Increased long chain acyl-Coa synthetase activity and fatty acid import is linked to membrane synthesis for development of picornavirus replication organelles.

All positive strand (+RNA) viruses of eukaryotes replicate their genomes in association with membranes. The mechanisms of membrane remodeling in infected cells represent attractive targets for designing future therapeutics, but our understanding of this process is very limited. Elements of autophagy and/or the secretory pathway were proposed to be hijacked for building of picornavirus replication organelles. However, even closely related viruses differ significantly in their requirements for components of these pathways. We demonstrate here that infection with diverse picornaviruses rapidly activates import of long chain fatty acids. While in non-infected cells the imported fatty acids are channeled to lipid droplets, in infected cells the synthesis of neutral lipids is shut down and the fatty acids are utilized in highly up-regulated phosphatidylcholine synthesis. Thus the replication organelles are likely built from de novo synthesized membrane material, rather than from the remodeled pre-existing membranes. We show that activation of fatty acid import is linked to the up-regulation of cellular long chain acyl-CoA synthetase activity and identify the long chain acyl-CoA syntheatse3 (Acsl3) as a novel host factor required for polio replication. Poliovirus protein 2A is required to trigger the activation of import of fatty acids independent of its protease activity. Shift in fatty acid import preferences by infected cells results in synthesis of phosphatidylcholines different from those in uninfected cells, arguing that the viral replication organelles possess unique properties compared to the pre-existing membranes. Our data show how poliovirus can change the overall cellular membrane homeostasis by targeting one critical process. They explain earlier observations of increased phospholipid synthesis in infected cells and suggest a simple model of the structural development of the membranous scaffold of replication complexes of picorna-like viruses, that may be relevant for other (+)RNA viruses as well.

Specific cleavage of the nuclear pore complex protein Nup62 by a viral protease.

Previous work has shown that several nucleoporins, including Nup62 are degraded in cells infected with human rhinovirus (HRV) and poliovirus (PV) and that this contributes to the disruption of certain nuclear transport pathways. In this study, the mechanisms underlying proteolysis of Nup62 have been investigated. Analysis of Nup62 in lysates from HRV-infected cells revealed that Nup62 was cleaved at multiple sites during viral infection. The addition of purified HRV2 2A protease (2A(pro)) to uninfected HeLa whole cell lysates resulted in the cleavage of Nup62, suggesting that 2A(pro) is a major contributor to Nup62 processing. The ability of purified 2A(pro) to cleave bacterially expressed and purified Nup62 demonstrated that 2A(pro) directly cleaves Nup62 in vitro. Site-directed mutagenesis of putative cleavage sites in Nup62 identified six different positions that are cleaved by 2A(pro) in vitro. This analysis revealed that 2A(pro) cleavage sites were located between amino acids 103 and 298 in Nup62 and suggested that the N-terminal FG-rich region of Nup62 was released from the nuclear pore complex in infected cells. Analysis of HRV- and PV-infected cells using domain-specific antibodies confirmed that this was indeed the case. These results are consistent with a model whereby PV and HRV disrupt nucleo-cytoplasmic trafficking by selectively removing FG repeat domains from a subset of nuclear pore complex proteins.

Inducible nitric oxide synthase in Theiler's murine encephalomyelitis virus infection.

