Persistent Replication of Severe Acute Respiratory Syndrome Coronavirus in Human Tubular Kidney Cells Selects for Adaptive Mutations in the Membrane Protein

Pacciarini, Filippo; Ghezzi, Silvia; Canducci, Filippo; Sims, Amy C.; Sampaolo, Michela; Ferioli, Elena; Clementi, Massimo; Poli, Guido; Conaldi, Pier Giulio; Baric, Ralph S.; Vicenzi, Elisa

doi:10.1128/jvi.00096-08

Cited by 55 publications

(54 citation statements)

References 54 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The membrane protein (M), the most abundant component of the virion, is key to viral particle assembly through interactions with spike (40) and nucleocapsid (41) as well as M-M interactions (42) and may, in addition, interact with the viral RNA (43,44) through the carboxy-terminal tail of the protein. Consistent with the role of M in virus assembly, an M protein mutant of SARS-CoV, selected by serial passage in primary human renal tubular epithelial cells, was associated with persistent infection and enhanced virus production (45). The combined introduction of two JHM.SD amino acid substitutions together into the carboxy-ter-minal endodomain of rWU M protein (rWU.M F155L,V224A ) impaired replication in L2 cells and quite dramatically in the mouse liver, although neither substitution alone conferred any detectable phenotype (Fig.…”

Section: Discussionmentioning

confidence: 66%

The nsp1, nsp13, and M Proteins Contribute to the Hepatotropism of Murine Coronavirus JHM.WU

Zhang

Cowley

et al. 2015

J Virol

View full text Add to dashboard Cite

Mouse hepatitis virus (MHV) isolates JHM.WU and JHM.SD promote severe central nervous system disease. However, while JHM.WU replicates robustly and induces hepatitis, JHM.SD fails to replicate or induce pathology in the liver. These two JHM variants encode homologous proteins with few polymorphisms, and little is known about which viral proteins(s) is responsible for the liver tropism of JHM.WU. We constructed reverse genetic systems for JHM.SD and JHM.WU and, utilizing these fulllength cDNA clones, constructed chimeric viruses and mapped the virulence factors involved in liver tropism. Exchanging the spike proteins of the two viruses neither increased replication of JHM.SD in the liver nor attenuated JHM.WU. By further mapping, we found that polymorphisms in JHM.WU structural protein M and nonstructural replicase proteins nsp1 and nsp13 are essential for liver pathogenesis. M protein and nsp13, the helicase, of JHM.WU are required for efficient replication in vitro and in the liver in vivo. The JHM.SD nsp1 protein contains a K194R substitution of Lys194, a residue conserved among all other MHV strains. The K194R polymorphism has no effect on in vitro replication but influences hepatotropism, and introduction of R194K into JHM.SD promotes replication in the liver. Conversely, a K194R substitution in nsp1 of JHM.WU or A59, another hepatotropic strain, significantly attenuates replication of each strain in the liver and increases IFN-␤ expression in macrophages in culture. Our data indicate that both structural and nonstructural proteins contribute to MHV liver pathogenesis and support previous reports that nsp1 is a Betacoronavirus virulence factor. IMPORTANCEThe Betacoronavirus genus includes human pathogens, some of which cause severe respiratory disease. The spread of severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) into human populations demonstrates the zoonotic potential of emerging coronaviruses, and there are currently no vaccines or effective antivirals for human coronaviruses. Thus, it is important to understand the virus-host interaction that regulates coronavirus pathogenesis. Murine coronavirus infection of mice provides a useful model for the study of coronavirus-host interactions, including the determinants of tropism and virulence. We found that very small changes in coronavirus proteins can profoundly affect tropism and virulence. Furthermore, the hepatotropism of MHV-JHM depends not on the spike protein and viral entry but rather on a combination of the structural protein M and nonstructural replicase-associated proteins nsp1 and nsp13, which are conserved among betacoronaviruses. Understanding virulence determinants will aid in the design of vaccines and antiviral strategies. Mouse hepatitis virus (MHV) is an enveloped, nonsegmented, positive-strand RNA virus that belongs to the order Nidovirales, family Coronaviridae, and genus Betacoronavirus (1). Betacoronaviruses can have significant effects on human (2) and animal ...

show abstract

Section: Discussionmentioning

confidence: 66%

The nsp1, nsp13, and M Proteins Contribute to the Hepatotropism of Murine Coronavirus JHM.WU

Zhang

Cowley

et al. 2015

J Virol

View full text Add to dashboard Cite

show abstract

“…As substitutions in S2 may alter properties of S1 and S including stabilizing S1-S2 association, altering fusion behavior, and changing tissue tropism (16), it is likely that the T1015N mutation enhances entry and/or egress phenotypes in vitro. Although the origin of the tissue-culture adaptation is unclear, other studies have demonstrated the rapid emergence of tissue-culture adaptations in the M and S glycoproteins of human and zoonotic strains of SARS-CoV (17,18). As our stock was derived from an inoculum used for primate experiments (19), it is possible that this tissue-culture adaptation altered MERS-CoV pathogenic outcomes after in vivo infection.…”

Section: Discussionmentioning

confidence: 99%

Reverse genetics with a full-length infectious cDNA of the Middle East respiratory syndrome coronavirus

