Optogenetic manipulation of medullary neurons in the locust optic lobe.

The locust is a widely used animal model for studying sensory processing and its relation to behavior. Due to the lack of genomic information, genetic tools to manipulate neural circuits in locusts are not yet available. We examined whether Semliki Forest virus is suitable to mediate exogenous gene expression in neurons of the locust optic lobe. We subcloned a channelrhodopsin variant and the yellow fluorescent protein Venus into a Semliki Forest virus vector and injected the virus into the optic lobe of locusts ( Schistocerca americana). Fluorescence was observed in all injected optic lobes. Most neurons that expressed the recombinant proteins were located in the first two neuropils of the optic lobe, the lamina and medulla. Extracellular recordings demonstrated that laser illumination increased the firing rate of medullary neurons expressing channelrhodopsin. The optogenetic activation of the medullary neurons also triggered excitatory postsynaptic potentials and firing of a postsynaptic, looming-sensitive neuron, the lobula giant movement detector. These results indicate that Semliki Forest virus is efficient at mediating transient exogenous gene expression and provides a tool to manipulate neural circuits in the locust nervous system and likely other insects. NEW & NOTEWORTHY Using Semliki Forest virus, we efficiently delivered channelrhodopsin into neurons of the locust optic lobe. We demonstrate that laser illumination increases the firing of the medullary neurons expressing channelrhodopsin and elicits excitatory postsynaptic potentials and spiking in an identified postsynaptic target neuron, the lobula giant movement detector neuron. This technique allows the manipulation of neuronal activity in locust neural circuits using optogenetics.

Filamin A promotes dynamin-dependent internalization of hyperpolarization-activated cyclic nucleotide-gated type 1 (HCN1) channels and restricts Ih in hippocampal neurons.

The actin-binding protein filamin A (FLNa) regulates neuronal migration during development, yet its roles in the mature brain remain largely obscure. Here, we probed the effects of FLNa on the regulation of ion channels that influence neuronal properties. We focused on the HCN1 channels that conduct Ih, a hyperpolarization-activated current crucial for shaping intrinsic neuronal properties. Whereas regulation of HCN1 channels by FLNa has been observed in melanoma cell lines, its physiological relevance to neuronal function and the underlying cellular pathways that govern this regulation remain unknown. Using a combination of mutational, pharmacological, and imaging approaches, we find here that FLNa facilitates a selective and reversible dynamin-dependent internalization of HCN1 channels in HEK293 cells. This internalization is accompanied by a redistribution of HCN1 channels on the cell surface, by accumulation of the channels in endosomal compartments, and by reduced Ih density. In hippocampal neurons, expression of a truncated dominant-negative FLNa enhances the expression of native HCN1. Furthermore, acute abrogation of HCN1-FLNa interaction in neurons, with the use of decoy peptides that mimic the FLNa-binding domain of HCN1, abolishes the punctate distribution of HCN1 channels in neuronal cell bodies, augments endogenous Ih, and enhances the rebound-response ("voltage-sag") of the neuronal membrane to transient hyperpolarizing events. Together, these results support a major function of FLNa in modulating ion channel abundance and membrane trafficking in neurons, thereby shaping their biophysical properties and function.

Synaptic scaffolding protein Homer1a protects against chronic inflammatory pain.

Glutamatergic signaling and intracellular calcium mobilization in the spinal cord are crucial for the development of nociceptive plasticity, which is associated with chronic pathological pain. Long-form Homer proteins anchor glutamatergic receptors to sources of calcium influx and release at synapses, which is antagonized by the short, activity-dependent splice variant Homer1a. We show here that Homer1a operates in a negative feedback loop to regulate the excitability of the pain pathway in an activity-dependent manner. Homer1a is rapidly and selectively upregulated in spinal cord neurons after peripheral inflammation in an NMDA receptor-dependent manner. Homer1a strongly attenuates calcium mobilization as well as MAP kinase activation induced by glutamate receptors and reduces synaptic contacts on spinal cord neurons that process pain inputs. Preventing activity-induced upregulation of Homer1a using shRNAs in mice in vivo exacerbates inflammatory pain. Thus, activity-dependent uncoupling of glutamate receptors from intracellular signaling mediators is a novel, endogenous physiological mechanism for counteracting sensitization at the first, crucial synapse in the pain pathway. Furthermore, we observed that targeted gene transfer of Homer1a to specific spinal segments in vivo reduces inflammatory hyperalgesia. Thus, Homer1 function is crucially involved in pain plasticity and constitutes a promising therapeutic target for the treatment of chronic inflammatory pain.

