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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Replication of viruses in species other than their natural hosts is frequently limited by entry and postentry barriers. The coronavirus that causes severe acute respiratory syndrome (SARS-CoV) utilizes the receptor angiotensin-converting enzyme 2 (ACE2) to infect cells. Here we compare human, mouse, and rat ACE2 molecules for their ability to serve as receptors for SARS-CoV. We found that, compared to human ACE2, murine ACE2 less efficiently bound the S1 domain of SARS-CoV and supported less-efficient S proteinmediated infection. Rat ACE2 was even less efficient, at near background levels for both activities. Murine 3T3 cells expressing human ACE2 supported SARS-CoV replication, whereas replication was less than 10% as efficient in the same cells expressing murine ACE2. These data imply that a mouse transgenically expressing human ACE2 may be a useful animal model of SARS.The severe acute respiratory syndrome coronavirus (SARSCoV) has been identified as the etiological agent of SARS, an acute pulmonary syndrome resulting in progressive respiratory failure and death in close to 10% of reported cases (2, 4, 10, 11). The SARS-CoV S protein, like that of other coronaviruses, mediates infection of receptor-expressing target cells (5,8). It was recently demonstrated that angiotensin-converting enzyme 2 (ACE2) is a functional receptor for SARS-CoV and shown that a 193-amino-acid receptor-binding domain of the S protein is sufficient to bind ACE2 with high affinity (13,20). A soluble form of ACE2, anti-ACE2 antibodies, and an antibody recognizing the ACE2-binding domain of the S protein each efficiently blocked SARS-CoV replication or infection by S protein-pseudotyped retrovirus (13,18). Moreover, the tissue distribution of ACE2 in the lungs, kidney, and gastrointestinal tract (7, 9) is consistent with the isolation of virus from each of these tissues in infected humans and animals (2, 10-12). Collectively, these studies suggest that ACE2 is the primary physiologically relevant receptor for SARS-CoV.SARS-CoV likely originated from one or more animal sources, and the virus can infect a number of species but does not appear to cause disease except in some primates, domestic cats, and ferrets (4,6,14,17,19). The virus has been shown to replicate in the respiratory tracts of mice challenged with SARS-CoV, but despite high challenge titers, the virus was cleared in all cases within 7 days (17). However, these mice raised neutralizing antibodies that were sufficient to prevent reinfection or infection of naïve mice following passive transfer of immune sera. These studies, as well as recent work demonstrating antibody-mediated protection in DNA-vaccinated mice (21), raise the possibility that a subunit vaccine may be sufficient for control of the virus. However, the lack of disease in mice makes them an imperfect model for evaluation of SARS vaccines and therapeutics.Rodents are of particular interest in the study of SARS, not only because they can provide convenient animal models of human disease but also because both mice a...

The human immunodeficiency virus type 1 (HIV-1) Nef protein has several independent functions that might contribute to efficient viral replication in vivo. Since HIV-1 adapts rapidly to its host environment, we investigated if different Nef properties are associated with disease progression. Functional analysis revealed that nef alleles obtained during late stages of infection did not efficiently downmodulate class I major histocompatibility complex but were highly active in the stimulation of viral replication. In comparison, functional activity in downregulation of CD4 and enhancement of HIV-1 infectivity were maintained or enhanced after AIDS progression. Our results demonstrate that various Nef activities are modulated during the course of HIV-1 infection to maintain high viral loads at different stages of disease progression. These findings suggest that all in vitro Nef functions investigated contribute to AIDS pathogenesis and indicate that nef variants with increased pathogenicity emerge in a significant number of HIV-1-infected individuals.

Two neutralizing epitopes were defined for MAbs to SARS-CoV S glycoprotein. Antibodies to both epitopes protected mice against SARS-CoV challenge. Clinical trials are planned to test MAb 201, a fully human MAb specific for the epitope within the receptor-binding region.