Further analysis of the role of calcium in rotavirus morphogenesis

doi:10.1016/0042-6822(87)90242-X

Virology

Volume 158, Issue 1, May 1987, Pages 103-111

https://doi.org/10.1016/0042-6822(87)90242-X Get rights and content

Abstract

Previously we reported that calcium plays an important role in the maturation of bovine rotavirus (M. S. Shahrabadi and P. W. K. Lee, 1986. Virology 152, 298–307). We now demonstrate that the formation of mature double-shelled (L) particles was strictly dependent on the concentration of calcium present in the growth medium. The formation of single-shelled (D) particles did not appear to be a calcium-mediated process. Subsequent labeling studies using ⁴⁵Ca revealed that calcium was incorporated into the L particles but not the D particles. The previously noted decreased level of the outer capsid protein VP7 (42K) in calcium-deprived cultures was now found to be due to the preferential degradation, and not to the impaired synthesis, of this protein in the absence of calcium. It was further demonstrated that calcium had a stabilizing effect on VP7 and that VP7 synthesized in the presence of calcium was not degraded upon subsequent calcium deprivation. Protein degradation during calcium deprivation was apparently limited to the mature form of VP7 since the unglycosylated precursor (pVP7), formed in the presence of tunicamycin, was found to be stable under this condition. Electron microscopic examination of infected cells revealed that in the presence of calcium, virus maturation took place by the budding of viral cores through the endoplasmic reticulum (ER). No such budding was observed in calcium-deprived cells. In these cells mature virions were absent and membrane fragments could be found associated with viral cores or single-shelled particles.