Long-term exposure to bis(2-ethylhexyl)phthalate (DEHP) inhibits growth of guppy fish (Poecilia reticulata).

Bis(2-ethylhexyl)phthalate (DEHP) is a widely used plasticizer that is a commonly found contaminant of aquatic environments. However, little is known about the long-term effects of DEHP on fish development, as previous studies yielded conflicting results and mostly investigated the effects of concentrations higher than those found in natural habitats. We thus aimed to investigate the effects of DHEP (i) at concentrations present in the environment, and (ii) under conditions that might accentuate any deleterious consequences (larvae rather than adult fish, use of higher temperature). Different concentrations of DEHP (0.1-10 microg l(-1)rpar; applied continuously for 91 days were tested on guppy fish that were less than one week old at the beginning of the treatment. As early as 14 days after the start of exposure, guppies treated with 10 microg l(-1) DEHP showed significantly reduced body length as compared with control fish. The inhibitory effect of DEHP was concentration-dependent and increased with time, leading to a maximal reduction in body length of 15 and 40% at 1 and 10 microg l(-1) DEHP, respectively. The effect was even more pronounced for body weight, which was diminished by up to 40 and 70% at 1 and 10 microg l(-1) DEHP, respectively. The reduction in growth was still significant at 91 days of DEHP treatment, whereas the Fulton's condition factor was unaffected. While DEHP significantly blocked growth in both male and female guppies, no shift in the sexual development was observed. These data show that DEHP, at concentrations present in aquatic environments, can profoundly affect development in fish.

The protooncogene Ski controls Schwann cell proliferation and myelination.

Schwann cell proliferation and subsequent differentiation to nonmyelinating and myelinating cells are closely linked processes. Elucidating the molecular mechanisms that control these events is key to the understanding of nerve development, regeneration, nerve-sheath tumors, and neuropathies. We define the protooncogene Ski, an inhibitor of TGF-beta signaling, as an essential component of the machinery that controls Schwann cell proliferation and myelination. Functional Ski overexpression inhibits TGF-beta-mediated proliferation and prevents growth-arrested Schwann cells from reentering the cell cycle. Consistent with these findings, myelinating Schwann cells upregulate Ski during development and remyelination after injury. Myelination is blocked in myelin-competent cultures derived from Ski-deficient animals, and genes encoding myelin components are downregulated in Ski-deficient nerves. Conversely, overexpression of Ski in Schwann cells causes an upregulation of myelin-related genes. The myelination-regulating transcription factor Oct6 is involved in a complex modulatory relationship with Ski. We conclude that Ski is a crucial signal in Schwann cell development and myelination.

Knockout of ERK1 MAP kinase enhances synaptic plasticity in the striatum and facilitates striatal-mediated learning and memory.

Extracellular signal-regulated kinases (ERK1 and 2) are synaptic signaling components necessary for several forms of learning. In mice lacking ERK1, we observe a dramatic enhancement of striatum-dependent long-term memory, which correlates with a facilitation of long-term potentiation in the nucleus accumbens. At the cellular level, we find that ablation of ERK1 results in a stimulus-dependent increase of ERK2 signaling, likely due to its enhanced interaction with the upstream kinase MEK. Consistently, such activity change is responsible for the hypersensitivity of ERK1 mutant mice to the rewarding properties of morphine. Our results reveal an unexpected complexity of ERK-dependent signaling in the brain and a critical regulatory role for ERK1 in the long-term adaptive changes underlying striatum-dependent behavioral plasticity and drug addiction.

HGF induces fibronectin matrix synthesis in melanoma cells through MAP kinase-dependent signaling pathway and induction of Egr-1.

