Rebecca Wattam 04-04-2003 ).83 The methods of cell invasion and viral multiplication within vertebrate cells are described for the family Arenaviridae, which includes Lassa Fever virus, Junin virus, Machupo virus, and Guanarito virus. wattam@vbi.vt.edu GP-1 Extracellular Ligand binding or carrier GP-1 is the peripheral membrane glycoprotein. It has 4 to 11 potential N-linked glycosylation sites. The GP-1 portion of the glycoprotein spike is responsible for initial binding to cellular receptors, and antibodies directed to epitopes on GP-1 can block viral binding and neutralize viral infectivity. GP-1 has only been described for LCMV, Lassa virus, Oliveros virus, and Mobala virus. Alpha dystroglycan Cell membrane Ligand binding or carrier Alpha dystroglycan is a cell surface protein. It is a highly versatile cellular receptor for proteins of the extracellular matrix that is fundamental in cell-mediated assembly and organization of basement membranes throughout the organism. It has broad tissue distribution, is highly conserved, and is also used by Mycobacterium leprae. Guanarito virus does not bind to this receptor. Virion Cell membrane Other The arenavirus binds to the alpha-dystroglycan cell surface protein. Virion, pH 6.2 Phagosome Other Following attachment, viruses are taken up in large (150-300 nm) smooth-walled vesicles that resemble phagocytic vesicles. This process is known as viropexis. This means that the virus membrane does not become part of the vesicle membrane. Virion, pH less than 6.2 Phagosome Other Intravesicular pH drops from 6.2 to 5.0. Initial evidence that arenaviruses are taken up and undergo a pH-dependent fusion event within host cells was provided by experiments showing that infection of cells by both the Old World arenavirus LCMV and the New World arenavirus Junin could by inhibited at the entry stage by agents that raise the enodosomal pH. Virion with GP-2 exposed Phagosome Other The change in pH causes GP-1 to become dissociated from the virion. It has been hypothesized that loss of GP-1 causes sequestered epitopes on GP-2 to be exposed, but the hypothetical ‘fusion protein’ that can mediated fusion of the virion with the vesicle membranes is currently unknown. The pH required to initiate this change is different depending upon the species. Virion bound to phagosome Phagosome Other The arenavirus membrane fuses with the vesicle membrane and virion contents are released into the cytoplasm (Meyer et al., 2002). Virion contents include both the L and S segments (which vary in the ratio that they are present in. Typically it is 2:5, with S always being the more abundant), host RNAs (these include the 5 and 5.8 ribosome RNAs and tRNAs, which are not essential for virion activity), Z mRNA, Z protein, and low levels of L protein (Meyer et al., 2002). Genomic segment L Cytoplasm Other The L RNA segment contains the L protein (polymerase) gene at the 3'-end in negative polarity and the zinc-binding (Z) protein at the 5'-end in message polarity (Peters et al., 1996). Genomic segment L Cytoplasm Other The L RNA segment contains the L protein (polymerase) gene at the 3'-end in negative polarity and the zinc-binding (Z) protein at the 5'-end in message polarity (Peters et al., 1996). Antigenomic segment L Cytoplasm Other Each RNA segment has an ambisence coding strategy, encoding two polypeptides in opposite orientation, separated by an intergenic region with a predicted folding of a stable hairpin structure. L mRNA Cytoplasm Other The L RNA, located at the 3’ end of the L genomic segment to the intergenic noncoding region, is transcribed. The gene for L is on the 3’ end of the L genomic segment and ends at an intergenic noncoding region (Meyer et al., 2002). Z mRNA Cytoplasm Other The L antigenomic segment is transcribed to produce Z mRNA. The gene for Z mRNA is on the 3’ end of the L antigenomic segment and ends at the intergenic noncoding region. L protein Cytoplasm Enzyme The L protein is the viral RNA-dependent RNA polymerase and has a reported molecular mass of 180-250 kDa. Motifs that are common to all RNA-dependent RNA polymerases and polymerases of segmented negative strand RNA viruses, as well as two regions found only in the polymerases of arenaviruses and Bunyaviruses are conserved in arenavirus L proteins. The L protein is a component of nucleocapsids, since polymerase activity has been associated with these structures. The L protein cannot be detected until 12-24 hours post-infection, and its concentration increases