Cell membrane Complex Enzyme Eukaryotic cell or cell component Microbe-host cell complex Microorganism or its component Other Other -- ion Pathway or action Protein Protein or gene Protein or gene complex Protein or protein complex Bacterial membrane or virus envelope Cell membrane Cytoplasm Eukaryotic cell or cell component Extracellular Golgi Golgi membrane Intercellular Intracellular Mitochondria Nucleocapsid/Cytoplasm Organelle Organelle -- Cell membrane Organelle -- Endoplasmic reticulum Organelle -- ER Organelle -- Golgi Organelle -- Golgi membrane Organelle -- Nucleus Organelle -- Phagolysosome Organelle -- Phagosome Organelle -- Ribosome Other Phagolysosome Phagosome Chaperone Defense, immunity protein Enzyme Enzyme activator Enzyme inhibitor Genomic S segment Infection Ligand binding or carrier Motor Nucleic acid binding Other Signal transducer Toxicity Transcription factor binding Transcription regulation Transporter Unknown IC Inferred by Curator IDA Inferred from Direct Assay IEA Inferred from Electronic Annotation IEP Inferred from Expression Pattern IGI Inferred from Genetic Interaction IMP Inferred from Mutant Phenotype IPI Inferred from Physical Interaction ISS Inferred from Sequence or Structural Similarity NAS Non-traceable Author Statement ND No biological Data available RCA inferred from Reviewed Computational Analysis TAS Traceable Author Statement NR Not Recorded Skoging-Nyberg U, Liljestrom P Journal of virology M-X-I motif of semliki forest virus capsid protein affects nucleocapsid assembly 2001 11312332 PubMed Strauss EG, Lenches EM, Strauss JH Journal of virology Molecular genetic evidence that the hydrophobic anchors of glycoproteins E2 and E1 interact during assembly of alphaviruses 2002 12239293 PubMed Loewy A, Smyth J, von Bonsdorff CH, Liljestrom P, Schlesinger MJ Journal of virology The 6-kilodalton membrane protein of Semliki Forest virus is involved in the budding process 1995 7983743 PubMed Strauss EG, Lenches EM, Strauss JH Journal of virology Molecular genetic evidence that the hydrophobic anchors of glycoproteins E2 and E1 interact during assembly of alphaviruses 2002 12239293 PubMed Hodgson LA, Ludwig GV, Smith JF Vaccine Expression, processing, and immunogenicity of the structural proteins of Venezuelan equine encephalitis virus from recombinant baculovirus vectors 19995 10195627 PubMed Brault AC, Powers AM, Holmes EC, Woelk CH, Weaver SC Journal of virology Positively charged amino acid substitutions in the e2 envelope glycoprotein are associated with the emergence of venezuelan equine encephalitis virus 2002 11799167 PubMed Suopanki J, Sawicki DL, Sawicki SG, Kaariainen L The Journal of general virology Regulation of alphavirus 26S mRNA transcription by replicase component nsP2 1998 9472615 PubMed Sawicki DL, Sawicki SG Archives of virology. Supplementum Alphavirus positive and negative strand RNA synthesis and the role of polyproteins in formation of viral replication complexes 1994 8032270 PubMed Netolitzky DJ, Schmaltz FL, Parker MD, Rayner GA, Fisher GR, Trent DW, Bader DE, Nagata LP The Journal of general virology Complete genomic RNA sequence of western equine encephalitis virus and expression of the structural genes 2000 10640553 PubMed Fata CL, Sawicki SG, Sawicki DL Journal of virology Modification of Asn374 of nsP1 suppresses a Sindbis virus nsP4 minus-strand polymerase mutant 2002 12163583 PubMed Froshauer S, Kartenbeck J, Helenius A The Journal of cell biology Alphavirus RNA replicase is located on the cytoplasmic surface of endosomes and lysosomes 1988 2904446 PubMed Frolov I, Schlesinger S Journal of virology Translation of Sindbis virus mRNA: analysis of sequences downstream of the initiating AUG codon that enhance translation 1996 8551579 PubMed Smerdou C, Liljestrom P Journal of virology Two-helper RNA system for production of recombinant Semliki forest virus particles 1999 9882310 PubMed Paredes A, Alwell-Warda K, Weaver SC, Chiu W, Watowich SJ Journal of virology Structure of isolated nucleocapsids from venezuelan equine encephalitis virus and implications for assembly and disassembly of enveloped virus 2003 12477868 PubMed Strauss JH, Strauss EG Microbiological reviews The