Rat dorsal root ganglia neurons as a model for Listeria monocytogenes infections in culture.

Neurotropism of Listeria monocytogenes was studied in rat dorsal root ganglia (DRG) and hippocampal neurons in culture. Using a system in which the DRG neurons can grow relatively free from other cells, it was observed that such DRG neurons, in contrast to hippocampal neurons, can be effectively infected by L. monocytogenes. The bacteria aligned along DRG axons, but not along hippocampal neurites. A mutant deficient in internalin, a protein required for entry into E-cadherin-expressing cells, did not interact with DRG neurons. Axonal migration of bacteria was studied in the DRG neurons grown in a double-chamber system, where either the neurites or the nerve cell bodies were exposed to the bacteria. The data suggest that L. monocytogenes can infect both axons and DRG nerve cell bodies, and that the bacteria can migrate in a retrograde as well as anterograde direction. These results support the notion that L. monocytogenes can spread via primary sensory neurons to the central nervous system. Infection of DRG primary sensory neurons, as employed in the present study, provides a model for analysis of bacterial and neuronal factors of importance for neurovirulence of L. monocytogenes.

Targeting of endoplasmic reticulum-associated proteins to axons and dendrites in rotavirus-infected neurons.

To analyze sorting and compartmentalization of molecules in neuronal endomembranes, the distribution of endogenous proteins associated with the endoplasmic reticulum (ER), intermediate compartment, the Golgi apparatus in cultures of dorsal root ganglion (DRG), and hippocampal neurons was compared with that of newly synthesized ER-associated rotavirus proteins. The endogenous ER-retained immunoglobulin heavy chain binding protein, protein disulfide isomerase, and a peptide containing the KDEL amino acid sequence appeared in the soma and dendrites up to their first branching, but not in axons. However, two other endogenous ER-associated proteins, calreticulin and calnexin, occurred in axons as well as in the somatodendritic domains. The ER-associated rotavirus proteins, VP7 and NSP4, were widely distributed in cell bodies and dendrites. The former appeared also in axons and its localization partially overlapped with that of calreticulin and calnexin. One intermediate compartment protein, ER-Golgi-intermediate compartment-protein-53 (ERGIC-53), extended beyond the first division of the dendrites and did not, as the small guanosine 5'-triphosphate (GTP)-binding protein rab2, appear in axons. The location of rab2 to small vesicles was distinct from that of rotavirus VP7. Cis/medial Golgi cistern proteins were restricted to the cell bodies and proximal dendrites. This study emphasizes the marked heterogeneity in the targeting to axons and dendrites of proteins associated with ER and intermediate compartments. Therefore, the composition of axonal ER-retained molecules differs from that in the soma and this variation may reflect differences in functions between the ER compartments. Viral proteins are useful reporters for such heterogeneities and rotavirus VP7 may be a tool to reveal sorting signals for targeting of vesicular proteins to axons via a nonclassical Golgi-independent mechanism. Such signals may also determine viral targeting to different regions of the brain.

Specific interactions between retrovirus Env and Gag proteins in rat neurons.

In this work we have studied the intracellular localization properties of the Gag and Env proteins of Moloney murine leukemia virus (MLV) and human immunodeficiency virus (HIV) in dorsal root ganglion (DRG) neurons of rat. These neurons form thick bundles of axons, which facilitates protein localization studies by immunofluorescence analyses. When such neuron cultures were infected with recombinant Semliki Forest virus particles carrying the gag genes of either retrovirus, the expressed Gag proteins were localized to both the somatic and the axonal regions of the DRG neurons. In contrast, the Env proteins were confined only to the somatic region. When the Gag and Env proteins were coexpressed, the Gag proteins were also excluded from the axons. This effect of the Env proteins was shown to be dependent on the concentration of the Gag proteins in the neuron and also to be specific for homologous pairs of retrovirus proteins. Therefore, the results suggest that there are specific interactions between the Env and the Gag proteins of MLV and HIV in the DRG neurons.

Assembly of viroplasm and virus-like particles of rotavirus by a Semliki Forest virus replicon.

In this study we have used an expression system based on Semliki Forest virus (SFV) to study assembly and intracellular localization of certain capsid proteins of rotavirus in neurons and mammalian epithelial cells. The complete genes of vp2 (vp2A) and vp6 (vp6A) of group A rotavirus (SA-11) and gene 5 encoding vp6 (vp6C) of porcine group C rotavirus (strain Cowden/AmC-1) were inserted into an SFV expression replicon. Transfection of BHK-21 cells with in vitro-made SFV transcripts resulted in a high level of expression of the heterologous genes. Cotransfection with helper RNA encoding the SFV structural proteins, but lacking the genomic RNA packing signal, resulted in production of recombinant infectious virus. Immunological and biochemical analysis revealed that vp6 was expressed to high levels in primary neurons and mammalian epithelial cells and that vp6 was retained as an authentic homotrimer, stabilized by noncovalent interactions with native antigenic determinants. Thin section electron microscopy analysis revealed that vp6 alone assembled into viroplasm-like structures in the cytoplasm. While coexpression of vp2 and vp6 of group A rotavirus resulted in formation of single-shelled-like particles, no evidence of intracellular assembly was found, suggesting that other viral proteins are required for intracellular formation of single-shelled particles. A notable observation was that the vp6 proteins of group A and C rotaviruses showed different immunofluorescence patterns in BHK-21 cells; vp6C displayed an intense punctate immunofluorescence pattern, while vp6A was characterized by a pronounced filamentous staining in close vicinity to the cytoskeleton.