dramatically during acute infection (Buchmeier, 2002). Z protein Cytoplasm Unknown Z mRNA encodes an 11 kDa protein known as Z. The Z protein contains a RING finger motif and binds zinc. It may be directly involved with regulation of gene expression in infected cells (Meyer et al., 2002). It has been suggested that it is both a structural component of the viral nucleocapsid, and that it acts as a cofactor in viral replication and/or transcription (Lee and de la Torre, 1993) Genomic segment S Cytoplasm Other The S segment encodes the nucleocapsid protein (NP) in negative, antimessage sense at the 3'-end and the viral glycoprotein precursor, GP-C in message sense at the 5'-end (Peters et al., 1996). Genomic segment S Cytoplasm Other The S segment encodes the nucleocapsid protein (NP) in negative, antimessage sense at the 3'-end and the viral glycoprotein precursor, GP-C in message sense at the 5'-end (Peters et al., 1996). Antigenomic segment S Cytoplasm Other Each RNA segment has an ambisence coding strategy, encoding two polypeptides in opposite orientation, separated by an intergenic region with a predicted folding of a stable hairpin structure. NP mRNA Cytoplasm Other The NP RNA, located at the 3’ end of the S genomic segment to the intergenic noncoding region, is transcribed. The first transcript produced by the incoming nucleoprotein complex is NP. NP mRNAs can be detected as early as 2 hours post infection. GP-C mRNA Cytoplasm Other The GP-C RNA, located at the 3’ end of the S antigenomic segment to the intergenic noncoding region, is transcribed. NP protein Cytoplasm Other NP protein can be detected after 6-12 hours depending upon the multiplicity of infection and sensitivity of the assay (Buchmeier, 2002). NP protein accumulation either precedes replication, or the two processes are contemporary, suggesting that the first transcript produced by the incoming nucleoprotein complex is NP and that this early message is transcribed by the incoming viral RNA polymerase (Buchmeier, 2002). NP protein is the most abundant structural protein of arenaviruses with a molecular mass of 60-68 kDa. It is a major protein of nucleocapsids and associates with virion RNA to form the string of bead-like structures observed by electron microscopy. The structure of NP is consistent with a globular, basic protein with the primary function of protecting viral RNA within the cell, and it can become phosphorylated in the latter stages of acute infection. NP may be important in regulating relative levels of transcription and replication in infected cells (Meyer et al., 2002, Lee and de la Torre, 1993). Overall, the structure of NP is consistent with a globular, basic protein with the primary function of protecting the viral RNA within the cell (Buchmeier, 2002). GP-C protein Cytoplasm Other The primary translation product from the GP mRNA is GP-C and it is cleaved post-translationally to yield the mature glycoproteins GP-1 and GP-2. GP-C protein Endoplasmic reticulum Other GP-C enters the endoplasmic reticulum and Golgi apparatus, where it is glycosylated. GP-C protein Golgi Other GP-C enters the endoplasmic reticulum and Golgi apparatus, where it is glycosylated. Glycosylated GP-C protein Golgi Other GP-C enters the endoplasmic reticulum and Golgi apparatus, where it is glycosylated. GP-1 protein Golgi Other Cleavage occurs 75-90 minutes after synthesis (Burns and Buchmeier, 1993). The GP-C is cleaved after conversion of high mannose oligosaccharides precursors to complex carbohydrates (Buchmeier, 2002, Burns and Buchmeier, 1993). GP-C is cleaved by a novel endoprotease, SKI-1/S1P, which, unlike furin, belongs to the pyrolysin group of subtilases and cleaves at non-basic residues. GP-C is the first viral glycoprotein known to be processed by SKI-1/S1P, and so far only three cellular proteins have been identified as substrates, the sterol regulatory element binding protein SREBP, the transcription factor ATF6, and the neurotrophic factor BDNF. GP-C is cleaved in the endoplasmic reticulum or in the cis-Golgi stacks of which SKI-1/S1P is a resident protease (Lenz et al., 2001). GP-2 protein Golgi Other Cleavage occurs 75-90 minutes after synthesis (Burns and Buchmeier, 1993). The GP-C is cleaved after conversion of high mannose oligosaccharides precursors to complex carbohydrates (Buchmeier, 2002, Burns and Buchmeier, 1993). GP-C is cleaved by a novel endoprotease, SKI-1/S1P, which, unlike furin, belongs to the pyrolysin group of subtilases and cleaves at non-basic