Our previous studies demonstrated that the mechanism of MV transmission in neurons differed dramatically from that in non-neuronal cells (Lawrence et al., 2000
). In fibroblasts, MV was highly cytolytic and resulted in robust release of infectious particles. In contrast, no virus-mediated lysis of infected primary hippocampal neurons was detected, nor could extracellular virus be recovered, despite extensive viral replication and spread. Electron microscopy studies revealed both a block in the ability of MV to bud from the neuronal plasma membrane as well as the presence of viral RNPs at synaptic junctions, indicating that MV adopts a trans-synaptic mode of spread in neurons (Lawrence et al., 2000
). Interestingly, trans-synaptic transmission was CD46-independent, a characteristic that appears to be unique to neurons, as CD46 was required for viral spread in fibroblast co-cultures (Makhortova, et. al, manuscript in press).
A synthetic tripeptide, FIP, that blocks MV fusion (Norrby, 1971
; Payan et al., 1984
; Richardson and Choppin, 1983
), inhibited both MV infection and spread in primary neurons expressing one of the human receptors, CD46. The mechanism by which FIP prevents fusion is not known, but may either be due to direct interference of the amino terminus of the F1 subunit with the target cell membrane, or to preventing the exposure of the fusogenic peptide (Norrby, 1971
; Richardson and Choppin, 1983
; Kelsey, 1991
The similarity of sequence between FIP and the neurotransmitter Substance P, coupled with previous reports that Substance P could interfere with MV replication in nonneuronal cells (Schroeder, 1986
; Richardson, Scheid, and Choppin, 1980
) prompted us to ask whether Substance P and other members of the neurotachykinin family could prevent viral translocation across the synaptic cleft. Indeed, all neurotachykinins blocked spread, a process that appeared to depend, at least in part, on the presence of the neurotachykinin receptor, NK-1. The majority of NK-1 deficient neonates, as well as NK-1+
adults mice treated with the specific antagonist, aprepitant, survived viral challenge without any symptoms of disease or viral replication in the CNS. Thus, we speculate that NK-1 may promote MV entry into CD46+
neurons by serving as a docking receptor for the MV-F protein, and that the ability of FIP and Substance P to block infection and spread may be due to competitive inhibition with the NK-1 receptor, a hypothesis currently under study in our laboratory.
The apparent ability of NK-1 to mediate viral spread at the neuronal synapse in the absence of an H receptor may be a unique attribute of this specialized membrane. MV-Ed trans-synaptic spread can occur in the absence of CD46, and primary mouse neurons do not express the other identified MV receptor, SLAM/CD150w. While these data indicate that trans-synaptic spread may not require a receptor for the H protein, we cannot rule out the possibility that a protein expressed at the post-synaptic membrane may serve as a unique H-receptor in neurons, specifically facilitating neuron-to-neuron transport across synapses. However, given the similarity in sequence and activity of Substance P and the fusion inhibitor FIP, we favor the idea that F, through binding to neurokinin receptors, facilitates transport of MV RNPs across the synapse in the absence of a need for H. Importantly, NK-1 is expressed in all mammals and on virtually all cells, including the Vero fibroblasts used as controls in our experiments (data not shown). Thus, we are also currently testing whether NK-1 is a necessary co-factor for MV entry into all permissive cells. Moreover, given that these studies were performed with MV-Edmonston, a vaccine strain, it will be important to address what role NK-1 plays in entry and spread of wild type isolates.
While our data implicate an important role for NK-1 in MV spread in neurons, some infected mice with deleted or impaired NK-1 did become infected, and neither FIP nor Substance P were able to fully ablate MV spread in primary neurons. This inability could be explained by: a) an insufficient concentration of these inhibitors at the synaptic membrane; b) a rapid off-rate of Substance P from NK-1 (Sarntinoranont, Iadarola, and Morrison, 2003
) and/or c) the potential for MV-F to utilize multiple neurokinin receptors, including NK-2 and NK-3, for entry. Because all three neurotachykinins inhibited MV spread to some degree (), and each serves as the ligand for three different neurokinin receptors, it remains possible that the other neurokinin receptors, NK-2 and NK-3, may serve as surrogates for MV entry under circumstances in which NK-1 is not present or active.
A recent paper provides additional support for the fusion-tachykinin relationship. The F protein of bovine respiratory syncytial virus (BRSV), a member of the Paramyxovirus
family, is post-translationally cleaved twice, unlike MV-F, which is cleaved once. The small peptide that is released from BRSV-F cleavage is converted to a biologically active tachykinin, called virokinin (Zimmer et al., 2003
). This soluble, virus-encoded peptide interacts with NK-1 and triggers the same downstream pathways as the bona fide
ligand, Substance P. Thus, fusion proteins or post-translationally cleaved products derived from the fusion proteins of multiple Paramyxoviruses
appear to interact with neurokinin receptors, though the benefit such an interaction confers to the virus is not yet understood.
Finally, these data may have implications for MV pathogenesis in the CNS. In the NSE-CD46 mouse model of neuronal MV infection, CNS disease occurs in infected, immunodeficient animals in the absence of neuronal death (Patterson et al., 2002
), suggesting that CNS disease in this mouse model is due to neuronal dysfunction rather than overt cell loss. We hypothesize that MV-mediated fusion of neuronal synapses could promote neuropathology through a number of possible mechanisms, including constitutive neurotransmitter signaling or loss of synaptic integrity. Continued use of this model system will allow us to determine how receptor interactions may contribute to neuropathology, and may identify novel candidates for antiviral therapies to prevent or limit MV-induced neuropathology in humans.