Identification of the polymerase polyprotein products p72 and p65 of the murine coronavirus MHV-JHM

Virus Research
Volume 45, Issue 2, December 1996, Pages 101-109

Hong-QiangGao, Jennifer J.Schiller, Susan C.Baker

Abstract

The RNA polymerase gene of murine coronavirus MHV-JHM encodes a polyprotein of greater than 750 kDa. This polyprotein is proposed to be processed by two papain-like cysteine proteinases, PCP-1 and PCP-2, and a poliovirus 3C-like proteinase domain, 3C-pro, to generate protein products. The amino-terminal product of the MHV polymerase polyprotein, p28, is generated by cleavage of the polyprotein by PCP-1. To identify the viral products downstream of p28, we generated a fusion-protein specific antiserum directed against the region adjacent to p28 and used the antiserum to detect virus-specific proteins from MHV-JHM infected cells. When this antiserum was used to immunoprecipitate radiolabeled proteins from MHV-JHM infected cell lysates, virus-specific proteins of 72 and 65 kDa were detected. Furthermore, pulse and chase experiments demonstrated that p72 is likely a precursor to the mature protein product, p65. To investigate which viral proteinase may be responsible for generating p72 and p65, we expressed the 5′-region of the MHV-JHM RNA polymerase gene including the two papain-like cysteine proteinase domains in an in vitro transcription/translation system and analyzed the translation products for proteolytic processing. We also cloned and expressed the 72 kDa region immediately downstream from p28, and tested the ability of in vitro translated PCP-1 and PCP-2 to cleave p72 to p65 in trans. Our results indicate that neither viral proteinase domain PCP-1 nor PCP-2 is capable of cleavage of p72 to produce p65 in vitro. The role of MHV proteinases in the processing of p72 and p65 is discussed.

Keywords

Coronavirus, Polymerase polyprotein, Proteolytic processing

The Common Cold

Primary Care: Clinics in Office Practice
Volume 23, Issue 4, 1 December 1996, Pages 657-675

George L.KirkpatrickMD

Abstract

The common cold has intrigued physicians and the general public for centuries. It has been defined as an acute epidemic respiratory disease characterized by mild coryzal symptoms of rhinorrhea, nasal obstruction, and sneezing. The nasal discharge is usually copious and thin during the first 2 days of illness, then it generally becomes more viscous and purulent.22 The disease is self-limited. Symptoms may persist for 2 days to more than 14 days; however, the cold may abort after only 1 day. Fever, cough, sore throat, or lacrimation may or may not be present. The common cold is of itself harmless, but bacterial invasion frequently follows the initial infection. It is these secondary invaders that may produce disorders of serious consequence.

Keywords

common cold, epidemic, respiratory

Syncytia Formation Induced by Coronavirus Infection Is Associated with Fragmentation and Rearrangement of the Golgi Apparatus

Virology
Volume 221, Issue 2, 15 July 1996, Pages 325-334

EHUD LAVI, QIAN WANG, SUSAN R.WEISS, NICHOLAS K.GONATAS

Abstract

Coronavirus mouse hepatitis virus (MHV) possesses a membrane glycoprotein (M) which is targeted to the Golgi apparatus (GA). We used immunocytochemistry with an organelle-specific antiserum to investigate the morphologic changes of the GA during infection of L2 murine fibroblasts with MHV-A59. Twenty-four hours after infection the GA was fragmented and translocated in the center of syncytia, while the microtubular network was also rearranged displaying radiating elements toward the center of syncytia. Two fusion-defective mutants, which contain an identical amino acid substitution in the cleavage signal sequence of the spike glycoprotein (S), induced fragmentation of the GA. However, the GA migrated only partially to the centers of syncytia during infection with these mutants. Revertant viruses, in which the above mutation was corrected, had fusion properties and GA staining similar to wtMHV-A59. Experiments with brefeldin A (BFA), which induces redistribution of the GA into the rough endoplasmic reticulum (RER), revealed that an intact GA for a period of 4–16 hr postinfection, is required for coronavirus replication and syncytia formation. Thus, during MHV infection, syncytia formation is associated with fragmentation of the GA, followed by a previously undescribed phenomenon of migration of the organelle into the centers of syncytia. The fragmentation of the GA, however, may occur without the formation of syncytia. Therefore, two distinct mechanisms may be responsible for the fragmentation of the GA and its subsequent migration to the center of syncytia.

Keywords

corona virus, mouse hepatitis, golgi apparatus, glycoprotein

Growth characteristics and protein profiles of prototype and wild-type rat coronavirus isolates grown in a cloned subline of mouse fibroblasts (L2p.176 cells)

Virus Research
Volume 41, Issue 1, March 1996, Pages 55-68

Diane J.Gaertner, Susan R.Compton, Deborah F.Winograd, Abigail L.Smith

Abstract

Rat coronaviruses (RCVs) infect laboratory rats and confound biomedical research results. In vitro systems developed so far have limited the growth in knowledge about RCVs by not permitting generation of plaque-cloned virus stocks, reliable isolation of RCVs from rat tissues, or growth of high titered stocks of all isolates. Due to the fact that less than 20% of L2(Percy) cells were becoming infected, sublines were produced and selected for maximal growth of RCVs. Screening of 238 cell sublines yielded L2p.176 cells which were highly susceptible to all RCVs tested; however, susceptibility declined after 30 passages in vitro. Low-passaged L2p.176 cells were used to isolate virus from natural outbreaks and to propagate individual RCV plaques into high titered stocks. Proteins from six RCV isolates were immunoblotted using polyclonal rat and mouse antibodies to sialodacryoadenitis virus and polyclonal monospecific rabbit and goat antibodies against the peplomer (S) and nucleocapsid (N) proteins of mouse hepatitis virus (MHV). Proteins of two prototype, one Japanese and three wild type RCVs were examined and found to be similar to those of MHV, although the exact sizes and ratios of protein forms were unique for most RCV isolats. This study reports the development of a continuous cell line which reliably supports RCVs opening an opportunity for further in vivo studies of the biology of these agents. As a first step in the characterization of RCVs, we have shown that RCV proteins are very similar to those of MHV.

Keywords

Rat coronavirus, Rat, Cell line, Cell culture, Virus strains, Sialodacryoadenitis virus