alphaviruses: gene expression, replication, and evolution 1994 7968923 PubMed Bernard KA, Klimstra WB, Johnston RE Virology Mutations in the E2 glycoprotein of Venezuelan equine encephalitis virus confer heparan sulfate interaction, low morbidity, and rapid clearance from blood of mice 200010 11021998 PubMed Schlesinger S and Schlesinger MJ. Field's Virology, Fourth Edition Volume One Togaviridae: The viruses and their replication 2001 E2 glycoprotein Function: Ligand binding or carrier. The viral glycoprotein E2 is the protein that interacts with the cellular receptors (Schlesinger and Schlesinger, 2001). Convincing evidence for the importance of E2 in binding virus to the cell surface came from experiments in which Sindbis virus mutants unable to bind to chicken embryo fibroblasts were altered in the E2 protein (Schlesinger and Schlesinger, 2001). E2 is the putative viral attachment protein for VEE and thus, interacts with the host cell (Bernard et al., 2000).(Schlesinger)(Bernard et al., 2000) Cell membrane receptor Function: Ligand binding or carrier. The alphaviruses have an enormous range that comprises both invertebrate hosts (mosquitoes and other hematophagous insects that serve as vectors) and vertebrate hosts (many species of birds and mammals). Within their hosts they replicate in a wide variety of cells including neurons and glial cells, striate and smooth muscle cells, cells of lymphoid origin, synovial cells, and brown fate cells. The question then arises whether the viruses are using the same receptor throughout this wide host range; if so, the same receptor must be expressed on the surface of many different mosquito, avian, and mammalian cells. Studies to address this issue have suggested that alphaviruses use protein receptors; that different alphaviruses may use the same receptor or different receptors; that more than one receptor can be used by one virus, leading to cases in which the major receptors used by one virus to enter different cells are different; that the nature of the receptors used determines in part the virulence of the virus; and that one or a few amino acid changes to the envelope glycoproteins can lead to utilization of different sets of receptors (Strauss and Strauss, 1994). Evidence is developing that the set of cellular receptors that an alphavirus can use can be altered by relatively minor changes in E1 and E2 (Strauss and Strauss, 1994).The mammalian receptor for VEE remains unknown, but a laminin-binding protein was identified as the putative host cell receptor for VEE in mosquito cell culture. A similar molecule on mammalian cells, the high-affinity laminin receptor, was suggested as a receptor for another alphavirus, Sindbis virus (SIN). SIN can also interact with heparan sulfate on mammalian cells. However, it has been demonstrated for SIN that HS binding is a cell culture adaptation and is correlated with attenuation in mice (Bernard et al., 2000).(Strauss et al., 1994)(Bernard et al., 2000) Intracellular virion Function: . Extensive data has been presented indicating that the normal pathway of entry for alphaviruses is endocytosis in clathrin-coated vesicles followed by transfer to endosomes, where the low pH leads to conformation reorganization of the E1-E2 heterodimer such that the fusion domain in E1 is exposed and the virus envelope fuses with the endosomal membrane (Strauss and Strauss, 1994).(Strauss et al., 1994) Fused virion Function: . Extensive data has been presented indicating that the normal pathway of entry for alphaviruses is endocytosis in clathrin-coated vesicles followed by transfer to endosomes, where the low pH leads to conformation reorganization of the E1-E2 heterodimer such that the fusion domain in E1 is exposed and the virus envelope fuses with the endosomal membrane (Strauss and Strauss, 1994). Low pH in the endosome is essential for infection to occur (Strauss and Strauss, 1994). Studies with Semliki Forest and Sindbis viruses have demonstrated that the low-pH environment of the endosome leads to a dissociation of the E1/E2 heterodimer and a concomitant trimerization of the E1 subunits, which acquire new epitopes and become trypsin resistant. These alterations suggest that the E1 trimer is the fusion-active form of the protein, with a structure possibly analogous to the low pH induced structure of the influenza virus HA2 protein (Schlesinger and Schlesinger, 2001).