Spike (S) proteins of coronaviruses, including the coronavirus that causes severe acute respiratory syndrome (SARS), associate with cellular receptors to mediate infection of their target cells. Here we identify a metallopeptidase, angiotensin-converting enzyme 2 (ACE2), isolated from SARS coronavirus (SARS-CoV)-permissive Vero E6 cells, that efficiently binds the S1 domain of the SARS-CoV S protein. We found that a soluble form of ACE2, but not of the related enzyme ACE1, blocked association of the S1 domain with Vero E6 cells. 293T cells transfected with ACE2, but not those transfected with human immunodeficiency virus-1 receptors, formed multinucleated syncytia with cells expressing S protein. Furthermore, SARS-CoV replicated efficiently on ACE2-transfected but not mock-transfected 293T cells. Finally, anti-ACE2 but not anti-ACE1 antibody blocked viral replication on Vero E6 cells. Together our data indicate that ACE2 is a functional receptor for SARS-CoV.

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Treatment of HIV-1-infected individuals with a combination of anti-retroviral agents results in sustained suppression of HIV-1 replication, as evidenced by a reduction in plasma viral RNA to levels below the limit of detection of available assays. However, even in patients whose plasma viral RNA levels have been suppressed to below detectable levels for up to 30 months, replication-competent virus can routinely be recovered from patient peripheral blood mononuclear cells and from semen. A reservoir of latently infected cells established early in infection may be involved in the maintenance of viral persistence despite highly active anti-retroviral therapy. However, whether virus replication persists in such patients is unknown. HIV-1 cDNA episomes are labile products of virus infection and indicative of recent infection events. Using episome-specific PCR, we demonstrate here ongoing virus replication in a large percentage of infected individuals on highly active anti-retroviral therapy, despite sustained undetectable levels of plasma viral RNA. The presence of a reservoir of 'covert' virus replication in patients on highly active anti-retroviral therapy has important implications for the clinical management of HIV-1-infected individuals and for the development of virus eradication strategies.

Infection of receptor-bearing cells by coronaviruses is mediated by their spike (S) proteins. The coronavirus (SARS-CoV) that causes severe acute respiratory syndrome (SARS) infects cells expressing the receptor angiotensin-converting enzyme 2 (ACE2).Here we show that codon optimization of the SARS-CoV S-protein gene substantially enhanced S-protein expression. We also found that two retroviruses, simian immunodeficiency virus (SIV) and murine leukemia virus, both expressing green fluorescent protein and pseudotyped with SARS-CoV S protein or S-protein variants, efficiently infected HEK293T cells stably expressing ACE2. Infection mediated by an S-protein variant whose cytoplasmic domain had been truncated and altered to include a fragment of the cytoplasmic tail of the human immunodeficiency virus type 1 envelope glycoprotein was, in both cases, substantially more efficient than that mediated by wild-type S protein. Using S-protein-pseudotyped SIV, we found that the enzymatic activity of ACE2 made no contribution to S-protein-mediated infection. Finally, we show that a soluble and catalytically inactive form of ACE2 potently blocked infection by S-proteinpseudotyped retrovirus and by SARS-CoV. These results permit studies of SARS-CoV entry inhibitors without the use of live virus and suggest a candidate therapy for SARS.A distinct coronavirus (SARS-CoV) has been identified as the etiological agent of severe acute respiratory syndrome (SARS), an acute pulmonary syndrome characterized by an atypical pneumonia that results in progressive respiratory failure and death in close to 10% of infected individuals (8,11,14,15). SARS-CoV is not closely related to any of the three previously defined genetic and serological coronavirus groups, although it may be distantly related to group 2 coronaviruses (21); the SARS-CoV spike (S) protein, a surface glycoprotein that mediates coronavirus entry into receptor-bearing cells, is also distinct from those of other coronaviruses (18,20). Reflecting this difference, SARS-CoV does not utilize any previously identified coronavirus receptors to infect cells. Rather, as our group have recently demonstrated, angiotensin-converting enzyme 2 (ACE2) serves as a functional receptor for this coronavirus (16,24,25).A quantitative system utilizing a well-characterized retroviral vector (1) for measuring SARS-CoV S-protein-mediated infection would obviate the need for specialized biosafety facilities for many studies, including those assessing humoral responses to potential vaccines. Here we show that simian immunodeficiency virus (SIV) pseudotyped with several codon-optimized S-protein variants could efficiently infect Vero E6 cells and HEK293T cells transiently or stably expressing ACE2. One such variant, truncated at its cytoplasmic tail and bearing instead a region of the tail of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (17), was especially efficient at mediating infection. Murine leukemia virus (MLV) pseudotyped with this S-protein variant also infected ACE2-ex...

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