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Molecular Biology VIRO: 00628 a0005 Molecular Biology Au3 K D Ryman and W B Klimstra, Louisiana State University Health Sciences Center at Shreveport, Shreveport, LA, USA S C Weaver, University of Texas Medical Branch, Galveston, TX, USA ã 2008 Elsevier Ltd. All rights reserved. Introduction s0005 Glossary g0005 Apoptosis A cascade of cellular responses to a The Togaviridae family includes the Alphavirus and Rubi- p0005 stimulus (e.g., virus infection) resulting in cell death. virus genera. The Alphavirus genus is comprised of 29 virus g0010 Cytopathic effect Destructive changes in cell species segregated into five antigenic complexes, most of morphology, structure, or metabolic processes which are transmitted between vertebrate hosts by mos- resulting from virus infection. quito vectors and are capable of replicating in a wide g0015 Full-length cDNA clone A copy of an RNA virus range of hosts including mammals, birds, amphibians, genome that has been reverse-transcribed into DNA reptiles, and arthropods. In contrast, the sole Rubivirus and placed into a subcloning vector. With positive- genus member is Rubella virus, which is limited to sense RNA viruses, infectious genomic RNA is human hosts and is primarily transmitted by respiratory, generally directly synthesized from bacteriophage congenital, or perinatal routes. Together, the viruses transcription promoters located upstream of the virus significant worldwide cause human and livestock disease. sequences. Alphavirus disease ranges from mild to severe febrile g0020 Host cell shut-off Interruption or cessation of host illness, to arthritis/arthralgia and fatal encephalitis, while transcription, translation, or other processes that Rubella virus is an important cause of congenital abnorm- occurs within virus-infected cells. alities and febrile illness frequently accompanied by g0025 Interferon A pro-inflammatory cytokine that is arthralgia/arthritis in the developing world. On the mole- produced after virus infection and stimulates antiviral cular level, the togaviruses are relatively uncomplicated, activities in cells expressing the IFNAR receptor small, enveloped virions with single-stranded, positive- complex. sense RNA genomes encoding two polyproteins, one g0030 Interferon-stimulated gene (ISG) A gene whose translated from the genomic RNA and the other from transcription is increased after interferon signaling. a subgenomic mRNA transcribed during replication. Some ISGs are directly antiviral and can inhibit virus However, their replication is very tightly regulated and transcription or translation. intimately associated with host cell metabolic processes. g0035 Positive-sense RNA genome The genomic Currently, an important aspect of togavirus research is material of the virus resembles a cellular messenger designed to exploit their molecular biology for therapeu- RNA and the open reading frame can be translated tic and gene therapy applications. Here, we summarize on host ribosomes. major aspects of togavirus structure, genome organization, g0040 Receptor Structures (e.g., protein, sulfated replicative cycle, and interactions with host cells. polysaccharide, phospholipid) found upon the surface of host cells that interact with virus attachment proteins and mediate attachment of virus particles to Virion Structure and Genome s0010 cell surfaces. These structures may also mediate Organization subsequent events in cell entry such as viral protein rearrangements leading to membrane fusion. Togavirus virions are small and enveloped, comprising an p0010 g0045 RepliconELSEVIER system A replication-competent but FIRSTicosahedral nucleocapsid PROOF composed of 240 capsid (C) propagation-incompetent form of a cloned virus. In protein monomers, cloaked in a lipid envelope studded many replicon systems, the replicon genome can be with membrane-anchored glycoprotein components, E1 packaged into a virus-like particle by trans- and E2. Their tightly packed, regular structure has been expression of structural proteins. revealed by high-resolution reconstruction of the parti- g0050 RNA-dependent RNA polymerase The primary cles by cryoelectron microscopy (Figure 1). The envelope RNA synthesis enzyme for RNA viruses which uses glycoproteins of alphaviruses are similarly arranged on viral genomic RNA as a template for transcription/ the outer surface of particles. E1 and E2 form a stable replication. heterodimer, and three heterodimers interact to form ‘spikes’ distributed on the virion surface in an icosahedral 1 VIRO: 00628 2 Togaviruses _ Molecular Biology 50-terminal two-thirds of the genome encode the four nonstructural proteins (nsPs 1–4); and (2) the 30 one-third of the genome encodes the three structural proteins (C, precursor E2 [PE2], and precursor E1 [6K/E1]). Over- all, the