The minor receptor group of human rhinovirus (HRV) includes HRV23 and HRV25, but the presence of a lysine in the VP1 HI loop is not sufficient for receptor binding

Abstract

Like all 10 minor receptor group human rhinoviruses (HRVs), HRV23 and HRV25, previously classified as major group viruses, are neutralized by maltose binding protein (MBP)-V33333 (a soluble recombinant concatemer of five copies of repeat 3 of the very-low-density lipoprotein receptor fused to MBP), bind to low-density lipoprotein receptor in virus overlay blots, and replicate in intercellular adhesion molecule 1 (ICAM-1)-negative COS-7 cells. From phylogenetic analysis of capsid protein VP1-coding sequences, they are also known to cluster together with other minor group strains. Therefore, they belong to the minor group; there are now 12 minor group and 87 major group HRV serotypes. Sequence comparison of the VP1 capsid proteins of all HRVs revealed that the lysine in the HI loop, strictly conserved in the 12 minor group HRVs, is also present in 9 major group serotypes that are neutralized by soluble ICAM-1. Despite the presence of this lysine, they are not neutralized by MBP-V33333 and fail to replicate in COS-7 cells and in HeLa cells in the presence of an ICAM-1-blocking antibody. These nine serotypes are therefore "true" major group viruses.

FIG. 1.

Alignment of VP1 sequences of all human rhinovirus serotypes possessing a lysine at the position equivalent to the one implicated in receptor recognition of HRV2 (36). Only regions corresponding to the BC, DE, and HI surface loops are depicted. Serotypes, except HRV23 and HRV25, originally classified as major group (35) are boxed. Residues identified in HRV2 as contacting the receptor are in reverse type. The lysine strictly conserved in all minor group HRVs is in italics and highlighted by the caret symbol. Minor group HRVs are arranged in clusters as revealed by phylogenetic analysis (16) (Fig. 4). HRV8 and -95 are bracketed to indicate their antigenic cross-reactivity; they might be considered a single serotype (16). Note that all HRVs shown belong to genus A (16, 27). Parts of the alignment of the entire VP1 sequences made with Clustal W (http://www.ebi.ac.uk/clustalw/index.html#) are shown.

FIG. 2.

HRV23 and HRV25 bind LDLR in virus overlay blots. Cell membranes were prepared from M4-LDLR806 cells overexpressing truncated human LDLR (28) and from wt M1 cells. Aliquots corresponding to 10⁶ cells were separated on sodium dodecyl sulfate-10% polyacrylamide gels under nonreducing conditions. The proteins were electrotransferred to a polyvinylidene difluoride membrane that was then incubated with radiolabeled virus. The membrane was exposed to Kodak X-ray film. Minor group rhinoviruses HRV1A, HRV2, and HRV62 were used as controls. The strong band corresponds to LDLR migrating with an apparent molecular mass of ∼120 kDa as deduced from marker proteins run on the same gel; the minor band underneath represents incompletely glycosylated LDLR.

FIG. 3.

HRV23 and HRV25 infect ICAM-1-deficient COS-7 cells. COS-7 cells grown in 96-well plates until 80% confluent were challenged with HRVs at a multiplicity of infection of 10. After incubation for 3 days at 34°C, cells were stained with crystal violet and photographed. The cytopathic effect resulted in detachment of the cells and lack of coloration. No infection was seen with the major group viruses HRV14, HRV16, and HRV85.

FIG. 4.

HRV23 and -25 cluster with established minor group serotypes (gray boxes numbered 1, 2, and 3), but other HI loop lysine-containing major group serotypes (arrows) do not. The neighbor-joining tree is based on amino acid sequences of entire VP1 capsid proteins of all genus A HRVs; HRV14, which is genus B, is included for comparison. Data are from reference . Bootstrapping values are indicated.