Featured Article - September 2008
Short description: J Virol 82, 5279-5294 (2008)
Severe acute respiratory syndrome (SARS) is a human respiratory disease caused by the SARS coronavirus (SARS-CoV). In 2002, a SARS outbreak started in Asia and rapidly spread to several continents, infecting more than 8 thousand people before it was contained in 2003 by public health measures. Most patients developed pneumonia and 9.6% died, a very high case-fatality rate. There is no effective treatment or vaccine for SARS.
SARS-CoV is an enveloped virus, with a positive strand RNA genome of 29.7 kb, encoding 16 non-structural proteins (nsp1 to nsp16), structural proteins (N, M, S and E) and accessory proteins (ORF3a, 6, 7a, 7b, 9b). The genome of SARS-CoV was quickly sequenced in 2003, but many aspects of SARS biology remained to be understood.
To explore the cellular events during early viral infection, Buchmeier, Kuhn and collegues took a proteomics approach, using structural genomics to understand the individual viral proteins and using mass spectrometry to identify proteins incorporated into virions. The authors filtered the list of proteins to remove those purified from uninfected cells, those identified from only one method of sample preparation, and those found on the surface of the virion only (and thus proteolytically sensitive). This led to 172 host proteins and 8 viral proteins: structural proteins N, M, S; non-structural proteins nsp3, nsp5, nsp2; accessory proteins ORF9b and ORF3a. While the structural proteins were well known to be part of the virion, the non-structural proteins were newly identified.
Among the host factors identified by mass spectrometry in the virions were proteins involved in vesicular trafficking, RNA-binding proteins, chaperones and two protein kinases, CSNK2 and DNA-activated protein kinase, but analysis of virion lysates using a kinase activity screen suggested other kinases might be incorporated as well.
The authors performed network analysis for the viral proteins, based on previously reported interactions. The protein that showed the most connections to other viral components was nsp3, a large multidomain protein containing a characterized papain-like proteinase domain, a poly(ADP-ribose)-binding domain and two ubiquitin-like folds. Most of nsp3 however has not been characterized, so the authors perform a phylogenetic analysis of nsp3 to identify potential domains, revealing 16 conserved domains. Some of these could be independently expressed in bacteria, allowing further characterization. This included a domain that is specific to SARS-CoV (SUD, for SARS unique domain), here shown to bind metals, via its conserved Cys residues, and nucleic acids in vitro. Another domain, NAB, was also shown to have nucleic-acid binding properties. Other nsp3 domains could not be expressed in bacteria, and were annotated using conservation-based statistics. The authors present a general model of nsp3 structure, based on the domain expression pattern and previous structural data. The model shows four transmembrane regions, and all but one domain of nsp3 are on one side of the membrane.
In conclusion, the extensive work performed here represents a first step to explore the roles of SARS CoV nsp3 protein in depth, as well as to characterize the cellular pathways involved in coronavirus assembly and budding. These might pave the way for therapeutic development, an important endeavor given the potential threat of SARS to global public health.
Benjamin W. Neuman, Jeremiah S. Joseph, Kumar S. Saikatendu, Pedro Serrano, Amarnath Chatterjee et al. Proteomics analysis unravels the functional repertoire of coronavirus nonstructural protein 3.
J Virol 82, 5279-5294 (2008). doi:10.1128/JVI.02631-07