residues. GP-C is the first viral glycoprotein known to be processed by SKI-1/S1P, and so far only three cellular proteins have been identified as substrates, the sterol regulatory element binding protein SREBP, the transcription factor ATF6, and the neurotrophic factor BDNF. GP-C is cleaved in the endoplasmic reticulum or in the cis-Golgi stacks of which SKI-1/S1P is a resident protease (Lenz et al., 2001). GP-1 protein Golgi membrane Other Following glycosylation, GPC is cleaved and transported to the cell membrane. The final movement of glycoproteins to the plasma membrane is presumably mediated by a vesicular transport process, as has been observed with other enveloped viruses. GP-2 protein Golgi membrane Other Following glycosylation, GPC is cleaved and transported to the cell membrane. The final movement of glycoproteins to the plasma membrane is presumably mediated by a vesicular transport process, as has been observed with other enveloped viruses. GP-1 and GP-2 protein Cell membrane Ligand binding or carrier Following glycosylation, GPC is cleaved and transported to the cell membrane. GP-1 is the peripheral membrane glycoprotein with 4 to 11 potential N-linked glycosylation sites. GP-2 homotetramers bind through ionic interaction with GP-1 homotetramers, which comprise the globular head of glycoprotein spikes. Virion begins budding Cell membrane Other Membrane changes seen at the sites of virus budding include an increase in density of both membrane lamellae in discrete areas large enough to form a viral envelope. Surface projections were observed on the exterior of emerging virus particles. In some cells observed at late stages of infection, extensive regions of the plasma membrane were involved in virus assembly (Compans, 1993). Virion Extracellular Other Virions bud from the plasma membrane of acutely infected cells in regions that display thickening of the membrane and aggregation of ribosome-like structures adjacent to budding virions. The assembly process is poorly understood. Nothing is known about the assembly of viral nucleocapsids and how the interaction with the glycoproteins occurs (Southern, 1996). Moreover, there is a lack of specificity in the assembly process (Compans, 1993). Newly synthesized genomic RNAs, NP protein, and L protein are assembled into nucleocapsids intracellulary. Arenaviruses also package host-cell ribosomes into virion particles, but the number of these ribosomes is highly variable (Buchmeier, 2002). Hydrogen ions Cytoplasm Other Following internalization of virions within vesicles, the vesicles enter the endocytic pathway and are acidified as they move through the cell. The intravesicular pH decreases from 6.2 in early endosomes to approximately 5.0 by the time the terminal compartments of the pathway (lysosomes) are reached. Fusion of the membrane of enveloped viruses with the vesicle membrane is triggered as the pH drops; the time and location of penetration of different viruses are determined by the pH dependence of the fusion activity (Kunz et al., 2002). SKI-1/S1P Extracellular Enzyme GP-C is cleaved by a novel endoprotease, SKI-1/S1P, which, unlike furin, belongs to the pyrolysin group of subtilases and cleaves at non-basic residues. GP-C is the first viral glycoprotein known to be processed by SKI-1/S1P, and so far only three cellular proteins have been identified as substrates, the sterol regulatory element binding protein SREBP, the transcription factor ATF6, and the neurotrophic factor BDNF. GP-C is cleaved in the endoplasmic reticulum or in the cis-Golgi stacks of which SKI-1/S1P is a resident protease. It is therefore cleaved at an earlier stage of the exocytotic transport route than, for instance, the influenza virus hemagglutinin. SKI-1/S1P Extracellular Enzyme GP-C is cleaved by a novel endoprotease, SKI-1/S1P, which, unlike furin, belongs to the pyrolysin group of subtilases and cleaves at non-basic residues. GP-C is the first viral glycoprotein known to be processed by SKI-1/S1P, and so far only three cellular proteins have been identified as substrates, the sterol regulatory element binding protein SREBP, the transcription factor ATF6, and the neurotrophic factor BDNF. GP-C is cleaved in the endoplasmic reticulum or in the cis-Golgi stacks of which SKI-1/S1P is a resident protease. It is therefore cleaved at an earlier stage of the exocytotic transport route than, for instance, the influenza virus hemagglutinin. The peripheral membrane glycoprotein of the virus, GP-1, binds to