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Replication of cowpea mosaic virus (CPMV) is associated with small membranous vesicles that are induced upon infection. The effect of CPMV replication on the morphology and distribution of the endomembrane system in living plant cells was studied by expressing green fluorescent protein (GFP) targeted to the endoplasmic reticulum (ER) and the Golgi membranes. CPMV infection was found to induce an extensive proliferation of the ER, whereas the distribution and morphology of the Golgi stacks remained unaffected. Immunolocalization experiments using fluorescence confocal microscopy showed that the proliferated ER membranes were closely associated with the electron-dense structures that contain the replicative proteins encoded by RNA1. Replication of CPMV was strongly inhibited by cerulenin, an inhibitor of de novo lipid synthesis, at concentrations where the replication of the two unrelated viruses alfalfa mosaic virus and tobacco mosaic virus was largely unaffected. These results suggest that proliferating ER membranes produce the membranous vesicles formed during CPMV infection and that this process requires continuous lipid biosynthesis.Many positive-stranded RNA viruses modify intracellular membranes of their host cells to create a membrane compartment in which RNA replication takes place. Modifications include proliferation and reorganization of different membranes, including the early and late endomembrane system (26,36,44,54), the nuclear envelope (11), the peroxisomal membrane (7), the chloroplasts (49), and the mitochondrial membrane (7). Furthermore, the importance of membranes for viral replication is evident from the observation that the activity of most purified viral RNA-dependent RNA polymerases (RdRps) depends on the presence of membranes and/or phospholipids (27,32,59). It was proposed that the membranes play both a structural role and a functional role in the replication complex. Although the modification of intracellular membranes seems an essential part of the viral replicative cycle, little is known about the mechanisms by which the virus converts intracellular membranes for its own use.Cowpea mosaic virus (CPMV), a bipartite positive-stranded RNA virus, is the type member of the comoviruses and bears strong resemblance to animal picornaviruses both in gene organization and in amino acid sequence of replicative proteins (1, 15). Both RNA1 and RNA2 express large polyproteins, which are proteolytically cleaved into the different cleavage products by the 24-kDa (24K) protease (Fig. 1). The proteins encoded by RNA1 are necessary and sufficient for replication, whereas RNA2 codes for the capsid proteins and the movement protein.Upon infection of cowpea plants with CPMV, a typical cytopathological structure is formed adjacent to the nucleus, consisting of an amorphous matrix of electron-dense material which is traversed by rays of small membranous vesicles (10). The membranous vesicles are closely associated with CPMV RNA replication, as was revealed by autoradiography in conjunction with electron mic...

In cowpea plant cells infected with cowpea mosaic virus, tubular structures containing virus particles are formed in the plasmodesmata between adjacent cells; these structures are supposedly involved in cell-to-cell spread of the virus. Here we show that similar tubular structures are also formed in cowpea protoplasts, from which the cell wall and plasmodesmata are absent. Between 12 and 21 h post-inoculation, tubule formation starts in the periphery of the protoplast at the level of the plasma membrane. Upon assembly, the viruscontaining tubule is enveloped by the plasma membrane and extends into the culture medium. This suggests that the tubule has functional polarity and makes it likely that a tubule 'grows' into a neighbouring cell in vivo. On average, 75 % of infected protoplasts were shown to possess tubular structures extending from their surface. The tubule wall was 3 to 4 nm thick and they were up to 20 gm in length, as shown by fluorescent light microscopy and negative staining electron microscopy. By analogy to infected plant cells, both the viral 58K/48K movement and capsid proteins were located in these tubules, as determined by immunofluorescent staining and immunogold labelling using specific antisera against these proteins. These results demonstrate that the formation of tubules is not necessarily dependent on the presence of plasmodesmata or the cell wall, and that they are composed, at least in part, of virus-encoded components.

From 1992 onwards, outbreaks of a previously unknown illness have been reported in Asian seabass (Lates calcarifer) kept in maricultures in Southeast Asia. The most striking symptom of this emerging disease is the loss of scales. It was referred to as scale drop syndrome, but the etiology remained enigmatic. By using a next-generation virus discovery technique, VIDISCA-454, sequences of an unknown virus were detected in serum of diseased fish. The near complete genome sequence of the virus was determined, which shows a unique genome organization, and low levels of identity to known members of the Iridoviridae. Based on homology of a series of putatively encoded proteins, the virus is a novel member of the Megalocytivirus genus of the Iridoviridae family. The virus was isolated and propagated in cell culture, where it caused a cytopathogenic effect in infected Asian seabass kidney and brain cells. Electron microscopy revealed icosahedral virions of about 140 nm, characteristic for the Iridoviridae. In vitro cultured virus induced scale drop syndrome in Asian seabass in vivo and the virus could be reisolated from these infected fish. These findings show that the virus is the causative agent for the scale drop syndrome, as each of Koch’s postulates is fulfilled. We have named the virus Scale Drop Disease Virus. Vaccines prepared from BEI- and formalin inactivated virus, as well as from E. coli produced major capsid protein provide efficacious protection against scale drop disease.

Infection of cowpea cells with cowpea mosaic virus (CPMV) is accompanied by the appearance of tubular structures containing virus-like particles which protrude from or penetrate the cell wall. Immunogold labelling of sections of infected cells using antisera against a CPMV M RNA translation products, and Protein A -g o l d , showed that the 58K and/or 48K tentative transport proteins of CPMV were located in or on these tubular structures. Furthermore, these proteins were detected in small electron-dense areas near the tail-end of the tubules. The possible function of these structures in virus movement from cell to cell is discussed.The bipartite genome of cowpea mosaic virus (CPMV) consists of two positive single-stranded RNA molecules (B and M RNA) that each contain only one large open reading frame and are translated into polyproteins which are cleaved by a B RNA-encoded protease into functional proteins (Goldbach & van Kammen, 1985;Vos et al., 1988). B RNA encodes all the functions necessary for replication of the RNAs (Goldbach et al., 1980;Eggen & van Kammen, 1988) and is able to replicate independently of the M RNA in cowpea protoplasts (Rezelman et al., 1982). The M RNA is translated in vitro into two overlapping polyproteins of Mr 105K and 95K (Vos et al., 1984), which are processed proteolytically to give the overlapping 58K and 48K non-structural proteins and, via a 60K precursor, the 37K and 23K coat proteins and Fig. 1). Rezelman et aL (1989) showed recently that these proteins are also produced in CPMV-infected cowpea protoplasts.Although the B RNA can replicate independently of M RNA in protoplasts, M RNA is essential for infection of plants (Rezelman et al., 1982). Using infectious transcripts of the M RNA, WeUink & van Kammen (1989) showed that mutations and deletions in the coding regions of both 58K/48K proteins and the capsid proteins prevented systemic infection in cowpea plants. These results suggest that both the 58K/48K proteins and the coat proteins of CPMV are indispensable for cell-tocell movement of the virus and also that CPMV is transported as particles and not as naked RNA.To study the possible involvement of M RNA products in virus movement we detected these products in sections of infected cells by the binding of specific antisera to antigens as shown by its reaction with Protein A-gold.To achieve a near-simultaneous infection of the cells in secondary leaves of cowpea plants (Vigna unguiculata cv. California Blackeye), plants were given a differential temperature treatment (DTT) after inoculation of the primary leaves with 1 mg/ml of purified CPMV in 0.01 M-phosphate buffer pH 7.0 (Dawson & Schlegel, 1976).