Severe Acute Respiratory Syndrome Coronavirus Protein 6 Is Required for Optimal Replication

Severe acute respiratory syndrome coronavirus (SARS-CoV) encodes several accessory proteins of unknown function. One of these proteins, protein 6 (p6), which is encoded by ORF6, enhances virus replication when introduced into a heterologous murine coronavirus (mouse hepatitis virus [MHV]) but is not essential for optimal SARS-CoV replication after infection at a relatively high multiplicity of infection (MOI). Here, we reconcile these apparently conflicting results by showing that p6 enhances SARS-CoV replication to nearly the same extent as when expressed in the context of MHV if cells are infected at a low MOI and accelerates disease in mice transgenic for the human SARS-CoV receptor.The genome of severe acute respiratory syndrome coronavirus (SARS-CoV) encodes several structural proteins, including the spike, nucleocapsid, membrane, and envelope proteins (13). Integrated between and within these structural proteins are eight accessory proteins (6, 8, 10, 15, 16, 18, 21-27). Our laboratory showed previously that one of these SARS-CoV-specific accessory proteins, encoded by ORF6, showed a clearly recognizable phenotype when introduced into a heterologous attenuated murine coronavirus, mouse hepatitis virus (MHV) strain J2.2-V-1 (rJ2.2.6). rJ2.2.6 grew more rapidly and to higher titers in tissue culture cells and in the murine central nervous system than control viruses, and the presence of p6 increased mortality in mice from 10 to 20% to 80% (7, 19, 20). However, the absence of p6 did not diminish SARS-CoV growth in tissue culture cells when cells were infected with 1 PFU/cell (31). In addition to a role in enhancing virus replication, when expressed in the context of a SARS-CoV infection or by transfection, p6 blocked interferon (IFN)-induced STAT1 nuclear translocation by retention of the nuclear import adaptor molecule karyopherin alpha 2 in the cytoplasm, indicating a role in thwarting innate immune effectors (5, 11). In contrast, p6 did not significantly diminish IFN sensitivity when expressed in the context of rJ2.2 (20).The results described above were puzzling, because p6 seemed to be required for the optimal replication of a heterologous coronavirus but not for that of SARS-CoV. Thus, the objective of this study was to determine whether p6 could enhance SARS-CoV replication in tissue culture cells under any conditions. For this purpose, we examined its function by comparing the growth of a recombinant SARS-CoV (rSARS-CoV) in which p6 was deleted (rSARS-CoVΔ6) with that of wild-type rSARS-CoV at a range of multiplicities of infection (MOIs). Normal mice infected with SARS-CoV readily cleared the infection, making it difficult to detect a role for p6 in vivo. However, mice that are transgenic for expression of the human receptor angiotensin-converting enzyme 2 (hACE2) are exquisitely sensitive to infection with SARS-CoV and are useful for identifying an in vivo role for p6 (14).