a cell surface protein, alpha dystroglycan. Following attachment, viruses are taken up in large (150-300 nm) smooth-walled vesicles that resemble phagocytic vesicles. This process is known as viropexis where the virus membrane does not become part of the vesicle membrane (Kunz et al., 2002). The initial pH within this vesicle is 6.2 Intravesicular pH drops from 6.2 to 5.0. The change in pH causes GP-1 to become dissociated from the virion. The pH required to initiate this change is different depending upon the species (Kunz et al., 2002). It has been hypothesized that loss of GP-1 causes sequestered epitopes on GP-2 to be exposed (Kunz et al., 2002). It has been hypothesized that loss of GP-1 causes sequestered epitopes on GP-2 to be exposed, but the hypothetical ‘fusion protein’ that can mediated fusion of the virion with the vesicle membranes is currently unknown (Kunz et al., 2002). The arenavirus membrane fuses with the vesicle membrane and virion contents are released into the cytoplasm (Meyer et al., 2002). Included within the arenavirus virion is the large (L) RNA genomic segment. The arenavirus membrane fuses with the vesicle membrane and virion contents are released into the cytoplasm (Meyer et al., 2002). Included within the arenavirus virion is the small (S) RNA genomic segment. The large (L) RNA genomic segment is replicated to produce an L antigenomic segment. The gene for the protein L is located at the 3’ end of the L genomic segment to the intergenic noncoding region. It is transcribed from the L genomic segment. The large (L) RNA antigenomic segment is replicated to produce an L genomic segment. The gene for the Z protein is located at the 3’ end of the L antigenomic segment and ends at the intergenic noncoding region. It is transcribed. The gene for L is on the 3’ end of the L genomic segment and ends at an intergenic noncoding region (Meyer et al., 2002). The L protein cannot be detected until 12-24 hours post-infection, and its concentration increases dramatically during acute infection (Buchmeier, 2002). The L antigenomic segment is transcribed to produce Z mRNA, which encodes an 11 kDa protein that has been assigned both structural and regulatory roles in the infectious cycle. The small (S) RNA genomic segment is replicated to produce an S antigenomic segment. The gene for the protein NP is located at the 3’ end of the S genomic segment to the intergenic noncoding region. It is transcribed from the S genomic segment. The first transcript produced by the incoming nucleoprotein complex is NP, with NP mRNAs detected as early as 2 hours post infection. The small (S) RNA antigenomic segment is replicated to produce an S genomic segment. The gene for the GP-C protein is located at the 3’ end of the S antigenomic segment and ends at the intergenic noncoding region. It is transcribed. The NP mRNA is translated to produce the NP protein. NP protein can be detected after 6-12 hours depending upon the multiplicity of infection and sensitivity of the assay (Buchmeier, 2002). NP protein accumulation either precedes replication, or the two processes are contemporary, suggesting that the first transcript produced by the incoming nucleoprotein complex is NP and that this early message is transcribed by the incoming viral RNA polymerase (Buchmeier, 2002). The primary translation product from the GP mRNA , GP-C, is cleaved post-translationally to yield the mature glycoproteins GP-1 and GP-2. GP-C enters the endoplasmic reticulum GP-C enters the Golgi apparatus. GP-C is glycosylated. Cleavage of GPC to form GP-1 and GP-2 occurs later in the secretory pathway, between the medial and trans-Golgi network (Burns and Buchmeier, 1993). Cleavage occurs 75-90 minutes after synthesis (Burns and Buchmeier, 1993). The GP-C is cleaved after conversion of high mannose oligosaccharides precursors to complex carbohydrates (Buchmeier, 2002, Burns and Buchmeier, 1993).GP-C is cleaved in the endoplasmic reticulum by the cellular subtilase SKI-1/S1P, an enzyme that has so far been observed to be involved in cholesterol metabolism (Lenz et al., 2000). Cleavage of GPC to form GP-1 and GP-2 occurs later in the secretory pathway, between the medial and trans-Golgi network (Burns and Buchmeier, 1993). Cleavage occurs 75-90 minutes after synthesis (Burns and Buchmeier, 1993). The GP-C is cleaved after conversion of high mannose oligosaccharides precursors to complex carbohydrates (Buchmeier, 2002, Burns and Buchmeier, 1993). GP-C is cleaved in the endoplasmic