Regulation of the initiation of coronavirus JHM infection in primary oligodendrocytes and L-2 fibroblasts.

Upon maturation, primary rat oligodendrocytes become resistant to coronavirus JHM (JHMV) infection at an early stage. Involvement of cAMP-dependent protein kinase (PK) in the regulation of oligodendrocyte differentiation has been established (S. Beushausen et al. (1987). J. Virol. 61, 3795-3803). An inducer which accelerates maturation, dibutyryl cyclic AMP (dbcAMP) also upregulates the expression of the regulatory subunit, R1 of PK1. Since (i) early block preventing infection of mature oligodendrocytes can be bypassed when transfection with genomic RNA is used and (ii) inhibitors of PKs counteract the dbcAMP effect, so as to alleviate the inhibition of JHMV, enhanced expression of R1 appeared to be connected with virus restriction. This idea was confirmed following upregulation of the R1 gene in fully permissive L-2 cells. There was a connection between an effect due to R1 and dephosphorylation of the nucleocapsid protein N by an endosomal phosphoprotein phosphatase (PPPase) having the properties of types 1 or 2A enzyme which occurs during penetration of inoculum virions. An inhibition in vitro (cell free) of N dephosphorylation by R1 together with evidence that in vivo (cell culture) overexpression of R1 inhibited the endosomal PPPase as well as replication of JHMV supports the hypothesis that uncoating of the JHMV inoculum occurs after dephosphorylation, a step obligatory for dissociation of the N protein from the genome. Thus inhibition by R prevents uncoating and thereby interferes with the commencement of replication. These observations intimate the existence of a novel mechanism controlling a virus infection of specific cell target(s) undergoing a process of differentiation and maturation in the central nervous system.

The murine coronavirus as a model of trafficking and assembly of viral proteins in neural tissue.

The replication of JHM, a murine coronavirus, provides a useful model of the assembly and dissemination of viral components in neuronal cells. Involvement of microtubules in virus trafficking is an important feature which may explain dissemination of the infection from primary cell targets at olfactory, hippocampal and cerebellar sites within the central nervous system, resulting in severe neuropathies.

Distribution and trafficking of JHM coronavirus structural proteins and virions in primary neurons and the OBL-21 neuronal cell line.

The neurotropic murine coronavirus JHM is capable of inducing various forms of neurologic diseases, including demyelination. Neurons have been shown to act as a repository site at the early stages of the disease process (O. Sorensen and S. Dales, J. Virol. 56:434-438, 1985). JHM virus (JHMV) replication and trafficking of viral proteins and virions in cultured rat hippocampal neurons and a neuronal cell line, OBL-21, were examined, with an emphasis placed on the role of the microtubular network. We show here that JHMV spread within the central nervous system occurs transneuronally and that virus protein trafficking was dependent upon microtubules. Viral trafficking occurred asymmetrically, involving both the somatodendritic and the axonal domains. Thus coronavirus can be disseminated from neurons at either the basolateral or the apical domains. A specific interaction between antibodies derived against the microtubule-associated protein tau and JHMV nucleocapsid protein (N) was observed, which can presumably be explained by an overall amino acid similarity of 44% and an identity of 20% between proteins N and tau, with optimal alignment at the microtubule binding domain of tau. Collectively, our data suggest an important role of the microtubule network in viral protein trafficking and distribution. They also draw attention to protein sequence mimicry of a cell component by this coronavirus as one strategy for making use of the host's functions on behalf of the virus.

Studies on the stability of a human adenovirus-rabies recombinant vaccine.

Human adenovirus type 5 containing the rabies virus glycoprotein gene (rHAd-RG1) has potential for the oral vaccination of animals. The stability of this recombinant was tested indoors and outdoors by measuring the loss in virus infectivity. Under indoor conditions the stability of the recombinant virus was studied in an egg yolk-containing commercial stabilizer and a simple buffered salt solution (EBSS; Earle's balanced salt solution) at 4 degrees C and room temperature (24-25 degrees C). Over 16 days, there was a more rapid loss in virus titer at room temperature than at 4 degrees C in both suspending media; however, these differences were slight and may be significant when the overall stability of the vaccine is considered. When the virus was mixed with either 10% (w/v) fox or skunk feces or EBSS, placed on stainless steel disks and the disks kept under ambient conditions (air temperature 24-25 degrees C; relative humidity 45-50%), there was a more rapid decline in virus titer in the fecal suspensions (3% remained after 72 h) than in EBSS (26% remained after 72 h). When bait-coated blister packs of the vaccine were placed in an outdoor location in the fall (October) season, there was a larger drop in the virus titer for vaccines placed in the sun (54% over 32 days) than for those in the shade (40% over 32 days). Incorporating proteinaceous stabilizers in the vaccine samples for outdoor study showed virus stability was not enhanced in their presence.(ABSTRACT TRUNCATED AT 250 WORDS)