Changes in rhinovirus protein 2C allow efficient replication in mouse cells

Abstract

Rhinovirus type 16 was found to replicate in mouse L cells that express the viral receptor, human intercellular adhesion molecule 1 (ICAM-1). However, infection of these cells at a low multiplicity of infection leads to no discernible cytopathic effect, and low virus titers are produced. A variant virus, 16/L, was isolated after alternate passage of rhinovirus 16 between HeLa and ICAM-1 L cells. Infection of mouse cells with 16/L leads to higher virus titers, increased production of RNA, and total cytopathic effect. Three amino acid changes were identified in the P2 region of virus 16/L, and the adaptation phenotype mapped to two changes in protein 2C. The characterization of a rhinovirus host range mutant will facilitate the investigation of cellular proteins required for efficient viral growth and the development of a murine model for rhinovirus infection.

FIG. 1.

One-step growth analysis. At the indicated times postinfection, cells were scraped into the medium, virus was released by freeze-thawing, and the virus titer was determined by plaque assay on HeLa cells. (A) Growth of HRV16 and 16/L in ICAM-L cells infected with 10 PFU/cell; (B) growth of HRV16 and 16/L in HeLa cells infected with 10 PFU/cell.

FIG. 2.

(A) Serial passage of rhinoviruses in mouse cells. Viral RNAs produced by in vitro transcription were introduced into HeLa cells by transfection (passage 1), and the viruses produced were serially passaged in ICAM-L cells (passages 2 to 4). Virus titers were quantified by plaque assay in HeLa cells after each passage. (B) One-step growth analysis of rhinoviruses on ICAM-L cells infected with 10 PFU/cell.

FIG. 3.

Trypan blue exclusion assay. ICAM-L cells were infected with the indicated viruses with 10 (A) or 1 (B) PFU/cell. At the indicated times after infection, cells were assayed for exclusion of trypan blue. Cells excluding trypan blue are viable and are expressed as a percentage of the total cells in each sample at each time point.

FIG. 4.

Positive-strand viral RNA production. ICAM-L cells (A) or HeLa cells (B) were infected with virus at 10 PFU/cell. At the indicated times after infection, total RNA was extracted and immobilized on a membrane. Plus-strand viral RNA was detected by hybridization with a ³²P-labeled RNA probe. RNA levels were quantified by using a PhosphorImager and ImageQuant software. At each time point, viral RNA levels were normalized to levels at time zero and to amounts of wild-type HRV16 RNA.

FIG. 5.

Homodimerization of 2BC proteins containing single or multiple amino acid changes determined by yeast two-hybrid assay. Plasmids pACT and pGBKT7 either alone or containing genes encoding the indicated proteins were introduced into yeast strain YGH1 by cotransformation. Empty vectors are designated by dashes (—). Interaction of the fusion proteins was quantified by using the Miller assay.

FIG. 6.

One-step growth analysis of HRV2, HRV39, and HRV14 in ICAM-L cells infected at 10 PFU/cell. At the indicated times postinfection, cells were scraped into the medium, virus was released by freeze-thawing, and the virus titer was determined by plaque assay on HeLa cells.

FIG. 7.

Alignment of the 2BC proteins of poliovirus, coxsackievirus, HRV2, and HRV16. Most structure-function information on 2BC comes from studies on poliovirus and coxsackievirus, and these domains were extrapolated to the rhinovirus proteins. Proteins 2B and 2C are labeled, and the cleavage site between the two is marked with a line. Outlined in purple are nucleoside triphosphate-binding motifs (36, 45). RNA-binding regions are outlined in blue (35), and membrane-binding regions are outlined in black (14, 17, 18, 44). Regions binding both membranes and RNA are outlined in orange. In the HRV16 sequence, red letters indicate amino acids that have changed in the mouse-adapted strain. In the HRV2 sequence, green letters indicate amino acids that have changed in the mouse-adapted strain. The amino acid in the adapted strain is shown below the parental sequence.