(Strauss et al., 1994)(Schlesinger) Uncoated virus Function: . Fusion leads to release of the nucleocapsid into the cytoplasm and must be followed by an uncoating event to permit the RNA to become accessible to ribosomes for initiation of translation. Several reports, based on in vivo and in vitro experiments, suggested that the binding of the nucleocapsid to ribosomes triggers the uncoating process. The capsid protein itself appears to have a ribosome-binding site between amino acids 94 and 106 of the capsid sequence, which is in the same region of the protein that interacts with viral RNA during encapsidation (Schlesinger and Schlesinger, 2001). The molecular details of this cycle of assembly, budding, infection, uncoating, and genome release remain largely unknown (Paredes et al., 2003).(Schlesinger)(Paredes et al., 2003) Genomic RNA Function: . The genome of alphaviruses is a capped and polyadenylated positive-strand RNA molecule of 11 to 12 kb (Smerdou and Liljestrom, 1999). In infected cells, the genome functions directly as an mRNA but only for the nsPs, which are synthesized as a polyprotein that is cleaved to produce four polypeptides. These proteins are required for the replication of the viral genomic RNA and for the transcription of the subgenomic RNA which is identical in sequence to the 3' one-third of the genome and codes for the viral structural proteins (Frolov and Schlesinger, 1996).(Smerdou et al., 1999)(Frolov et al., 1996) Nonstructural polyprotein Function: . Replication and transcription of alphavirus RNAs occur on cellular membranes (Schlesinger and Schlesinger, 2001). The RNA replicase of Semliki Forest and Sindbis virus (two closely related alphaviruses) is located in complex ribonucleoprotein structures associated with the cytoplasmic surface of modified secondary lysosomes and endosomes. These nucleoprotein complexes often form a bridge between the membrane of the endocytic vacuole and the rough endoplasmic reticulum where the synthesis of the structural proteins of these viruses occurs (Froshauer et al., 1988).The four alphavirus nonstructural proteins (nsPs) are numbered according to their gene order and are translated from the 5' two-thirds of the genome as polyprotein P123 or P1234 (Fata et al., 2002). The nonstructural proteins (nsP1, nsP2, nsP3 and nsP4) are also synthesized as a polyprotein and processed into the four nsPs by an nsP2 protease. Two versions of the nonstructural polyprotein are synthesized in alphavirus-infected cells, due to frequent read through of an opal codon between the nsP3 and nsP4 genes in several alphaviruses. The nsPs function in a complex with host factors to replicate the genome and transcribe the subgenomic mRNA (Netolitzky et al., 2000).(Schlesinger)(Froshauer et al., 1988)(Fata et al., 2002)(Netolitzky et al., 2000) nsP1 Function: . nsP1 appears to be specifically required for initiation of (or continuation of) synthesis of minus-strand RNA (Strauss and Strauss, 1994). nsP1 is also thought to be the enzyme, or a component of the enzyme, that caps the genomic and subgenomic RNAs during transcription (Strauss and Strauss, 1994). A third activity of nsP1 is its modulation of the activity of the proteinase activity of nsP2. Polyproteins containing nsP1 cleave the bond between nsP2 and nsP3 very poorly (Strauss and Strauss, 1994). The nsP1 protein appears to be involved in the association of the alphavirus replicase to membranes (Schlesinger and Schlesinger, 2001).Replication and transcription of alphavirus RNAs occur on cellular membranes (Schlesinger and Schlesinger, 2001). The RNA replicase of Semliki Forest and Sindbis virus (two closely related alphaviruses) is located in complex ribonucleoprotein structures associated with the cytoplasmic surface of modified secondary lysosomes and endosomes. These nucleoprotein complexes often form a bridge between the membrane of the endocytic vacuole and the rough endoplasmic reticulum where the synthesis of the structural proteins of these viruses occurs (Froshauer et al., 1988).