The matrix fibronectin protein is a multifunctional adhesive molecule that promotes migration and invasiveness of many tumors including melanomas. Increased fibronectin synthesis has been associated with the metastatic potential of melanoma cells; however, the molecular mechanisms underlying fibronectin overexpression during melanoma development are poorly understood. We report that hepatocyte growth factor/scatter factor (HGF) induces fibronectin expression and its extracellular assembly on the surface of melanoma cells through activation of mitogen-activated protein (MAP) kinase pathway, and induction and transcriptional activation of Early growth response-1 (Egr-1). Inhibition of B-RAF/MAP kinase pathway by dominant-negative mutants and by U0126-abrogated HGF-induced Egr-1, and chromatin immunoprecipitation showed that Egr-1 is bound to the fibronectin promoter in response to HGF. Exogenously expressed Egr-1 increased fibronectin levels, while blockage of Egr-1 activation by expression of the Egr-1 corepressor NAB2 interfered with the upregulation of fibronectin synthesis induced by HGF, indicating that Egr-1 exerts a significant role in fibronectin expression in response to HGF. Finally, analysis of the expression pattern of fibronectin in melanoma cells demonstrated that fibronectin levels are correlated with constitutive MAP kinase signaling. Our data define a novel mechanism that might have important implications in regulation of melanoma progression by autocrine HGF signaling or by constitutive activation of MAP kinase pathway.

Advanced modular self-inactivating lentiviral expression vectors for multigene interventions in mammalian cells and in vivo transduction.

In recent years, lentiviral expression systems have gained an unmatched reputation among the gene therapy community for their ability to deliver therapeutic transgenes into a wide variety of difficult-to-transfect/transduce target tissues (brain, hematopoietic system, liver, lung, retina) without eliciting significant humoral immune responses. We have cloned a construction kit-like self-inactivating lentiviral expression vector family which is compatible to state-of-the-art packaging and pseudotyping technologies and contains, besides essential cis-acting lentiviral sequences, (i) unparalleled polylinkers with up to 29 unique sites for restriction endonucleases, many of which recognize 8 bp motifs, (ii) strong promoters derived from the human cytomegalovirus immediate-early promoter (P(hCMV)) or the human elongation factor 1alpha (P(hEF1)(alpha)), (iii) P(hCMV-) or P(PGK-) (phosphoglycerate kinase promoter) driven G418 resistance markers or fluorescent protein-based expression tracers and (iv) tricistronic expression cassettes for coordinated expression of up to three transgenes. In addition, we have designed a size-optimized series of highly modular lentiviral expression vectors (pLenti Module) which contain, besides the extensive central polylinker, unique restriction sites flanking any of the 5'U3, R-U5-psi+-SD, cPPT-RRE-SA and 3'LTR(DeltaU3) modules or placed within the 5'U3 (-78 bp) and 3'LTR(DeltaU3) (8666 bp). pLentiModule enables straightforward cassette-type module swapping between lentiviral expression vector family members and facilitates the design of Tat-independent (replacement of 5'LTR by heterologous promoter elements), regulated and self-excisable proviruses (insertion of responsive operators or LoxP in the 3'LTR(DeltaU3) element). We have validated our lentiviral expression vectors by transduction of a variety of insect, chicken, murine and human cell lines as well as adult rat cardiomyocytes, rat hippocampal slices and chicken embryos. The novel multi-purpose construction kit-like vector series described here is compatible with itself as well as many other (non-viral) mammalian expression vectors for straightforward exchange of key components (e.g. promoters, LTRs, resistance genes) and will assist the gene therapy and tissue engineering communities in developing lentiviral expression vectors tailored for optimal treatment of prominent human diseases.

Alphaviral vectors for gene transfer into neurons.

Alphaviruses are small, enveloped positive-strand RNA viruses that have been successfully transformed into expression vectors in the case of Semliki Forest virus (SFV), Sindbis virus (SIN), and Venezuelan equine encephalitis virus. Compared to other viral vectors, their advantages are easy and fast generation of recombinant viral particles, rapid onset, and high-level transgene expression. When applied to neuronal tissue, SFV and SIN vectors possess the additional advantage of efficiently and preferentially transducing neurons rather than non-neuronal cells. This article gives an overview of the biology of SFV and SIN, their generation into expression vectors, and their application in neurobiology, with particular emphasis on the transduction of hippocampal neurons. In addition, it describes the more recent development of alphaviral vectors with decreased or absent cytotoxicity and lowered transgene expression, temperature-controllable gene expression, and altered host-cell specificity in the central nervous system (CNS). Finally, the review evaluates the use of SFV and SIN vectors in hippocampal tissue cultures vs recombinant lentivirus, adenovirus type 5, adeno-associated virus type 2, and measles virus.

Homer/Vesl proteins and their roles in CNS neurons.

