Role of Endocytosis and Low pH in Murine Hepatitis Virus Strain A59 Cell Entry

ABSTRACT Infection by the coronavirus mouse hepatitis virus strain A59 (MHV-A59) requires the release of the viral genome by fusion with the respective target membrane of the host cell. Fusion is mediated by the viral S protein. Here, the entry pathway of MHV-A59 into murine fibroblast cells was studied by independent approaches. Infection of cells assessed by plaque reduction assay was strongly inhibited by lysosomotropic compounds and substances that interfere with clathrin-dependent endocytosis, suggesting that MHV-A59 is taken up via endocytosis and delivered to acidic endosomal compartments. Infection was only slightly reduced in the presence of substances inhibiting proteases of endosomal compartments, precluding that the endocytic uptake is required to activate the fusion potential of the S protein by its cleavage. Fluorescence confocal microscopy of labeled MHV-A59 confirmed that virus is taken up via endocytosis. Bright labeling of intracellular compartments suggests their fusion with the viral envelope. No fusion with the plasma membrane was observed at neutral pH conditions. However, when virus was bound to cells and the pH was lowered to 5.0, we observed a strong labeling of the plasma membrane. Electron microscopy revealed low pH triggered conformational alterations of the S ectodomain. Very likely, these alterations are irreversible because low-pH treatment of viruses in the absence of target membranes caused an irreversible loss of the fusion activity. The results imply that endocytosis plays a major role in MHV-A59 infection and the acidic pH of the endosomal compartment triggers a conformational change of the S protein mediating fusion.

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Protection from lethal coronavirus infection by affinity-purified spike glycoprotein of murine hepatitis virus, strain A59

Virology ◽

10.1016/0042-6822(90)90057-x ◽

1990 ◽

Vol 174 (1) ◽

pp. 87-94 ◽

Cited By ~ 29

Author(s):

Claude Daniel ◽

Pierre J. Talbot

Keyword(s):

Virus Strain ◽

Hepatitis Virus ◽

Spike Glycoprotein ◽

Murine Hepatitis Virus ◽

Coronavirus Infection

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Characterization of the Coronavirus Mouse Hepatitis Virus Strain A59 Small Membrane Protein E

Journal of Virology ◽

10.1128/jvi.74.5.2333-2342.2000 ◽

2000 ◽

Vol 74 (5) ◽

pp. 2333-2342 ◽

Cited By ~ 102

Author(s):

Martin J. B. Raamsman ◽

Jacomine Krijnse Locker ◽

Alphons de Hooge ◽

Antoine A. F. de Vries ◽

Gareth Griffiths ◽

...

Keyword(s):

Virus Strain ◽

Hepatitis Virus ◽

Expression System ◽

Mouse Hepatitis Virus ◽

Viral Envelope ◽

Membrane Structures ◽

Electron Microscopic ◽

E Protein ◽

Infected Cells ◽

Virus Expression

ABSTRACT The small envelope (E) protein has recently been shown to play an essential role in the assembly of coronaviruses. Expression studies revealed that for formation of the viral envelope, actually only the E protein and the membrane (M) protein are required. Since little is known about this generally low-abundance virion component, we have characterized the E protein of mouse hepatitis virus strain A59 (MHV-A59), an 83-residue polypeptide. Using an antiserum to the hydrophilic carboxy terminus of this otherwise hydrophobic protein, we found that the E protein was synthesized in infected cells with similar kinetics as the other viral structural proteins. The protein appeared to be quite stable both during infection and when expressed individually using a vaccinia virus expression system. Consistent with the lack of a predicted cleavage site, the protein was found to become integrated in membranes without involvement of a cleaved signal peptide, nor were any other modifications of the polypeptide observed. Immunofluorescence analysis of cells expressing the E protein demonstrated that the hydrophilic tail is exposed on the cytoplasmic side. Accordingly, this domain of the protein could not be detected on the outside of virions but appeared to be inside, where it was protected from proteolytic degradation. The results lead to a topological model in which the polypeptide is buried within the membrane, spanning the lipid bilayer once, possibly twice, and exposing only its carboxy-terminal domain. Finally, electron microscopic studies demonstrated that expression of the E protein in cells induced the formation of characteristic membrane structures also observed in MHV-A59-infected cells, apparently consisting of masses of tubular, smooth, convoluted membranes. As judged by their colabeling with antibodies to E and to Rab-1, a marker for the intermediate compartment and endoplasmic reticulum, the E protein accumulates in and induces curvature into these pre-Golgi membranes where coronaviruses have been shown earlier to assemble by budding.