capped and polyadenylated 9.5 kB Rubella virus genome is similarly organized (Figure 2), but the 50-proximal open reading frame (ORF) encodes only two nsPs (P150 and P90) and three structural proteins are encoded by the 30 ORF (C, E1, and E2). (a) (b) Infection and Replication Cycle s0015 The togavirus genome encodes very few but, therefore, p0020 necessarily multifunctional proteins. The replication of (c) (d) these viruses is very tightly regulated and as little as a single nucleotide change can alter susceptibility to cellu- lar defense mechanisms. The following section outlines molecular aspects of togavirus infection and replication pertinent to the virus–cell interaction. Replication pro- cesses of viruses of the family Togaviridae have been most extensively studied for the alphaviruses, Sindbis virus (SINV) and Semliki Forest virus (SFV), and extrapolated to the other family members. (e) (f) f0005 Figure 1 Structure of New World (VEEV) and Old World (SINV) Virus Attachment, Entry, and Uncoating s0020 alphaviruses determined by image reconstructions of electron micrographs. Isosurface view along a threefold axis of VEEV The entry pathways of Rubella virus have undergone only p0025 (a) and SINV (b) reconstructions showing outer spike trimers limited study, but there is some evidence that a glycolipid (yellow) and envelope skirt region (blue). Isosurface molecule serves as an attachment receptor on some cell representations of VEEV (c) and SINV (d) nucleocapsids viewed types. The nature of the receptor for naturally circulating, along a threefold-symmetry axis. Cross-sections through VEEV (e) and SINV (f) perpendicular to the threefold axis and in plane ‘wild-type’ alphaviruses has been examined more ex- with a vertical fivefold axis showing trimers (yellow), skirt region tensively, but remains controversial, as is the case with (blue), virus membrane (red), nucleocapsid (green), and RNA many arthropod-borne viruses. Typically, arthropod-borne genome (white). Scale bar corresponds to 100 A˚ . Reproduced viruses are thought to interact either with a single, evolu- from Paredes et al. (2001) Journal of Virology 75: 9532–9537. tionarily conserved molecule expressed on most cells, or to utilize different receptors in different hosts and with lattice that mirrors the symmetry of the nucleocapsid. cells derived from different tissues. The 67 kD high- The regularity of the spike distribution is determined by affinity laminin receptor (HALR) has been identified as the direct hydrophobic interaction of the E2 glycoprotein an initial attachment receptor for cell culture-adapted, tail with a pocket on the surface of the C-protein. E2 laboratory strains of SINV and an antigenically related projects outward from the virion to form the spikes that molecule has similarly been identified for Venezuelan interact with host-cell attachment receptors, while the equine encephalitis virus (VEEV). In addition, receptor E1 glycoprotein, which mediates fusion with host cell activity for particular cell types has been attributed to membranes,ELSEVIER appears to lie parallel to the lipid envelope. FIRSTseveral other, as yet PROOF uncharacterized, proteins. However, The E2 and E1 proteins are glycosylated at one to four more recent studies have indicated that efficient cell positions, depending on the virus strain, during transit binding by many laboratory strains of alphaviruses is through the host-cell secretory apparatus. due to interaction with heparan sulfate (HS), a sulfated p0015 The encapsidated alphavirus genome consists of glycosaminoglycan molecule, and that this phenotype is a nonsegmented, single-stranded, positive-sense RNA conferred by positively charged amino acid substitutions 0 molecule of approximately 11–12 kB with a 5 -terminal in E2 that accompany adaptation to cultured cells. Cur- 0 methylguanylate cap and 3 polyadenylation, resembling rently, it is unclear whether or not the 67 kDa HALR or cellular mRNAs (Figure 2). The genome is divided into other identified receptor proteins act in concert with 0 0 two major regions flanked by the 5 and 3 nontranslated HS to bind cell culture-adapted alphavirus strains or if regions (NTRs) and divided by an internal NTR: (1) the any of these molecules function in the natural replication VIRO: 00628 T _ Molecular Biology 3 0 1 2 3 4 5 6 7 8 9 10 11 12 Kilobases (kB) Genomic promoter Subgenomic promoter Alphavirus 5 NTR 3 NTR nsP1nsP2 nsP3 nsP4 C PE2 6KE1 AAA Subgenomic promoter Rubivirus Genomic promoter 5 NTR 3 NTR C E2 E1 P150 P90 AAA 7-methylguanylate cap Conserved cis -acting sequence element (CSE) RNA helicase motif Region of homology RdRp replicase motif f0010 Figure 2 Comparison of Alphavirus (SINV) and Rubivirus (RUB) genome organization. Regions of nucleotide homology and
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