Since their initial discovery in 1997, Homer/Vesl proteins have become increasingly investigated as putative regulators of receptor and ion-channel function in the central nervous system. Within a relatively brief period, numerous research reports have described manifold effects of Homer proteins, including the modulation of the trafficking of type I metabotropic glutamate receptors (mGluRs), axonal pathfinding, mGluR coupling to calcium and potassium channels, agonist-independent mGluR activity, ryanodine receptor regulation, locomotor activity, and behavioral plasticity. This review summarizes our current knowledge on the induction, expression, and structure of the various forms of Homer proteins, as well as their roles in neuronal function. In addition, we provide an outlook on novel developments with regard to the involvement of Homer-1a in hippocampal synaptic function.

Egr-1 upregulates the Alzheimer's disease presenilin-2 gene in neuronal cells.

Regulation of the Alzheimer's disease (AD)-related gene, presenilin-2 (PSEN2), was analyzed in neuronal (SK-N-SH) and non-neuronal (human embryonic kidney 293, HEK293) cells. We show that the PSEN2 regulatory region includes two separate promoter elements, each located upstream of multiple transcription start sites in the first and second exons. The stronger upstream promoter, P1, has housekeeping characteristics: it resides in a CpG island, is TATA-less, and up to 83% of PSEN2-P1 activity depends on a stimulating protein 1 (Sp1) site at the most 5' initiation site. However, the downstream promoter P2 includes neuronal-specific elements and two sites for early growth response gene-1 (Egr-1), a transcription factor upregulated in learning paradigms and implicated in neuronal plasticity, in response to injury. We show that Egr-1 binds to PSEN-P2, and that PSEN-P2 activity is increased threefold by overexpression of Egr-1, and by 12-O-tetradecanoylphorbol-13-acetate (TPA), which induces physiological Egr-1 levels. Egr-1 represses PSEN2-P1 activity by 50% in neuronal cells, suggesting it partially shifts promoter usage from PSEN2-P1 to PSEN2-P2. This could lead to a relative increase in shorter exon 2 transcripts, which may be more efficiently translated than exon 1 transcripts. Identification of PSEN2 as an Egr-1 target suggests a link between PSEN2 expression and Egr-1-related processes, which may impact on understanding PSEN-2's physiological function and its role in Alzheimer's disease.

Egr-1 target genes in human endothelial cells identified by microarray analysis.

Early growth response factor 1 (Egr-1) is a key transcriptional factor to mediate gene expression after vascular injury. To better understand the role of Egr-1 in vasculature, we globally profiled Egr-1 target genes in human endothelial cells using adenoviral gene transfer and Affymetrix oligonucleotide-based microarray technology. More than 300 genes regulated by >/=3-fold with Egr-1 overexpression were identified and, partially, confirmed by Northern and Western blotting, including genes for transcriptional regulators, signaling proteins, cell cycle regulatory proteins, growth factors, and cytokines. Among them, thymus-expressed chemokine (TECK) and IP-30 were dramatically induced by Egr-1, but TNFalpha-related apoptosis inducing ligand (TRAIL) was significantly repressed by Egr-1, suggesting that Egr-1 is a key mediator of inflammation and apoptosis in vascular cells. These data provide novel Egr-1 target genes and contribute to the understanding of the role of Egr-1 in vasculature.

Alphaviral gene transfer in neurobiology.

Semliki Forest virus (SFV), Sindbis virus (SIN), and Venezuelan equine encephalitis virus are simple, enveloped plus-strand RNA viruses belonging to the Alphavirus genus of the Togaviridae family. They have been developed into expression vectors that infect a wide host cell range and cause rapid and high-level transgene expression. Their easy and fast generation, classification into biosafety levels 1 and 2, and preferential transduction of neurons in cell and tissue cultures makes them an increasingly used gene transfer system. This review summarizes the alphaviral replication and expression, the replicon system, and its application in neurobiology. Alphaviral vectors can introduce multiple transgenes into host cells, and mutants with low or absent cytotoxicity and increased or decreased transgene expression levels are available. Temperature-dependent mutants permit to control the host cell specificity as well as the on- and offset of gene expression. These features, together with the transduction characteristics revealed in a direct comparison of alphaviral and other viral vectors in hippocampal slice cultures, make SFV and SIN vectors a powerful tool for neurobiological studies.

Tracing inputs to inhibitory or excitatory neurons of mouse and cat visual cortex with a targeted rabies virus.

