Amino Acid Changes in Proteins 2B and 3A Mediate Rhinovirus Type 39 Growth in Mouse Cells

Abstract

Many steps of viral replication are dependent on the interaction of viral proteins with host cell components. To identify rhinovirus proteins involved in such interactions, human rhinovirus 39 (HRV39), a virus unable to replicate in mouse cells, was adapted to efficient growth in mouse cells producing the viral receptor ICAM-1 (ICAM-L cells). Amino acid changes were identified in the 2B and 3A proteins of the adapted virus, RV39/L. Changes in 2B were sufficient to permit viral growth in mouse cells; however, changes in both 2B and 3A were required for maximal viral RNA synthesis in mouse cells. Examination of infected HeLa cells by electron microscopy demonstrated that human rhinoviruses induced the formation of cytoplasmic membranous vesicles, similar to those observed in cells infected with other picornaviruses. Vesicles were also observed in the cytoplasm of HRV39-infected mouse cells despite the absence of viral RNA replication. Synthesis of picornaviral nonstructural proteins 2C, 2BC, and 3A is known to be required for formation of membranous vesicles. We suggest that productive HRV39 infection is blocked in ICAM-L cells at a step posttranslation and prior to the formation of a functional replication complex. The observation that changes in HRV39 2B and 3A proteins lead to viral growth in mouse cells suggests that one or both of these proteins interact with host cell proteins to promote viral replication.Picornaviruses are small RNA viruses with a single-stranded, positive-sense genome of approximately 7 to 8 kb enclosed in a nonenveloped, icosahedral capsid. These viruses are responsible for a wide range of diseases including foot-and-mouth disease, hepatitis, poliomyelitis, and the common cold. Following cell entry, the viral genome is translated as a single polyprotein that is co-and posttranslationally cleaved by viral proteases, yielding both precursor proteins and end products. Many of the precursor proteins are essential for virus replication, with functions distinct from the end products (29,40,42). Four structural proteins produced from the P1 region of the genome form the viral capsid. Nonstructural proteins of the P2 and P3 region of the genome encode all of the viral proteins responsible for genomic RNA replication.Viral genome replication occurs on the surface of cytoplasmic vesicles induced by the synthesis of viral proteins 2C and 3A and the precursor 2BC (70). These vesicles are formed in conjunction with massive cell membrane proliferation (4, 7) and increased cellular synthesis of phospholipids (32). With the exception of the two viral proteases 2A pro and 3C pro , all of the nonstructural proteins have been found on the surface of these vesicles and are thought to interact in the replication complex (26,59,61,70). The vesicles assemble into a rosette, and the replication complex forms on the inner face of the rosette (13, 26). Salt-induced dissociation of the rosette demonstrated that the individual vesicles themselves can also support replication initiation and elongation (26). Neither t...

Complete genome sequence analysis of Seneca Valley virus-001, a novel oncolytic picornavirus

“…In FMDV and human rhinoviruses, these proteins have been shown to be involved in host-cell tropism and virulence (Lomax & Yin, 1989;Beard & Mason, 2000;Knowles et al, 2001;Pacheco et al, 2003;Harris & Racaniello, 2005).…”

Adaptation of an ICAM-1-Tropic Enterovirus to the Mouse Respiratory Tract

“…Previous mouse adaptation strategies with HRVs in vitro revealed adaptation mutations mapping to the coding region for the viral P2 proteins, 2B and 2BC (24,47), or the P3 protein, 3A (23). However, in our in vitro approach in ICAMLa-4 cells or in hICAM-1 tg mice in vivo, adaptation mutations occurred in the coding region for the VP2 and VP3 capsid proteins (Fig.…”

“…Since our data indicated that CAV21 enters hICAM-1 tg mouse RT epithelial cells in an hICAM-1-mediated event, CAV21's obvious growth deficit in mouse lungs may be due to host/cell-type-specific restrictions of virus replication. Although, in contrast to hICAM-1-tropic HRVs (23,24), CAV21 grows efficiently in mouse L fibroblasts expressing hICAM-1 (ICAM-L5) ( Fig. 2A, B), these are nonrepresentative targets that may not accurately predict virus competency in RT epithelium.…”

“…Growth of HRV2 improved upon genetic adaptations after serial passage in mouse L fibroblasts (47), which were shown to map to the nonstructural proteins 2B/2C (32). Similarly, poor replication of HRVs 16 and 39 in mouse L fibroblasts expressing hICAM-1 significantly improved upon acquisition of adaptation mutations in viral nonstructural proteins 2B, 2C, or 3A (23,24).…”

Age-Dependent Poliovirus Replication in the Mouse Central Nervous System Is Determined by Internal Ribosome Entry Site-Mediated Translation

“…While the block to replication of HRV14 and HRV16 in mouse L cells is relieved upon synthesis of hICAM-1, HRV39 fails to replicate (17). Passage of HRV39 in mouse cells producing hICAM-1 led to the identification of a virus, HRV39/L, that can replicate in these cells (16). Amino acid changes in viral proteins 2B and 3A mediate HRV39 growth in mouse cells.…”