Peter L. Collins

Respiratory Syncytial Virus Infection of Human Airway Epithelial Cells Is Polarized, Specific to Ciliated Cells, and without Obvious Cytopathology

ABSTRACT Gene therapy for cystic fibrosis (CF) lung disease requires efficient gene transfer to airway epithelial cells after intralumenal delivery. Most gene transfer vectors so far tested have not provided the efficiency required. Although human respiratory syncytial virus (RSV), a common respiratory virus, is known to infect the respiratory epithelium, the mechanism of infection and the epithelial cell type targeted by RSV have not been determined. We have utilized human primary airway epithelial cell cultures that generate a well-differentiated pseudostratified mucociliary epithelium to investigate whether RSV infects airway epithelium via the lumenal (apical) surface. A recombinant RSV expressing green fluorescent protein (rgRSV) infected epithelial cell cultures with high gene transfer efficiency when applied to the apical surface but not after basolateral inoculation. Analyses of the cell types infected by RSV revealed that lumenal columnar cells, specifically ciliated epithelial cells, were targeted by RSV and that cultures became susceptible to infection as they differentiated into a ciliated phenotype. In addition to infection of ciliated cells via the apical membrane, RSV was shed exclusively from the apical surface and spread to neighboring ciliated cells by the motion of the cilial beat. Gross histological examination of cultures infected with RSV revealed no evidence of obvious cytopathology, suggesting that RSV infection in the absence of an immune response can be tolerated for >3 months. Therefore, rgRSV efficiently transduced the airway epithelium via the lumenal surface and specifically targeted ciliated airway epithelial cells. Since rgRSV appears to breach the lumenal barriers encountered by other gene transfer vectors in the airway, this virus may be a good candidate for the development of a gene transfer vector for CF lung disease.

Characterization of the 10 proteins of human respiratory syncytial virus: identification of a fourth envelope-associated protein.

A total of 13 respiratory syncytial (RS) virus specific polypeptides were identified by pulse-chase metabolic labeling of infected HEp-2 cells. Ten of the 13 proteins were shown to be unique. They were the L, G, F (F1, F2), N, P, M, 24K, 14K, 11K and 9.5K proteins. These conclusions were based on peptide mapping and on previous work showing that each of 10 polypeptides are coded for by a unique mRNA. The seven largest proteins, L, G, F (F1, F2), N, P, M and 24K were identified clearly as virion structural proteins. The 24K protein was characterized by detergent and salt dissociation studies as an envelope-associated protein, bringing to four (G, F (F1, F2), M and 24K) the number of membrane associated proteins for RS virus. A fourth membrane-associated protein has not been described previously for any other paramyxovirus. Of the three smallest proteins, the 14K and 11K were characterized as non-structural proteins. The 9.5K protein was detected in low amounts in highly purified preparations of virions.

Correct sequence for the major nucleocapsid protein mRNA of respiratory syncytial virus.

A nucleotide sequence for the mRNA of the major nucleocapsid (N) protein gene of respiratory syncytial virus was reported previously (N. Elango and S. Venkatesen, 1983, Nucleic Acids Res. 11, 5941-5951). However, we have been unable to confirm part of this sequence as N mRNA-specific and suggest that the published sequence represents that of an aberrant chimeric transcript. Here we present an alternative sequence for the N mRNA and provide data supporting its authenticity. The corrected N mRNA sequence contains 1197 rather than 1427 nucleotides exclusive of poly(A), and encodes a protein of 391 rather than 467 amino acids. The calculated molecular weight for the 391-amino acid protein described by the sequence presented here is 42,600, in agreement with the molecular weight of 42,000 determined for the RS viral N protein by gel electrophoresis. In addition, we present sequence data from dicistronic RNAs that span the junction between the 1B protein and N cistrons, and the junction between the N and phosphoprotein (P) cistrons.

The 1A protein gene of human respiratory syncytial virus: nucleotide sequence of the mRNA and a related polycistronic transcript

The 1A mRNA is the smallest mRNA of human respiratory syncytial (RS) virus and encodes a single protein of approximate molecular weight 9500 as estimated previously by gel electrophoresis. The nucleotide sequence of the 1A mRNA, determined from several full-length cDNA clones, is reported. The 1A mRNA consists of 405 nucleotides, exclusive of poly(A), with relatively long nontranslated regions at the 5 and 3 ends (84 and 126 nucleotides, respectively). The sequences at the 5 and 3 termini of the 1A mRNA conform to the previously described conserved consensus sequences for RS virus mRNAs. The major open reading frame of the 1A mRNA codes for a hydrophobic polypeptide of 64 amino acids with a calculated molecular weight of 7536. The 5 terminus of the 1A mRNA was mapped and sequenced by primer extension under conditions for sequencing by partial chain termination. These experiments also identified a population of polycistronic RNA having the general structure: 5 M protein mRNA-1A mRNA 3. This polytranscript was sequenced in order to determine the intergenic sequence. In the polytranscript, the nucleotide sequence of the M gene is followed by, in mRNA sense, six A residues and the intergenic sequence 5 ... UAUACACNN (N represents unidentified nucleotide).

Nucleotide sequences of the 1B and 1C nonstructural protein mRNAs of human respiratory syncytial virus

The genes encoding the 1C and 1B mRNAs of human respiratory syncytial (RS) virus are first in the order of viral transcription and encode nonstructural (NS) proteins of approximate molecular weights 14,000 and 11,000, respectively, estimated by gel electrophoresis. The complete nucleotide sequences of the 1C and 1B mRNAs determined from several full-length cDNA clones are described. The 1C and 1B mRNAs contain 528 and 499 nucleotides, respectively, exclusive of poly(A), and encode proteins of 139 and 124 amino acids. The calculated molecular weights of the predicted NS1C and NS1B proteins are 15,567 and 14,674, respectively. Both mRNA sequences contain the 5-terminal sequence, 5 GGGGCAAAU . . . , and the 3-terminal sequence, 5 . . . AGUAUA(N)1-4-poly(A), that were identified previously as conserved among six other RS viral mRNAs. In addition, a dicistronic readthrough RNA having the general structure 5 1C mRNA-intergenic sequence-1B mRNA 3 was identified by dideoxynucleotide sequencing of intracellular poly(A)+ RNA using a DNA primer derived from a 1B-cDNA clone. In the dicistronic RNA, the nucleotide sequences of the 1C and 1B cistrons are separated by, in mRNA sense, four A residues and the intergenic sequence 5 . . . CUUAACAGAAGACAAAAAN . . . 3 (N represents unidentified nucleotide). The significance of these sequences is discussed.

Human Respiratory Syncytial Virus Genome and Gene Products

Human respiratory syncytial (RS) virus is recognized as the major cause of serious lower respiratory tract disease in young children [1,2]. In 1978, it was classified in the genus Pneumovirus of the Paramyxoviridae family with the proviso that because so little was known about the virus at that time, future information might alter this classification [3].