(Strauss et al., 1994)(Schlesinger)(Froshauer et al., 1988) nsP23 Function: . The first cleavage of P123 produces nsP1 and P23, and complexes containing these products would be expected to exist, at least transiently (Strauss and Strauss, 1994). The possibility that a complex containing nsP1, nsP23 and nsP4 also functions as a minus strand replicase has not been ruled out. Complexes containing nsP1 and P23 are more efficient in plus-strand synthesis than P123-containing complexes but not as efficient as the complex containing the fully processed nonstructural proteins, and it is possible that they represent an intermediate state for the replicase in which both plus and minus stands can be made (Strauss and Strauss, 1994).Replication and transcription of alphavirus RNAs occur on cellular membranes (Schlesinger and Schlesinger, 2001). The RNA replicase of Semliki Forest and Sindbis virus (two closely related alphaviruses) is located in complex ribonucleoprotein structures associated with the cytoplasmic surface of modified secondary lysosomes and endosomes. These nucleoprotein complexes often form a bridge between the membrane of the endocytic vacuole and the rough endoplasmic reticulum where the synthesis of the structural proteins of these viruses occurs (Froshauer et al., 1988).(Strauss et al., 1994)(Schlesinger)(Froshauer et al., 1988) nsP2 Function: . The nsP2 sequence encodes two enzymatic activities. The N-half of the Sindbis protein has sequence motifs for helicase and NTP-binding activity that are conserved within the superfamily; hepatitis E, rubella, and furoviruses have a helicase domain partly resembling that of coronaviruses (Sawicki and Sawicki, 1994). The C-half of the Sindbis nsP2 sequence is encoded in genomes of animal virus members of the superfamily and contains a papain-like protease activity that has been shown to be responsible for processing the viral nonstructural polyproteins and allowing RNA replication (Sawicki and Sawicki, 1994). For alphaviruses, nsP2 also plays an essential role in the internal initiation of transcription of the subgenomic mRNA and in the regulation of negative strand synthesis. Recently it was found that nsP2 contains a nuclear localiztion signal and may contribute to the shut down of host transcription in infected cells (Sawicki and Sawicki, 1994).Replication and transcription of alphavirus RNAs occur on cellular membranes (Schlesinger and Schlesinger, 2001). The RNA replicase of Semliki Forest and Sindbis virus (two closely related alphaviruses) is located in complex ribonucleoprotein structures associated with the cytoplasmic surface of modified secondary lysosomes and endosomes. These nucleoprotein complexes often form a bridge between the membrane of the endocytic vacuole and the rough endoplasmic reticulum where the synthesis of the structural proteins of these viruses occurs (Froshauer et al., 1988).(Sawicki et al., 1994)(Schlesinger)(Froshauer et al., 1988) nsP3 Function: . The N-terminus of alphavirus nsP3 contains a sequence motif of unknown function found also in the rubella and hepatitis E genome and related to a domain in coronaviruses. A role of this"X" domain in the regulation of polyprotein processing has been suggested. For alphaviruses, only the N-half of the nsP3 is a component of transcriptionally active replication complexes and plays a role in the formation of the replication complex for negative strand synthesis (Sawicki and Sawicki, 1994). The functions of nsP3 have remained undefined, although it was necessary for the RNA positive phenotype of Sindbis virus (Suopanki et al., 1998).Replication and transcription of alphavirus RNAs occur on cellular membranes (Schlesinger and Schlesinger, 2001). The RNA replicase of Semliki Forest and Sindbis virus (two closely related alphaviruses) is located in complex ribonucleoprotein structures associated with the cytoplasmic surface of modified secondary lysosomes and endosomes. These nucleoprotein complexes often form a bridge between the membrane of the endocytic vacuole and the rough endoplasmic reticulum where the synthesis of the structural proteins of these viruses occurs (Froshauer et al., 1988).