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Effect of eicosanoids on induction of procoagulant activity by murine hepatitis virus strain 3 in vitro

Prostaglandins ◽

10.1016/0090-6980(91)90013-6 ◽

1991 ◽

Vol 42 (6) ◽

pp. 501-513 ◽

Cited By ~ 6

Author(s):

S.W. Chung ◽

S.B. Sinclair ◽

L.S. Fung ◽

E.H. Cole ◽

G.A. Levy

Keyword(s):

Virus Strain ◽

Hepatitis Virus ◽

Procoagulant Activity ◽

Murine Hepatitis Virus

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Enhanced Virulence Mediated by the Murine Coronavirus, Mouse Hepatitis Virus Strain JHM, Is Associated with a Glycine at Residue 310 of the Spike Glycoprotein

Journal of Virology ◽

10.1128/jvi.77.19.10260-10269.2003 ◽

2003 ◽

Vol 77 (19) ◽

pp. 10260-10269 ◽

Cited By ~ 51

Author(s):

Evelena Ontiveros ◽

Taeg S. Kim ◽

Thomas M. Gallagher ◽

Stanley Perlman

Keyword(s):

Virus Strain ◽

Hepatitis Virus ◽

Neurological Diseases ◽

Mouse Hepatitis Virus ◽

Neutralizing Antibody ◽

Lateral Spread ◽

S Protein ◽

Acute Encephalitis ◽

The Central Nervous System

ABSTRACT The coronavirus, mouse hepatitis virus strain JHM, causes acute and chronic neurological diseases in rodents. Here we demonstrate that two closely related virus variants, both of which cause acute encephalitis in susceptible strains of mice, cause markedly different diseases if mice are protected with a suboptimal amount of an anti-JHM neutralizing antibody. One strain, JHM.SD, caused acute encephalitis, while infection with JHM.IA resulted in no acute disease. Using recombinant virus technology, we found that the differences between the two viruses mapped to the spike (S) glycoprotein and that the two S proteins differed at four amino acids. By engineering viruses that differed by only one amino acid, we identified a serine-to-glycine change at position 310 of the S protein (S310G) that recapitulated the more neurovirulent phenotype. The increased neurovirulence mediated by the virus encoding glycine at position S310 was not associated with a different tropism within the central nervous system (CNS) but was associated with increased lateral spread in the CNS, leading to significantly higher brain viral titers. In vitro studies revealed that S310G was associated with decreased S1-S2 stability and with enhanced ability to mediate infection of cells lacking the primary receptor for JHM (“receptor-independent spread”). These enhanced fusogenic properties of viruses encoding a glycine at position 310 of the S protein may contribute to spread within the CNS, a tissue in which expression of conventional JHM receptors is low.

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A novel potentially recombinant rodent coronavirus with a polybasic cleavage site in the spike protein

Journal of Virology ◽

10.1128/jvi.01173-21 ◽

2021 ◽

Author(s):

Xin Li ◽

Liang Wang ◽

Peipei Liu ◽

Hongying Li ◽

Shuting Huo ◽

...

Keyword(s):

Rural Areas ◽

Urban Areas ◽

Cleavage Site ◽

One Health ◽

Hepatitis Virus ◽

Southern China ◽

Genome Sequences ◽

Murine Hepatitis Virus ◽

S Protein ◽

Pcr Screening

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has reignited global interest in animal coronaviruses and their potential for human transmission. While bats are thought to be the wildlife reservoir of SARS-CoV and SARS-CoV-2, the widespread human coronavirus OC43 is thought to have originated in rodents. We sampled 297 rodents and shrews representing eight species in three municipalities of southern China. We report coronavirus prevalence of 23.3% and 0.7% in Guangzhou and Guilin, respectively, with samples from urban areas having significantly higher coronavirus prevalence than those from rural areas. We obtained three coronavirus genome sequences from Rattus norvegicus , including a Betacoronavirus RCoV-GCCDC3, an Alphacoronavirus RCoV-GCCDC5 and a novel Betacoronavirus RCoV-GCCDC4. Recombination analysis suggests that there was a potential recombinant event involving RCoV-GCCDC4, Murine hepatitis virus and Longquan Rl rat coronavirus. Furthermore, we uncovered a polybasic cleavage site RARR in the spike (S) protein of RCoV-GCCDC4, which is dominant in RCoV. These findings provide further information on the potential for inter-species transmission of coronaviruses and demonstrate the value of a One Health approach to virus discovery. Importance Surveillance of viruses among rodents within rural and urban areas of South China identified three rodent coronaviruses RCoV-GCCDC3, 4 and 5, one of which was identified as a novel potentially recombinant coronavirus with a polybasic cleavage site in the spike (S) protein. Through reverse transcription PCR screening of coronaviruses, we found that coronavirus prevalence in urban areas is much higher than that in rural areas. Subsequently, we obtained three coronavirus genome sequences by deep sequencing. After different method-based analyses, we found that RCoV-GCCDC4 was a novel potentially recombinant coronavirus with a polybasic cleavage site in S protein, dominant in RCoV. This newly identified coronavirus RCoV-GCCDC4 with its potentially recombinant genome and polybasic cleavage site provides a new insight into the evolution of coronaviruses. Furthermore, our results provide further information on the potential for inter-species transmission of coronaviruses and demonstrate the necessity of a One Health approach for zoonotic disease surveillance.

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