BACKGROUND: Cortical inhibition plays a critical role in controlling and modulating cortical excitation, and a more detailed understanding of the neuronal circuits contributing to each will provide more insight into their roles in complex cortical computations. Traditional neuronal tracers lack a means for easily distinguishing between circuits of inhibitory and excitatory neurons. To overcome this limitation, we have developed a technique for retrogradely labeling inputs to local clusters of inhibitory or excitatory neurons, but not both, using neurotropic adenoassociated and lentiviral vectors, cell-type-specific promoters, and a modified rabies virus. RESULTS: Applied to primary visual cortex (V1) in mouse, the cell-type-specific tracing technique labeled thousands of presynaptically connected neurons and revealed that the dominant source of input to inhibitory and excitatory neurons is local in origin. Neurons in other visual areas are also labeled; the percentage of these intercortical inputs to excitatory neurons is somewhat higher (~20%) than to inhibitory neurons (<10%), suggesting that intercortical connections have less direct control over inhibition. The inputs to inhibitory neurons were also traced in cat V1, and when aligned with the orientation preference map revealed for the first time that long-range inputs to inhibitory neurons are well tuned to orientation. CONCLUSIONS: These novel findings for inhibitory and excitatory circuits in the visual cortex demonstrate the efficacy of our new technique and its ability to work across species, including larger-brained mammals such as the cat. This paves the way for a better understanding of the roles of specific cell types in higher-order perceptual and cognitive processes.

Distinct regional and subcellular localization of the actin-binding protein filamin A in the mature rat brain.

Filamin A (FLNa) is an actin-binding protein that regulates cell motility, adhesion, and elasticity by cross-linking filamentous actin. Additional roles of FLNa include regulation of protein trafficking and surface expression. Although the functions of FLNa during brain development are well studied, little is known on its expression, distribution, and function in the adult brain. Here we characterize in detail the neuroanatomical distribution and subcellular localization of FLNa in the mature rat brain, by using two antisera directed against epitopes at either the N' or the C' terminus of the protein, further validated by mRNA expression. FLNa was widely and selectively expressed throughout the brain, and the intensity of immunoreactivity was region dependent. The most intensely FLNa-labeled neurons were found in discrete neuronal systems, including basal forebrain structures, anterior nuclear group of thalamus, and hypothalamic parvocellular neurons. Pyramidal neurons in neocortex and hippocampus and magnocellular cells in basolateral amygdaloid nucleus were also intensely FLNa immunoreactive, and strong FLNa labeling was evident in the pontine and medullary raphe nuclei and in sensory and spinal trigeminal nuclei. The subcellular localization of FLNa was evaluated in situ as well as in primary hippocampal neurons. Punctate expression was found in somata and along the dendritic shaft, but FLNa was not detected in dendritic spines. These subcellular distribution patterns were recapitulated in hippocampal and neocortical pyramidal neurons in vivo. The characterization of the expression and subcellular localization of FLNa may provide new clues to the functional roles of this cytoskeletal protein in the adult brain.

Alphaviruses: Semliki forest virus and Sindbis virus vectors for gene transfer into neurons.

Alphaviral vectors based on Semliki Forest virus and Sindbis virus infect many host cell types, causing rapid and high-level transgene expression. In the CNS, Semliki Forest virus and Sindbis virus exhibit an outstanding preference for neurons rather than glial cells, compared to other viruses. Generation of high-titer virus stocks is rapid (less than two days) and typically requires biosafety level 1 or 2 containment. Wild-type vectors are cytotoxic, permitting short-term transgene expression. However, mutant vectors with decreased cytotoxicity, to prolong host cell survival, have been developed. They also increase transgene expression and cellular co-infection, permitting heteromeric protein expression in individual cells. In addition, mutants with temperature-dependent control of transgene expression and altered host cell preference to target interneurons and astrocytes rather than principal neurons are available. Other alphavirus vectors based on Venezuelan equine encephalitis and Eastern equine encephalitis virus replicons have been engineered, too. Alphavirus vectors have been successfully used not only in neuroscience, but also for other applications including drug discovery, structural biology, vaccine development, and cancer therapy.

Induction of early growth response-1 mediates microglia activation in vitro but is dispensable in vivo.