(Sawicki et al., 1994)(Suopanki et al., 1998)(Schlesinger)(Froshauer et al., 1988) nsP4 Function: . Alphavirus nsP4 contains conserved sequences that encode the RNA-dependent RNA polymerase domain. It functions in elongation of both positive and negative strand RNAs, in promoter recognition, and in host range (Sawicki and Sawicki, 1994). NsP4 is the catalytic subunit of the polymerase (Suopanki et al., 1998).Replication and transcription of alphavirus RNAs occur on cellular membranes (Schlesinger and Schlesinger, 2001). The RNA replicase of Semliki Forest and Sindbis virus (two closely related alphaviruses) is located in complex ribonucleoprotein structures associated with the cytoplasmic surface of modified secondary lysosomes and endosomes. These nucleoprotein complexes often form a bridge between the membrane of the endocytic vacuole and the rough endoplasmic reticulum where the synthesis of the structural proteins of these viruses occurs (Froshauer et al., 1988).(Sawicki et al., 1994)(Suopanki et al., 1998)(Schlesinger)(Froshauer et al., 1988) Replication complex Function: . After infection by alphaviruses, RNA replicase complexes are assembled on modified endosomal and lysosomal membranes. Attempts to purify the replicase complexes have met with only limited success: these partially purified complexes are membrane associated; contain viral nonstructural proteins nsP1, nsPe, nsP3 and nsP4, as well as a cellular 120 kDa protein and other cellular proteins; and are capable of limited RNA synthesis. These complexes are formed early in infection and are stable throughout the infection cycle (Strauss and Strauss, 1994).(Strauss et al., 1994) Negative strand RNA Function: . During RNA replication, a minus-strand copy of the genome RNA is produced that is an exact complement except for the presence of an unpaired G at the 3' end. This full-length minus strand serves as a template not only for the production of additional genomic RNA, but also for transcription of the subgenomic RNA (Strauss and Strauss, 1994). In vertebrate cells, minus-strand RNA is made only early after infection. Minus-strand RNA is synthesized until about 6 hours after infection at 30 C or about 4 hours at 40 C, and its synthesis then ceases abruptly; after this time, only plus-strand RNAs are produced (Strauss and Strauss, 1994).Replication and transcription of alphavirus RNAs occur on cellular membranes (Schlesinger and Schlesinger, 2001). The RNA replicase of Semliki Forest and Sindbis virus (two closely related alphaviruses) is located in complex ribonucleoprotein structures associated with the cytoplasmic surface of modified secondary lysosomes and endosomes. These nucleoprotein complexes often form a bridge between the membrane of the endocytic vacuole and the rough endoplasmic reticulum where the synthesis of the structural proteins of these viruses occurs (Froshauer et al., 1988).(Strauss et al., 1994)(Schlesinger)(Froshauer et al., 1988) Genomic positive-strand RNA Function: . During RNA replication, a minus-strand copy of the genome RNA is produced that is an exact complement except for the presence of an unpaired G at the 3' end. This full-length minus strand serves as a template not only for the production of additional genomic RNA, but also for transcription of the subgenomic RNA (Strauss and Strauss, 1994). In vertebrate cells, minus-strand RNA is made only early after infection. Minus-strand RNA is synthesized until about 6 hours after infection at 30 C or about 4 hours at 40 C, and its synthesis then ceases abruptly; after this time, only plus-strand RNAs are produced (Strauss and Strauss, 1994).Replication and transcription of alphavirus RNAs occur on cellular membranes (Schlesinger and Schlesinger, 2001). The RNA replicase of Semliki Forest and Sindbis virus (two closely related alphaviruses) is located in complex ribonucleoprotein structures associated with the cytoplasmic surface of modified secondary lysosomes and endosomes. These nucleoprotein complexes often form a bridge between the membrane of the endocytic vacuole and the rough endoplasmic reticulum where the synthesis of the structural proteins of these viruses occurs (Froshauer et al., 1988).