Severe acute respiratory syndrome coronavirus (SARS-CoV) is a highrisk infectious pathogen. In the proposed model of respiratory failure, SARS-CoV down-regulates its receptor, angiotensin-converting enzyme 2 (ACE2), but the mechanism involved is unknown. We found that the spike protein of SARS-CoV (SARS-S) induced TNF-␣-converting enzyme (TACE)-dependent shedding of the ACE2 ectodomain. The modulation of TACE activity by SARS-S depended on the cytoplasmic domain of ACE2, because deletion mutants of ACE2 lacking the carboxyl-terminal region did not induce ACE2 shedding or TNF-␣ production. In contrast, the spike protein of HNL63-CoV (NL63-S), a CoV that uses ACE2 as a receptor and mainly induces the common cold, caused neither of these cellular responses. Intriguingly, viral infection, judged by real-time RT-PCR analysis of SARS-CoV mRNA expression, was significantly attenuated by deletion of the cytoplasmic tail of ACE2 or knock-down of TACE expression by siRNA. These data suggest that cellular signals triggered by the interaction of SARS-CoV with ACE2 are positively involved in viral entry but lead to tissue damage. These findings may lead to the development of anti-SARS-CoV agents.shedding ͉ cytoplasmic tail ͉ HNL63-CoV ͉ TNF-␣ S evere acute respiratory syndrome coronavirus (SARS-CoV) is an infectious pathogen known to have caused acute respiratory distress in Ͼ8,000 patients with a mortality rate of Ϸ10% (1). Although outbreaks of SARS-CoV are now well controlled, the mechanism of severe respiratory failure in infected patients is unknown. Angiotensin-converting enzyme 2 (ACE2), an ACE homolog that functions as a positive regulator of the reninangiotensin system (RAS) (2, 3), was identified as a receptor of SARS-CoV (4). A possible indicator of a severe clinical outcome, the spike protein of SARS-CoV (SARS-S) was found to downregulate ACE2 expression (5). ACE2 knockout (KO) mice were also shown to be susceptible to severe respiratory failure after chemical challenge (5, 6), and ACE2 has been shown to moderate ACE-induced intracellular inflammation, suggesting that the mechanism of ACE2 down-regulation may explain the molecular basis of SARS-CoV-related severe respiratory distress.HNL63-CoV, a CoV (7) that causes the common cold, was recently found to use ACE2 for viral infection (8), and it was further shown that the spike protein of HNL63-CoV (NL63-S) binds ACE2 directly (9). NL63-S and SARS-S show 21% identity (10), whereas that between NL63-S and the spike protein of HCoV-229E, which uses CD13 [a completely different carboxy (C)-peptidase] as a cellular receptor (11), is 55%. It is important to note that, despite their phylogenetically distinct properties and their producing different clinical outcomes, SARS-CoV and HNL63-CoV both use ACE2.Based on these observations, we hypothesized that the functional modulation of ACE2 may be differentially induced by SARS-S and NL63-S. To test this prediction, we first clarified the mechanism of SARS-S-induced ACE2 down-regulation, and then compared the cellular respon...

Recent observations imply that HIV-1 infection induces chromosomal DNA damage responses. However, the precise molecular mechanism and biological relevance are not fully understood. Here, we report that HIV-1 infection causes double-strand breaks in chromosomal DNA. We further found that Vpr, an accessory gene product of HIV-1, is a major factor responsible for HIV-1-induced double-strand breaks. The purified Vpr protein promotes double-strand breaks when incubated with isolated nuclei, although it does not exhibit endonuclease activity in vitro. A carboxyl-terminally truncated Vpr mutant that is defective in DNA-binding activity is less capable of Vpr-dependent double-strand break formation in isolated nuclei. The data suggest that double-strand breaks induced by Vpr depend on its DNA-binding activity and that Vpr may recruit unknown nuclear factor(s) with positive endonuclease activity to chromosomal DNA. This is the first direct evidence that Vpr induces double-strand breaks in HIV-1-infected cells. We discuss the possible roles of Vpr-induced DNA damage in HIV-1 infection and the involvement of Vpr in further acquired immunodeficiency syndrome-related tumor development. (Cancer Res 2006; 66(2): 627-31)

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We focus on the role of Vpr in inducing DNA double-strand breaks (DSBs) in the host cell. Based on the summarized findings of Vpr-induced DSBs and the finding of Vpr in the plasma of HIV-1-positive patients, we discuss the roles of Vpr in viral infection, especially viral infection of resting macrophages. We also describe the possible involvement of Vpr in non-AIDS-defining cancers, which represent an emerging crisis in HIV-1-positive patients.

Supplementary Method from HIV-1 Vpr Induces DNA Double-Strand Breaks

Supplementary Method from HIV-1 Vpr Induces DNA Double-Strand Breaks

<div>Abstract<p>Recent observations imply that HIV-1 infection induces chromosomal DNA damage responses. However, the precise molecular mechanism and biological relevance are not fully understood. Here, we report that HIV-1 infection causes double-strand breaks in chromosomal DNA. We further found that Vpr, an accessory gene product of HIV-1, is a major factor responsible for HIV-1–induced double-strand breaks. The purified Vpr protein promotes double-strand breaks when incubated with isolated nuclei, although it does not exhibit endonuclease activity <i>in vitro</i>. A carboxyl-terminally truncated Vpr mutant that is defective in DNA-binding activity is less capable of Vpr-dependent double-strand break formation in isolated nuclei. The data suggest that double-strand breaks induced by Vpr depend on its DNA-binding activity and that Vpr may recruit unknown nuclear factor(s) with positive endonuclease activity to chromosomal DNA. This is the first direct evidence that Vpr induces double-strand breaks in HIV-1–infected cells. We discuss the possible roles of Vpr-induced DNA damage in HIV-1 infection and the involvement of Vpr in further acquired immunodeficiency syndrome–related tumor development. (Cancer Res 2006; 66(2): 627-31)</p></div>

<div>Abstract<p>Recent observations imply that HIV-1 infection induces chromosomal DNA damage responses. However, the precise molecular mechanism and biological relevance are not fully understood. Here, we report that HIV-1 infection causes double-strand breaks in chromosomal DNA. We further found that Vpr, an accessory gene product of HIV-1, is a major factor responsible for HIV-1–induced double-strand breaks. The purified Vpr protein promotes double-strand breaks when incubated with isolated nuclei, although it does not exhibit endonuclease activity <i>in vitro</i>. A carboxyl-terminally truncated Vpr mutant that is defective in DNA-binding activity is less capable of Vpr-dependent double-strand break formation in isolated nuclei. The data suggest that double-strand breaks induced by Vpr depend on its DNA-binding activity and that Vpr may recruit unknown nuclear factor(s) with positive endonuclease activity to chromosomal DNA. This is the first direct evidence that Vpr induces double-strand breaks in HIV-1–infected cells. We discuss the possible roles of Vpr-induced DNA damage in HIV-1 infection and the involvement of Vpr in further acquired immunodeficiency syndrome–related tumor development. (Cancer Res 2006; 66(2): 627-31)</p></div>