We investigated the role of inducible nitric oxide synthase (iNOS) in Theiler's murine encephalomyelitis virus (TMEV) infection of susceptible (SJL) and resistant (C57BL/6 [B6]) strains of mice. TMEV is an excellent model of virus-induced demyelinating disease, such as multiple sclerosis (MS). Previous studies of others have suggested that NO may play a role in the pathogenesis of demyelinating disease. The presence and level of iNOS were determined in the brains and spinal cords of SJL and B6 TMEV-infected mice by the following methods: (i) PCR amplification of iNOS transcripts, followed by Southern blotting with an iNOS-specific probe, and (ii) immunohistochemical staining with an anti-iNOS-specific affinity-purified rabbit antibody. iNOS-specific transcripts were determined in the brains and spinal cord of both SJL and B6 TMEV-infected mice on days 0 (control), days 3, 6, and 10 (encephalitic stage of disease), and days 39 to 42, 66, and 180 (demyelinating phase) postinfection (p.i.). iNOS-specific transcripts were found in the brains and spinal cords of both SJL and B6 TMEV-infected mice at 6, 10, and 39 (SJL) days p.i., but they were absent in mock-infected mice and in TMEV-infected SJL and B6 mice at 0, 3, 66, and 180 days p.i. Immunohistochemical staining confirmed the presence of iNOS protein in both TMEV-infected SJL and B6 mice at days 6 and 10 p.i., but not at days 0, 3, 66, and 180 days p.i. Weak iNOS staining was also observed in TMEV-infected SJL mice at 42 days p.i. iNOS-positive staining was found in reactive astrocytes surrounding areas of necrotizing inflammation, particularly in the midbrain. Weak iNOS staining was also observed in cells of the monocyte/macrophage lineage in areas of parenchymal inflammation and necrosis (mesencephalon) and in leptomeningeal and white matter perivascular infiltrates of the spinal cord. Rod-shaped microglia-like cells and foamy macrophages (myelin-laden) were iNOS negative. These results suggest that NO does not play a direct role in the late phase of demyelinating disease in TMEV-infected mice.

A mechanism of quinolinic acid formation by brain in inflammatory neurological disease. Attenuation of synthesis from L-tryptophan by 6-chlorotryptophan and 4-chloro-3-hydroxyanthranilate.

Quinolinic acid (QUIN), kynurenic acid (KYNA) and L-kynurenine (L-KYN) are neuroactive kynurenine pathway metabolites that accumulate in inflammatory neurological diseases. These increases were attributed to the induction of indoleamine-2,3-dioxygenase (IDO), the enzyme that converts L-tryptophan into L-KYN. Direct conversion of L-tryptophan into QUIN by brain tissue occurs in conditions of CNS inflammation, but not by normal brain tissue. To investigate whether increased activity of enzymes distal to IDO may determine L-KYN conversion to QUIN, rhesus macaques were inoculated with poliovirus directly into the spinal cord, as a model of focal inflammatory neurological disease (FASEB J. 6, 2977-2989, 1992). Induction of spinal cord IDO (35.9-fold) accompanied smaller, but proportional increases in kynurenine-3-hydroxylase (2.4-fold) and kynureninase (2.3-fold) activities, which were correlated to CSF and tissue QUIN levels, as well as to measures of inflammatory lesions. 3-Hydroxyanthranilate-3,4-dioxygenase activity was unchanged. Cerebrospinal fluid KYNA levels increased in proportion to both IDO activity and L-KYN accumulation, though kynurenine aminotransferase activity was unaffected. Cerebrospinal fluid neopterin, a marker of macrophage and immune activation, accumulated in proportion to the responsive enzymes and metabolites. The cell types involved in producing QUIN were investigated in vitro. Human foetal brain cultures consisting of astrocytes and neurons converted large quantities of [13C6]L-tryptophan into L-KYN when stimulated by gamma-interferon, but very little [13C6]QUIN was formed unless macrophages (THP-1 cells) were first added to the cultures (to model a key component of brain inflammation). [13C6]L-Tryptophan was converted into [13C6]QUIN by either gamma-interferon stimulated macrophages, or following intracisternal administration into poliovirus-infected macaques. Inhibitors of the kynurenine pathway, 6-chlorotryptophan and 4-chloro-3-hydroxyanthranilic acid, attenuated [13C6]QUIN formation by macrophages, and when co-infused with [13C6]L-tryptophan into poliovirus-infected macaques. These results suggest roles for increased activities of IDO, kynurenine-3-hydroxylase and kynureninase in accelerating the synthesis of QUIN, L-KYN and KYNA in conditions of brain inflammation. Macrophage infiltrates, and perhaps microglia, are important sources of QUIN, whereas constitutive brain cells and macrophages are sources of L-KYN. Drugs that inhibit kynurenine pathway enzymes attenuate QUIN formation in the CNS, and provide tools to examine the consequences of reduced QUIN accumulation.