(Strauss et al., 1994)(Schlesinger)(Froshauer et al., 1988) 26 S mRNA Function: . The 26S RNA is identical in sequence to the 3'-third of the genome and codes for the structural proteins of the virus. Transcription of 26S RNA takes place from an internal promoter (Suopanki et al., 1998).Replication and transcription of alphavirus RNAs occur on cellular membranes (Schlesinger and Schlesinger, 2001). The RNA replicase of Semliki Forest and Sindbis virus (two closely related alphaviruses) is located in complex ribonucleoprotein structures associated with the cytoplasmic surface of modified secondary lysosomes and endosomes. These nucleoprotein complexes often form a bridge between the membrane of the endocytic vacuole and the rough endoplasmic reticulum where the synthesis of the structural proteins of these viruses occurs (Froshauer et al., 1988).(Suopanki et al., 1998)(Schlesinger)(Froshauer et al., 1988) Structural polypeptide Function: . The negative-sense RNA is used for the generation of genomic RNA as well as a subgenomic mRNA (26S) that is homologous to the 3' one-third of the genome. The subgenomic RNA is translated directly into a structural polyprotein in ER that is proteolytically cleaved into the capsid, E2, and E1 envelope glycoproteins (Brault et al., 2002B).(Brault et al., 2002) Capsid protein Function: . As translation of the 26S message proceeds, the capsid protein is removed by its autoprotease activity and assumes a cytoplasmic localization (Hodgson et al., 1999). Capsid protein binds specifically to the viral genomic RNA, and this binding is believed to be important for the initiation of nucleocapsid formation and possibly for the stimulation of RNA synthesis (Strauss and Strauss, 1994).(Hodgson et al., 1999)(Strauss et al., 1994) PE2/6K/E1 polyprotein Function: . PE2, a precursor to E2, and E1 are synthesized on the rough endoplasmic reticulum as a precursor polyprotein and are cleaved by host cell signalase, releasing a small peptide called 6K (Strauss et al., 1995).(Strauss et al., 2002) PE2 protein Function: . PE2, a precursor to E2, and E1 are synthesized on the rough endoplasmic reticulum as a precursor polyprotein and are cleaved by host cell signalase, releasing a small peptide called 6K (Strauss et al., 1995). Both PE2 and E1 are type I integral membrane proteins that have a membrane-spanning anchor at or near the C terminus (Strauss and Strauss, 1994).(Strauss et al., 2002)(Strauss et al., 1994) 6K protein Function: . The two genes encoding the glycoproteins of the alphaviruses are separated by a sequence of 155 nucleotides that codes for a very hydrophobic 6 kDa polypeptide called 6K. This small protein is expressed as part of the polyprotein translated from a subgenomic 26S mRNA containing all the structural genes for these viruses, and the 6K protein can be detected shortly after its synthesis in amounts equivalent to those of the virus capsid proteins and the glycoprotein. During translocation of the glycoproteins across the membrane of the endoplasmic reticulum, the signal peptidase of the infected cell frees the 6K protein from the nascent polyprotein. These events lead to a membrane-topological orientation for the 6K protein that has the amino terminus in the ER lumen followed by a transmembrane segment, then a short sequence containing a cluster of cysteines at the cytoplasmic face of the membrane and then another transmembrane segment and the carboxyl terminus of 6K in the ER lumen. This latter segment has been shown to function as a signal sequence for translocation of the virus E1 glycoprotein (Loewy et al., 1995). The 6K peptide is co-translationally removed by signalase cleavages at both its N and C termini, resulting in the separation of the PE2 and E1 glycoproteins (Hodgson et al., 1999).(Loewy et al., 1995)(Hodgson et al., 1999) E1 protein Function: . PE2, a precursor to E2, and E1 are synthesized on the rough endoplasmic reticulum as a precursor polyprotein and are cleaved by host cell signalase, releasing a small peptide called 6K (Strauss et al., 1995). Both PE2 and E1 are type I integral membrane proteins that have a membrane-spanning anchor at or near the C terminus (Strauss and Strauss, 1994).