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Rhesus rotavirus VP6 regulates ERK-dependent calcium influx in cholangiocytes
2016, Virology
Citation Excerpt :
Ludert et al. (1987) observed that calcium driven viral uncoating does not occur when calcium concentration is equilibrated across the plasma membrane. Furthermore, Shahrabadi et al. showed that calcium reduction leads to impaired viral replication and titer (Shahrabadi et al., 1987). Our results are consistent with these observations, showing that impairment of calcium influx through the blockade of L-type calcium channels with Verapamil results in decreased viral titer.
The Rhesus rotavirus (RRV) induced murine model of biliary atresia (BA) is a useful tool in studying the pathogenesis of this neonatal biliary obstructive disease. In this model, the mitogen associated protein kinase pathway is involved in RRV infection of biliary epithelial cells (cholangiocytes). We hypothesized that extracellular signal-related kinase (ERK) phosphorylation is integral to calcium influx, allowing for viral replication within the cholangiocyte. Utilizing ERK and calcium inhibitors in immortalized cholangiocytes and BALB/c pups, we determined that ERK inhibition resulted in reduced viral yield and subsequent decreased symptomatology in mice. In vitro, the RRV VP6 protein induced ERK phosphorylation, leading to cellular calcium influx. Pre-treatment with an ERK inhibitor or Verapamil resulted in lower viral yields. We conclude that the pathogenesis of RRV-induced murine BA is dependent on the VP6 protein causing ERK phosphorylation and triggering calcium influx allowing replication in cholangiocytes.
Intracellular disassembly of infectious rotavirus particles by depletion of Ca<sup>2+</sup> sequestered in the endoplasmic reticulum at the end of virus cycle
2007, Virus Research
Rotavirus infection is characterized by a number of Ca²⁺ dependent virus–cell interactions. The structure of rotavirus triple-layered particles (TLP) is dependent on Ca²⁺ concentration. Acquisition of the capsid outer layer requires a high Ca²⁺ concentration inside the ER. Infection modifies Ca²⁺ homeostasis of the cell, increasing ER Ca²⁺ content, which may be advantageous to virus replication. We studied the role of sequestered Ca²⁺ on the stabilization of already mature viral particles within the ER. Thapsigargin (TG), a SERCA pump inhibitor, added for 30 min at the end of infection depleted ER Ca²⁺ and reduced the titer of already mature TLP accumulated in the cell. Another inhibitor, cyclopiazonic acid, and two Ca²⁺ ionophores (A23187 and ionomycin) in the presence of EGTA had similar effects. TG eliminated the peak of radiolabeled TLP, increasing that of DLP in CsCl gradients. Electron microscopy revealed accumulation of clustered particles in the ER, which had lost their integrity. The [Ca²⁺] in the ER of infected cells is important for virus maturation and for maintaining the integrity of mature TLP. Viral particles in this compartment may be potentially infectious, already containing VP7 and VP4.
Cytoplasmic calcium measurement in rotavirus enterotoxin-enhanced green fluorescent protein (NSP4-EGFP) expressing cells loaded with Fura-2
2003, Cell Calcium
The green fluorescent protein (GFP) and its analogs are standard markers of protein expression and intracellular localization of proteins. The fluorescent properties of GFP complicate accurate measurement of intracellular calcium using calcium sensitive fluorophores, which show a great degree of spectral overlap with GFP, or their K_d values are too high for accurate measurement of subtle changes in cytoplasmic calcium concentrations. Here we describe a simple modification of the standard microscope-based Fura-2 calcium-imaging technique which permits the quantitative measurement of intracellular calcium levels in cells expressing enhanced green fluorescent protein (EGFP) fusion proteins. Longpass emission filtering of the Fura-2 signal in cells expressing an EGFP fusion protein is sufficient to eliminate the EGFP–Fura-2 emission spectra overlap and allows quantitative calibration of intracellular calcium. To validate this technique, we investigated the ability of rotavirus enterotoxin NSP4-EGFP to elevate intracellular calcium levels in mammalian HEK 293 cells. We show here that inducible intracellular expression of NSP4-EGFP fusion protein elevates basal intracellular calcium more than two-fold by a phospholipase C (PLC) independent mechanism.
Influence of calcium on the early steps of rotavirus infection
2002, Virology
The structure of rotaviruses and many steps of their replication cycle depend on the concentration of calcium in the microenvironment. In this work, to learn about the role of calcium during the early steps of the infection, we characterized the effect of increasing the calcium concentration in the medium on the infectivity of rotaviruses. We found that a fivefold increase in the calcium concentration of the cell culture medium results in an increased viral titer in all rotavirus strains tested. The effect of this divalent ion seems to be mainly on the viral particle and not on the surface of the cell. Analysis of the intrinsic fluorescence spectra of purified triple-layered particles revealed that changes in the environment of tryptophan residues occurred as calcium concentration increased, suggesting that conformational changes in the viral particle might be responsible for the effect of this ion on the viral infectivity.
Purified recombinant rotavirus VP7 forms soluble, calcium-dependent trimers
2000, Virology
Rotavirus is a major cause of severe, dehydrating childhood diarrhea. VP7, the rotavirus outer capsid glycoprotein, is a target of protective antibodies and is responsible for the calcium-dependent uncoating of the virus during cell entry. We have purified, characterized, and crystallized recombinant rhesus rotavirus VP7, expressed in insect cells. A critical aspect of the purification is the elution of VP7 from a neutralizing monoclonal antibody column by EDTA. Gel filtration chromatography and equilibrium analytical ultracentrifugation demonstrate that, in the presence of calcium, purified VP7 trimerizes. Trimeric VP7 crystallizes into hexagonal plates. Preliminary X-ray analysis suggests that the crystal packing reproduces the hexagonal component of the icosahedral lattice of VP7 on triple-layered rotavirus particles. These data indicate that the rotavirus outer capsid assembles from calcium-dependent VP7 trimers and that dissociation of these trimers is the biochemical basis for EDTA-induced rotavirus uncoating and loss of VP7 neutralizing epitopes.
Role of Ca<sup>2+</sup> in the replication and pathogenesis of rotavirus and other viral infections
2000, Cell Calcium
Ca²⁺plays a key role in many pathological processes, including viral infections. Rotavirus, the major etiological agent of viral gastroenteritis in children and young animals, provides a useful model to study a number of Ca²⁺dependent virus–cell interactions. Rotavirus entry, activation of transcription, morphogenesis, cell lysis, particle release, and the distant action of viral porteins are Ca²⁺-dependent processes. In the extracellular medium, Ca²⁺stabilizes the structure of the viral capsid. During entry into the cell the low cytoplasmic Ca²⁺concentration induced the solubilization of the outer protein layer of the capsid and transcriptase activation. Viral protein synthesis modifies Ca²⁺homeostasis which, in turn, favours viral morphogenesis and induces cell death. The generation of diarrhea is a multifactorial process involving Ca²⁺-dependent secretory processes of mediators and water and electrolytes, as well as the induction of cell death in the different cell types that compose the intestinal epithelium. The discovery of the non-structural viral protein NSP4 as a viral enterotoxin and the possible participation of the enteric nervous system in the pathogenesis of diarrhea represent significant advances in its understanding. Ca²⁺also plays a role in the replication cycles and pathogenesis of other viral diseases such as poliovirus, Cocksackie virus, cytomegalovirus, vaccinia and measles virus and HIV.

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Further analysis of the role of calcium in rotavirus morphogenesis

Abstract

Lancet

Virology

Virus Res.

Virology

Ultrastructural study of rotavirus replication in cultured cells

J. Gen. Virol.

Rotavirus isolation and cultivation in the presence of trypsin

J. Clin. Microbiol.

Characterization of two particle types of calf rotavirus

J. Gen. Virol.

Activation of rotavirus RNA polymerase by calcium chelation

Arch. Virol.

Human virus gastroenteritis

Microbiol. Rev.

Proteolytic enhancement of rotavirus infectivity: Molecular mechanism

J. Virol.

Rotaviruses: a review

Curr. Top. Microbiol. Immunol.

Acute gastroenteritis associated with roovrus-like particles

J. Clin. Pathol.