reticulum by the cellular subtilase SKI-1/S1P, an enzyme that has so far been observed to be involved in cholesterol metabolism (Lenz et al., 2000). The final movement of glycoproteins to the plasma membrane is presumably mediated by a vesicular transport process, as has been observed with other enveloped viruses. The final movement of glycoproteins to the plasma membrane is presumably mediated by a vesicular transport process, as has been observed with other enveloped viruses. GP-1 is transported to the cell membrane. GP-1 is the peripheral membrane glycoprotein with 4 to 11 potential N-linked glycosylation sites. GP-2 is the integral membrane glycoprotein, which contains one to 4 potential N-linked glycosylation sites and a membrane spanning domain. The carboxyl terminus of GP-2 contains charged residues and is thought to interact with NP in the cytosol and within the interior of the virion. GP-2 homotetramers bind through ionic interaction with GP-1 homotetramers, which comprise the globular head of glycoprotein spikes. GP-1 is transported to the cell membrane. GP-1 is the peripheral membrane glycoprotein with 4 to 11 potential N-linked glycosylation sites. GP-2 is the integral membrane glycoprotein, which contains one to 4 potential N-linked glycosylation sites and a membrane spanning domain. The carboxyl terminus of GP-2 contains charged residues and is thought to interact with NP in the cytosol and within the interior of the virion. GP-2 homotetramers bind through ionic interaction with GP-1 homotetramers, which comprise the globular head of glycoprotein spikes. Membrane changes seen at the sites of virus budding include an increase in density of both membrane lamellae in discrete areas large enough to form a viral envelope. Surface projections were observed on the exterior of emerging virus particles. In some cells observed at late stages of infection, extensive regions of the plasma membrane were involved in virus assembly (Compans, 1993). Virions bud from the plasma membrane of acutely infected cells in regions that display thickening of the membrane and aggregation of ribosome-like structures adjacent to budding virions. The assembly process is poorly understood. Nothing is known about the assembly of viral nucleocapsids and how the interaction with the glycoproteins occurs (Southern, 1996). Moreover, there is a lack of specificity in the assembly process (Compans, 1993). Virions are released after completion of budding by pinching off at the plasma membrane (Compans, 1993). Newly synthesized genomic RNAs, NP protein, Z protein, and L protein are assembled into nucleocapsids intracellulary. Arenaviruses also package host-cell ribosomes into virion particles, but the number of these ribosomes is highly variable (Buchmeier, 2002). Aggregates of electro-dense granules are found free in the cytoplasm of infected cells, as well as in close opposition to the cytoplasmic face of the plasma membrane near patches of the glycoprotein spikes. After association of these structures with the plasma membrane, budding takes place by pinching off of the lipid bilayer and subsequent separation of the virion and plasma membranes (Buchmeier, 2002). The time from cell surface binding to new virions budding off takes between 16 and 24 hours (Meyer et al., 2002). Arenavirus pathway Lenz O, ter Meulen J, Klenk HD, Seidah NG, Garten W Proc Natl Acad Sci U S A 2001 98 22 12701 12705 Buchmeier MJ Arenaviruses: Protein structure and function Oldstone MBA 2002 159-173 Springer-Verlag Kunz S, Borrow P, Oldstone MBA Receptor structure, binding, and cell entry of arenaviruses Oldstone MBA 2002 111-137 Springer-Verlag Lee KJ, de la Torre JC Reverse genetics of arenaviruses Oldstone MBA 2002 175-193 Springer-Verlag Meyer BJ, de la Torre JC, Southern PJ Arenaviruses: Genomic RNAs, transcription, and replication Oldstone MBA 2002 139-157 Springer-Verlag Burns JW, Buchmeier MJ Glycoproteins of the arenaviruses Salvato MS 1993 17-35 Plenum Press Compans RW Arenavirus ultrastructure and morphogenesis Salvato MS 1993 3-16 Plenum Press Kolakofsky D, Garcin D The unusual mechanism of arenavirus RNA synthesis Salvato MS 1993 103-112 Plenum Press Southern PJ Arenaviridae: the viruses and their replication Fields BN, Knipe DM, Howley PM 1996 1505-1519 Lippincott-Raven Peters CJ, Buchmeier M, Rollin PE, Ksiazek TG Arenaviruses Fields BN, Knipe DM, Howley PM 1996 1521-1551 Lippincott-Raven TEXT TEXT TEXT TEXT TEXT TEXT TEXT TEXT

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