(Strauss et al., 2002)(Strauss et al., 1994) PE2/6K/E1 complex Function: . The E1, E2 (in the form of its precurosor p62), and 6K proteins form heterotrimers prior to exit from the endoplasmic reticulum (Lowey et al., 1995). The pE2, E1, and 6K proteins appear to move as a cohort through the secretory vesicles from their site of synthesis on the endoplasmic reticulum to their ultimate location at the plasma membrane of the cell. This process takes about 30 minutes at physiologic temperatures and leads to several posttranslational modifications(Schlesinger and Schlesinger, 2001).(Loewy et al., 1995)(Schlesinger) PE2/6K/E1 complex Function: . The pE2, E1, and 6K proteins appear to move as a cohort through the secretory vesicles from their site of synthesis on the endoplasmic reticulum to their ultimate location at the plasma membrane of the cell. This process takes about 30 minutes at physiologic temperatures and leads to several posttranslational modifications(Schlesinger and Schlesinger, 2001).(Schlesinger) E2/6K/E1 complex Function: . PE2, a precursor to E2, and E1 are synthesized on the rough endoplasmic reticulum as a precursor polyprotein and are cleaved by host cell signalase, releasing a small peptide called 6K . PE2 and E1 quickly associate to form a heterodimer that is transported through the Golgi apparatus to the cell plasma membrane. During transport, PE2 is cleaved to E2 and the hydrophobic domains of both E2 and E1 are fatty acylated (Strauss et al., 2002).During transport to the cell surface, alphavirus glycoproteins are potentially exposed to slightly acidic pH which could trigger the low-pH-induced changes in the E2-E1 heterodimer, thereby inactivating it. The greater stability of PE2-E1 heterodimer supports a model in which the stable PE2-E1 heterodimers are transported through the cell and the cleavage of PE2 just before arrival at the cell surface activates virus infectivity (Strauss and Strauss, 1994).The E1, E2 (in the form of its precurosor p62), and 6K proteins form heterotrimers prior to exit from the endoplasmic reticulum. During transport to the Golgi vesicles, the cysteines in 6K are modified by covalent attachment of palmitic acid. Although the 6K protein is on the surface of infected cells and complexed with E1-E2 heterodimers, very little 6K is incorporated into the budded particles (Loewy et al., 1995).(Strauss et al., 2002)(Strauss et al., 1994)(Loewy et al., 1995) E3 protein Function: . Cleavage of PE2 is effected by a host cell proteinase and is a late event in vertebrate cells, occurring after the proteins reach the trans Golgi network but before arrival at the cell surface, but has been reported to be an early and continuous event in mosquito cells (Strauss and Strauss, 1994).During transport, PE2 is cleaved to E32 and E2 by furin or a furin-like enzyme, resulting in the formation of E2-E1 heterodimers and the activation of the fusion activity of E1, which is required for virus entry. In most alphaviruses, E3 is not retained in the complex and is not a component of the virion (Strauss et al., 1995).(Strauss et al., 1994)(Strauss et al., 2002) E2/6K/E1 complex Function: . During transport to the cell surface, alphavirus glycoproteins are potentially exposed to slightly acidic pH which could trigger the low-pH-induced changes in the E2-E1 heterodimer, thereby inactivating it. The greater stability of PE2-E1 heterodimer supports a model in which the stable PE2-E1 heterodimers are transported through the cell and the cleavage of PE2 just before arrival at the cell surface activates virus infectivity (Strauss and Strauss, 1994).The E1, E2 (in the form of its precurosor p62), and 6K proteins form heterotrimers prior to exit from the endoplasmic reticulum. During transport to the Golgi vesicles, the cysteines in 6K are modified by covalent attachment of palmitic acid. Although the 6K protein is on the surface of infected cells and complexed with E1-E2 heterodimers, very little 6K is incorporated into the budded particles (Loewy et al., 1995). The viral glycoprotein E2 is the protein that interacts with the cellular receptors (Schlesinger and Schlesinger, 2001).