Rotavirus causes selective vimentin reorganization in monkey kidney CV-1 cells.

The effect of rotavirus infection on cytoskeletal organization was examined in cultured African green monkey kidney (CV-1) cells. Rhesus rotavirus caused significant and selective changes in the organization of the vimentin filament network without having any effect on microtubules or actin. Double-immunofluorescence studies showed that at 6 h post-infection, and in the absence of cytopathic effect, the normal arrays of vimentin fibres radiating from multiple sites around the nucleus were lost. Vimentin fibres became irregularly distributed in the cytoplasm and were totally disrupted in the later stages of infection. Vimentin reorganization occurred independent of extracellular Ca2+ levels.

Microtubule-associated protein 2 appears in axons of cultured dorsal root ganglia and spinal cord neurons after rotavirus infection.

The immunohistochemical distribution of microtubule-associated protein 2 (MAP2), being normally restricted to nerve cell bodies and dendrites, became altered in rat dorsal root ganglia and spinal cord neurons in cultures infected with rhesus rotavirus. MAP2 appeared in axons of both sources of neurons as displayed with monoclonal antibodies to MAP2a + b and MAP2a + b + c at 48 hr post-infection (p.i.). Other cytoskeletal elements, i.e., tau, MAP1, MAP5, neurofilament, actin, and tubulin, did not reveal any alterations in the rotavirus-infected neurons. One of the rotavirus cytosolic proteins, the inner capsid protein vp6, was expressed in axons at 48 hr p.i. simultaneously with the appearance of MAP2, while two other viral proteins, vp4 and NS28, remained in the nerve cell bodies. By quantitative enzyme-linked immunosorbent assay (ELISA) a binding of single-shelled rotaviruses, which express vp6 on their surfaces, to purified MAP2 was found. There was no binding of these viral particles to tau or tubulin proteins. This study indicates that a selective interaction between certain viral and neuronal cytoskeletal proteins can occur and that a non-cytolytic viral infection can cause alterations in the polarized sorting of neuronal proteins.

The endoplasmic reticulum-associated VP7 of rotavirus is targeted to axons and dendrites in polarized neurons.

Rotavirus, which matures and is retained in the endoplasmic reticulum, was used to examine how polarized dorsal root ganglion and spinal cord neurons distributed cytoplasmic and endoplasmic reticulum-associated proteins. A remarkable observation was that NS28, a trans-endoplasmic reticulum-membrane protein which functions as a receptor for budding particles, remained in the cell body during the whole course of infection (48 h) while the VP7 glycoprotein, which is endoplasmic reticulum associated and usually retained in the endoplasmic reticulum, was targeted to axons already 4 h post infection. VP7 was furthermore transported in an endo-beta-N-acetylglucosaminidase H sensitive form through the secretory pathway. The segregated appearances of NS28 and the endo-beta-N-acetylglucosaminidase H sensitive VP7 indicate that VP7 enters a transport compartment separate from NS28. Brefeldin A treatment rapidly disintegrated the Golgi apparatus of the neurons and rapidly blocked axonal transport of Sendai virus glycoproteins, while axonal transport of rotavirus VP7 was not blocked, suggesting that VP7 uses an intracellular pathway in neurons which does not involve the Golgi apparatus.

Segregation of viral structural proteins in cultured neurons of rat spinal ganglia and cord.

Cultured spinal ganglion and spinal cord neurons were used to examine the intraneuronal distribution of five structural proteins of Sendai virus by immunohistochemistry. In spinal ganglion cells the internal, cytosolic viral proteins (the nucleocapsid, polymerase and matrix proteins) were confined to the perikarya, while the envelope glycoproteins (the haemagglutinin-neuraminidase and fusion proteins) also appeared in the axon-like processes. All five proteins occurred in the dendrite-like processes of spinal cord neurons. In both types of neuron the cytosolic viral proteins showed a pattern of distribution similar to that observed for the microtubule-associated protein MAP2. The segregated occurrence of the viral envelope and cytosolic proteins in axons may prevent virus assembly in axons and limit long-distance spread of paramyxoviruses in the nervous system.