Several recent reports indicate that cholesterol might play an important role in human immunodeficiency virus type 1 (HIV-1) replication. We investigated the effects of HIV-1 infection on cholesterol biosynthesis and uptake using microarrays. HIV-1 increased gene expression of cholesterol genes in both transformed T-cell lines and primary CD4 ؉ T cells. Consistent with our microarray data, 14 C-labeled mevalonate and acetate incorporation was increased in HIV-1-infected cells. Our data also demonstrate that changes in cholesterol biosynthesis and uptake are only observed in the presence of functional Nef, suggesting that increased cholesterol synthesis may contribute to Nef-mediated enhancement of virion infectivity and viral replication.
Prion protein (PrP) is a glycosylphosphatidylinositol (GPI) anchored cell surface protein expressed by many cells, including those of the mammalian nervous system. At present the physiologic functions of PrP remain unclear. Deletion of Prnp, the gene encoding PrP in mice, has been shown to alter normal synaptic and electrophysiologic activities, indicating a potential role in seizure susceptibility. However, published efforts to link PrP with seizures, using both in vivo and in vitro models, are conflicting and difficult to interpret due to use of various mouse backgrounds and seizure induction techniques. Here we investigated the role of PrP in kainic acid (KA)-induced seizure sensitivity, using three types of mice. In contrast to previous published results, Prnp−/− mice on the C57BL/10SnJ background had a significant decrease in KA-induced seizure susceptibility. In genetic complementation experiments using a PrP-expressing transgene, genes derived from strain 129/Ola, which flanked the Prnp−/− locus in C57BL/10SnJ mice, rather than Prnp itself, appeared to account for this effect. Furthermore, using coisogenic 129/Ola mice differing only at Prnp, this difference was not reproduced when comparing PrP-negative and PrP-positive mice. In contrast, substrains of PrP-expressing C57BL mice, showed large variations in KA-induced seizure sensitivity. The magnitude of these differences in susceptibility was larger than that associated with the presence of the Prnp gene, suggesting extensive influence of genes other than Prnp on seizure sensitivity in this system.
Specimens from a patient of the 2000 Israel West Nile virus epidemic were analyzed by reverse transcription-polymerase chain reaction. Products corresponding to E, NS3, and NS5 sequences were amplified from cerebellar but not from cortical samples. Phylogenetic analyses indicated a closer relationship of this isolate to 1996 Romanian and 1999 Russian than to 1998-99 Israeli or 1999 New York isolates.
Changes in the envelope proteins of retroviruses can alter the ability of these viruses to infect the central nervous system (CNS) and induce neurological disease. In the present study, nine envelope residues were found to influence neurovirulence of the Friend murine polytropic retrovirus Fr98. When projected on a threedimensional model, these residues were clustered in two spatially separated groups, one in variable region B of the receptor binding site and the other on the opposite side of the envelope. Further studies indicated a role for these residues in virus replication in the CNS, although the residues did not affect viral entry.Amino acid sequence variations in retroviral envelope proteins play a critical role in altering viral pathogenesis and the host response. Human immunodeficiency virus envelope variants vary in chemokine induction in vitro and may influence neurovirulence in vivo (4,18,19). Similarly, simian immunodeficiency virus, feline immunodeficiency virus, and murine retrovirus envelopes play a role in regulating virus infection, host response, and neurological disease (1,3,8,11,15). Determining which amino acid residues are required for neurovirulence may provide important information on how retroviruses induce damage in the central nervous system (CNS).The neurovirulence of the polytropic murine retrovirus Fr98 is encoded within the SphI-ClaI restriction sites of the viral genome, which contain the 3Ј end of the polymerase and most of the viral envelope gene (16). The polytropic Fr54, which differs from Fr98 by multiple nucleotide substitutions in the SphI-ClaI region, does not induce neurovirulence, despite neuroinvasion and infection of similar brain cell types (16). Two separate areas of the SphI-ClaI region influence neurovirulence, one region within the SphI-EcoRI (SE) restriction sites and one within the EcoRI-ClaI (EC) restriction sites (6). These regions mediate pathogenesis by separate mechanisms, as viruses encoding only the SE or EC region of the Fr98 genome induce disease more slowly than Fr98 does (6).Previous studies mapped the residues in the EC region responsible for neurovirulence to two residues at positions 165 and 168 (17) in the receptor binding domain (RBD) (5). However, the Fr98 residues in the SE region which are associated with neurovirulence have not been identified. In the present study, we analyzed which amino acids encoded by the SE fragment of the Fr98 envelope gene were necessary or sufficient for the induction of neurological disease.A common restriction site, BbsI, found in the 5Ј end of the envelope gene for both Fr54 and SE was used to generate a chimeric virus, BE, that coded for Fr98 residues in the envelope region, but not in the polymerase gene. Newborn inbred Rocky Mountain White (IRW) mice injected with BE by intraperitoneal inoculation developed clinical signs of ataxia and/or seizures at 20 to 50 days postinoculation (Fig. 1A), similar to that of mice injected with SE (17). Thus, the neurovirulent determinants of SE were encoded within the BE reg...
Prion protein (PrP) is expressed on a wide variety of cells and plays an important role in the pathogenesis of transmissible spongiform encephalopathies. However, its normal function remains unclear. Mice that do not express PrP exhibit deficits in spatial memory and abnormalities in excitatory neurotransmission suggestive that PrP may function in the glutamatergic synapse. Here we show that transport of D-aspartate, a non-metabolized L-glutamate analog, through excitatory amino acid transporters (EAATs) was faster in astrocytes from PrP knockout (PrPKO) mice than in astrocytes from C57BL/10SnJ wildtype (WT) mice. Experiments using EAAT subtype-specific inhibitors demonstrated that in both WT and PrPKO astrocytes, the majority of transport was mediated by EAAT1. Furthermore, PrPKO astrocytes were more effective than WT astrocytes at alleviating L-glutamate-mediated excitotoxic damage in both WT and PrPKO neuronal cultures. Thus, in this in vitro model, PrPKO astrocytes exerted a functional influence on neuronal survival and may therefore influence regulation of glutamatergic neurotransmission in vivo.
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