(Strauss et al., 1994)(Loewy et al., 1995)(Schlesinger) Nucleocapsid Function: . Nucleocapsids assembled in the cell cytopolasm are thought to diffuse freely to the plasma membrane, where they are bound by the viral glycoproteins present at the cell surface (Strauss and Strauss, 1994). The assembly of nucleocapsids includes both oligomerization of the capsid protein and selection of the genomic RNA for encapsidation. Although the subgenomic RNA is present in molar excess, it is the genomic RNA that is packaged into nucleocapsids and virions (Schlesinger and Schlesinger, 2001).(Strauss et al., 1994)(Schlesinger) Budding virion Function: . The final stages in the replication of alphaviruses occur when nucleocapsids interact with the host-cell plasma membrane at sites occupied by viral transmembrane glycoproteins. This nucleation event leads to binding of additional virus glycoproteins and a bending of the membrane around the nucleocapsid until the bilayers meet and fuse to release the enveloped particle (Schleinger and Schlesinger, 2001). Several independent studies have led to the conclusion that most of the viral glycoprotein present at the cell surface is bound to nucleocapsids after the first few hours of infection (Strauss and Strauss, 1994).The current view is that new virus particles are formed at the plasma membrane via interactions between intracellular capsid proteins and the cytoplasmic tails of the E2 spike proteins. In this model, an Y-X-L motif in the cytoplasmic domain of E2 interacts with a defined hydrophobic cavity of the capsid protein (Skoging-Nyberg and Liljestrom, 2001).(Schlesinger)(Strauss et al., 1994)(Skoging-Nyberg et al., 2001) Virion Function: . The molecular details of this cycle of assembly, budding, infection, uncoating, and genome release remain largely unknown (Paredes et al., 2003). Infection of vertebrate cells by most alphaviruses leads to inhibition of host-cell protein synthesis and cell death. Mechanisms that have been proposed for shut-off of host-cell proteins synthesis include inhibition of host factors required for protein synthesis, with preferential translation of viral mRNAs, changes in the ionic environment of infected cells, which favors translation of viral RNAs, and direct inhibition by Capsid protein (Schlesinger and Schlesinger, 2001).Cell death in alphavirus-infected vertebrate cells occurs by apoptosis, but in mosquito cells, the mechanism appears to be different and may more closely resemble necrosis (Schlesinger and Schlesinger, 2001).(Paredes et al., 2003)(Schlesinger) Signalase Function: Enzyme. The 6K peptide is co-translationally removed in ER by signalase cleavages at both its N and C termini, resulting in the separation of the PE2 and E1 glycoproteins (Hodgson et al., 1999).(Hodgson et al., 1999) Signalase Function: Enzyme. The 6K peptide is co-translationally removed in ER by signalase cleavages at both its N and C termini, resulting in the separation of the PE2 and E1 glycoproteins (Hodgson et al., 1999).(Hodgson et al., 1999) Furin-like enzyme Function: Enzyme. During transport, PE2 is cleaved to E32 and E2 by furin or a furin-like enzyme, resulting in the formation of E2-E1 heterodimers and the activation of the fusion activity of E1, which is required for virus entry (Strauss et al., 1995).(Strauss et al., 2002) Furin-like enzyme Function: Enzyme. During transport, PE2 is cleaved to E32 and E2 by furin or a furin-like enzyme, resulting in the formation of E2-E1 heterodimers and the activation of the fusion activity of E1, which is required for virus entry (Strauss et al., 1995).(Strauss et al., 2002) Replication complex Function: . After infection by alphaviruses, RNA replicase complexes are assembled on modified endosomal and lysosomal membranes. Attempts to purify the replicase complexes have met with only limited success: these partially purified complexes are membrane associated; contain viral nonstructural proteins nsP1, nsPe, nsP3 and nsP4, as well as a cellular 120 kDa protein and other cellular proteins; and are capable of limited RNA synthesis. These complexes are formed early in infection and are stable throughout the infection cycle (Strauss and Strauss, 1994).(Strauss et al., 1994)