Scobey¹,

Yount²,

Sims³

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

275

341

View full text Add to dashboard Cite

Severe acute respiratory syndrome with high mortality rates (~50%) is associated with a novel group 2c betacoronavirus designated Middle East respiratory syndrome coronavirus (MERS-CoV). We synthesized a panel of contiguous cDNAs that spanned the entire genome. Following contig assembly into genome-length cDNA, transfected full-length transcripts recovered several recombinant viruses (rMERS-CoV) that contained the expected marker mutations inserted into the component clones. Because the wild-type MERS-CoV contains a tissue culture-adapted T1015N mutation in the S glycoprotein, rMERS-CoV replicated ~0.5 log less efficiently than wild-type virus. In addition, we ablated expression of the accessory protein ORF5 (rMERS•ORF5) and replaced it with tomato red fluorescent protein (rMERS-RFP) or deleted the entire ORF3, 4, and 5 accessory cluster (rMERS-ΔORF3-5). Recombinant rMERS-CoV, rMERS-CoV•ORF5, and MERS-CoV-RFP replicated to high titers, whereas MERS-ΔORF3-5 showed 1-1.5 logs reduced titer compared with rMERS-CoV. Northern blot analyses confirmed the associated molecular changes in the recombinant viruses, and sequence analysis demonstrated that RFP was expressed from the appropriate consensus sequence AACGAA. We further show dipeptidyl peptidase 4 expression, MERS-CoV replication, and RNA and protein synthesis in human airway epithelial cell cultures, primary lung fibroblasts, primary lung microvascular endothelial cells, and primary alveolar type II pneumocytes, demonstrating a much broader tissue tropism than severe acute respiratory syndrome coronavirus. The availability of a MERS-CoV molecular clone, as well as recombinant viruses expressing indicator proteins, will allow for high-throughput testing of therapeutic compounds and provide a genetic platform for studying gene function and the rational design of live virus vaccines.

show abstract

“…8A), and sporadic mutations were identified in either nsp3 or nsp14. Among these mutations, previous studies had suggested that a mutation at position 11 (E11K) in the MA15 M glycoprotein gene enhanced virus yields in cells, suggesting a key role in improving the efficiency of virus maturation and release in different hosts (46). Mutations in the mouse hepatitis virus (MHV) nsp14 gene, encoding a putative RNA proofreading function and 7-0-methyltransferase, were also shown to attenuate pathogenesis in mice, supporting the role of nsp14 as a putative virulence allele (9,13,50,68).…”

Section: Discussionmentioning

confidence: 99%

Molecular Determinants of Severe Acute Respiratory Syndrome Coronavirus Pathogenesis and Virulence in Young and Aged Mouse Models of Human Disease

Frieman

Yount

Agnihothram

et al. 2012

J Virol

144

166

View full text Add to dashboard Cite

SARS coronavirus (SARS-CoV) causes severe acute respiratory tract disease characterized by diffuse alveolar damage and hyaline membrane formation. This pathology often progresses to acute respiratory distress (such as acute respiratory distress syndrome [ARDS]) and atypical pneumonia in humans, with characteristic age-related mortality rates approaching 50% or more in immunosenescent populations. The molecular basis for the extreme virulence of SARS-CoV remains elusive. Since young and aged (1-year-old) mice do not develop severe clinical disease following infection with wild-type SARS-CoV, a mouse-adapted strain of SARS-CoV (called MA15) was developed and was shown to cause lethal infection in these animals. To understand the genetic contributions to the increased pathogenesis of MA15 in rodents, we used reverse genetics and evaluated the virulence of panels of derivative viruses encoding various combinations of mouse-adapted mutations. We found that mutations in the viral spike (S) glycoprotein and, to a much less rigorous extent, in the nsp9 nonstructural protein, were primarily associated with the acquisition of virulence in young animals. The mutations in S likely increase recognition of the mouse angiotensin-converting enzyme 2 (ACE2) receptor not only in MA15 but also in two additional, independently isolated mouse-adapted SARS-CoVs. In contrast to the findings for young animals, mutations to revert to the wild-type sequence in nsp9 and the S glycoprotein were not sufficient to significantly attenuate the virus compared to other combinations of mouse-adapted mutations in 12-month-old mice. This panel of SARS-CoVs provides novel reagents that we have used to further our understanding of differential, age-related pathogenic mechanisms in mouse models of human disease.

show abstract

Persistent Replication of Severe Acute Respiratory Syndrome Coronavirus in Human Tubular Kidney Cells Selects for Adaptive Mutations in the Membrane Protein

Cited by 55 publications

References 54 publications

The nsp1, nsp13, and M Proteins Contribute to the Hepatotropism of Murine Coronavirus JHM.WU

The nsp1, nsp13, and M Proteins Contribute to the Hepatotropism of Murine Coronavirus JHM.WU

Reverse genetics with a full-length infectious cDNA of the Middle East respiratory syndrome coronavirus

Molecular Determinants of Severe Acute Respiratory Syndrome Coronavirus Pathogenesis and Virulence in Young and Aged Mouse Models of Human Disease

Contact Info

Product

Resources

About