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J. Virol. doi:10.1128/JVI.00096-08
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Persistent Replication of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) in Human Tubular Kidney Cells Selects for Adaptive Mutations in the Membrane Protein

Viral Pathogens and Biosafety Unit, San Raffaele Scientific Institute, Milano, Italy; Laboratory of Microbiology and Virology, San Raffaele Scientific Institute, Milano, Italy; University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America; AIDS Immunopathogenesis Unit, San Raffaele Scientific Institute, Milano, Italy; Department of Medicine and Public Health, University of Insubria, Varese, Italy; Vita-Salute San Raffaele University, School of Medicine, Milano, Italy; Laboratory of Clinical Pathology, Microbiology and Virology, Mediterranean Institute for Transplantation and Advanced Specialised Therapies, University of Pittsburgh Medical Center, Palermo, Italy

^* To whom correspondence should be addressed. Email: vicenzi.elisa{at}hsr.it.

	Abstract

SARS is a systemic disease characterized by both lung pathology and widespread extra-pulmonary virus dissemination causing multiple organ injuries. In this regard, renal dysfunction is an ominous sign in patients with SARS. Indeed, clusters of SARS-CoV particles have been detected in the cytoplasm of renal tubular epithelial cells in post-mortem studies explaining the presence of infectious virus in the urine of SARS patients. In order to investigate the potential SARS-CoV kidney tropism, we have evaluated the susceptibility of human renal cells of tubular and glomerular origin to in vitro SARS-CoV infection. Immortalized cultures of differentiated proximal tubular epithelial cells (PTEC), glomerular mesangial (MC) and glomerular epithelial cells (podocytes) were found to express the SARS-CoV receptor angiotensin converting enzyme-2 (ACE2) on their surface. Productive infection, however, occurred only in PTEC but not in glomerular cells. A transient infection with poor virus production was observed in MC, whereas podocytes were not permissive to SARS-CoV infection. In contrast to the cytopathic infection of the Vero E6 cell line, SARS-CoV did not cause overt cytopathic effects in PTEC or MC. Of interest, PTEC, but not MC, maintained stable levels of SARS-CoV production in serial subcultures suggesting a state of persistent state of infection. In this regard, a SARS-CoV variant with increased replication capacity in PTEC was selected after 4 serial subculture passages. This SARS-CoV variant acquired a single non-conservative amino acid change from glutamic acid (E) to alanine (A) at position 11 in the viral membrane (M) protein. The E11A point mutation was sufficient for enhanced SARS-CoV replication and persistence in PTEC when introduced in a SARS-CoV recombinant infectious clone. These findings indicate that human PTEC may represent a site of SARS-CoV productive and persistent replication favoring the emergence of viral variants with increased replication capacity at least in these kidney cells.