We have previously identified activation of microglia and induction of the early growth response gene 1 (Egr1) in the retina of retinoschisin-deficient (Rs1h(-/Y)) mice. We hypothesized that microglial expression of Egr1 might support retinal microgliosis. To test this, Egr1 transcript levels were determined in RNAs isolated from early postnatal retinas and primary microglia from Rs1h(-/Y) mice and wild-type controls. Egr1 mRNA expression was strongly induced in retinoschisin-deficient retinas as well as in ex vivo isolated microglia. Increased microglial Egr1 protein expression was concordantly detected in retinal sections of Rs1h(-/Y) mice using immunohistochemistry. Prominent activation-dependent Egr1 mRNA and protein expression was also confirmed in murine BV-2 microglia. Using binding site prediction and chromatin immunoprecipitation, we identified that the Egr1 promoter itself and the microglial marker genes Clec7a and Caspase11 are direct transcriptional targets of Egr1. Over-expression of Egr1 in BV-2 cells by adenoviral infection promoted Clec7a and Caspase11 mRNA synthesis, whereas expression of the Egr1 repressor NAB2 blocked the transcription of these genes. To analyze whether Egr1 was absolutely required for microglial marker expression in vivo, transcript levels were quantified in Rs1h(-/Y)/Egr1(-/-) retinas. No significant differences in activation marker expression could be measured in retinal tissue from Rs1h(-/Y)/Egr1(-/-) mice compared to Rs1h(-/Y) mice, suggesting that lack of Egr1 does not impair transcription of microglia genes in vivo. Taken together, our findings suggest that increased Egr1 expression is present in activated retinal microglia and contributes to their activation. However, up-regulation of Egr1 is not absolutely required for retinal microglia activation in vivo.

Alphaviruses: Semliki Forest virus and Sindbis virus vectors for gene transfer into neurons.

Alphaviral vectors based on Semliki Forest virus and Sindbis virus infect many host cell types, causing rapid and high-level transgene expression. Compared to other viruses used to infect CNS cell and tissue preparations, Semliki Forest virus and Sindbis virus exhibit an outstanding preference for neurons rather than glial cells. High-titer vector generation typically requires biosafety level 1 or 2 containment and occurs in less than 2 days. Wild-type vectors are cytotoxic, permitting short-term transgene expression. However, mutant vectors with decreased cytotoxicity, to prolong host cell survival, have been developed. They also increase transgene expression and cellular coinfection, permitting heteromeric protein expression in individual cells. Other mutants with temperature-dependent control of transgene expression and altered host cell preference to target interneurons and astrocytes rather than principal neurons are available. Because of these advantages, alphaviral vectors are increasingly used in neurobiological and other studies, including structural biology, vaccine development, and cancer treatment.

Semliki Forest virus vectors with mutations in the nonstructural protein 2 gene permit extended superinfection of neuronal and non-neuronal cells.

Semliki Forest virus (SFV) vectors are widely used in neurobiological studies because they efficiently infect neurons. As with any viral vector, they possess a limited cloning capacity, so infection with different SFV vectors may be required to introduce multiple transgenes into individual cells. However, this approach is limited by superinfection exclusion. The authors examined marker expression in baby hamster kidney cells, mouse cortical neurons, and rat hippocampal neurons using different fluorophore-encoding vectors that are based on the wild-type SFV4 strain and on the less cytopathic SFV4(PD) mutant, which carries two point mutations in nonstructural protein 2. For every fluorophore tested, SFV4(PD) gave higher (up to 22-fold) expression compared to SFV4. In infections using two and three different vectors, SFV4 caused relatively few multifluorescent baby hamster kidney cells when applied at 0-s, 15-min, or 2-h intervals. In contrast, SFV4(PD) permitted significantly enhanced marker coexpression, resulting in 46% doubly and 21% triply fluorescent baby hamster kidney cells, and 67% to 8% doubly fluorescent cortical and hippocampal neurons. At 15-min or 2-h addition intervals, SFV4(PD) still permitted 23% to 36% doubly fluorescent baby hamster kidney cells. The increased efficiency of SFV4(PD) in coexpressing separate markers from different viral particles suggests that mutations in nonstructural protein 2 affect alphaviral superinfection exclusion. The results demonstrate that SFV4(PD) is well-suited to coexpress multiple proteins in neuronal and non-neuronal cells. This capability is particularly valuable to express the various components of heteromeric protein complexes, especially when the individual cDNAs cannot be combined into single SFV particles.