wahlberg, et al., 1992; Wahlberg and Garoff, 1992; Bron, et al., 1993; Justman, et al., 1993).

E1 is likely to be responsible for membrane fusion activity. Residues ca 75-91 is probably the hydrophobic fusion domain.
Mutagenesis of the putative fusion domain of SFV E1 (DYQCKVYTGVYPFMWGGAYCFCD) corresponding to SIN E1 75-97 alters cell fusion at different pHs (Levy-Mintz and Kielian, 1991):

	transport to cell surface	threshold pH for fusion	pH for maximal fusion	maximal efficiency
wt	yes	6.5	6.2	>90%
D 75 to A	yes	5.8	5.0	70%
K 79 to Q	like wt
G 83 to A	yes	6.1	5.3	>80%
delete 83-92	hung up in ER
P 86 to D	hung up in ER
M 88 to L	like wt
G 91 to A	yes	5.4	5.0	70%
G 91 to D	yes	no fusion activity
G 91t o P	hung up in ER

Other mutations:

ts-23 (D)
• 10380 G to A, Ala 106 to Thr.
• 10864 G to A, Arg 272 to Gln.
  Both reverted to wild type in revertant.

ts-10 (D)

• 10590 AAG to GGG, Lys 176 to Gly. Revertant ts 10R has CGG for Arg.

E1 Lys 227 to Met partially suppresses ts-103 of E2 (residue344).

E1 terminal ArgArg are cytoplasmic. Although conserved, no function for the dipepide has been found (Barth, et al., 1992).

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3' Untranslated Sequence

Terminal 19 nt are conserved (Ou, et al., 1981; Pfeffer, et al., 1998) and are required for DI replication (Levis, et al., 1986). They are presumed to be recognized for initiation of (-) strand synthesis (Strauss et al. 1990).

Mutations in the terminal 19 nt decrease growth rates (Kuhn, et al., 1990).

The 3' UT also contains repeats (Ou, et al., 1982; Pfeffer, et al., 1998). Their function, if any, is unknown. Their deletion causes decreased virus yields, especially in mosquito cells (Kuhn, et al., 1990).

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Poly-A Sequence

References

Frothingham (1955). "Tissue culture applied to the study of Sindbis virus." Am. J. Trop. Med. Hyg. 4: 863-871.

Hurlbut, H. S. (1953). "The experimental transmission of coxsackie-like viruses by mosquitoes." J. Egypt. Med. Assoc. 36: 495-498.

Strauss, JH, RJ Kuhn, HGM Niesters and EG Strauss (1990). Functions of the 5'-terminal and 3'-terminal sequences of the Sindbis virus genome in replication. New Aspects of Positive-Strand RNA Viruses. MA Brinton and FX Heinz. Washington, D.C., American Society of Microbiology: 61-66.

Taylor, RM and HS Hurlbut (1953). "Isolation of coxsackie-like viruses from mosquitoes." J. Egypt. Med. Assoc. 36: 489-494.

Taylor, RM, HS Hurlbut, TH Work, JR Kingsbury and TE Frothingham (1955). "Sindbis virus: A newly recognized arthropod-transmitted virus." Am. J. Trop. Med. Hyg. 4: 844-846.