Human immunodeficiency virus type 1 assembly and lipid rafts: Pr55(gag) associates with membrane domains that are largely resistant to Brij98 but sensitive to Triton X-100.

The assembly and budding of human immunodeficiency virus type 1 (HIV-1) at the plasma membrane are directed by the viral core protein Pr55(gag). We have analyzed whether Pr55(gag) has intrinsic affinity for sphingolipid- and cholesterol-enriched raft microdomains at the plasma membrane. Pr55(gag) has previously been reported to associate with Triton X-100-resistant rafts, since both intracellular membranes and virus-like Pr55(gag) particles (VLPs) yield buoyant Pr55(gag) complexes upon Triton X-100 extraction at cold temperatures, a phenotype that is usually considered to indicate association of a protein with rafts.

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.

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.

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.

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.

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.

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.

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.

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.