Construction of recombinant pseudorabies viruses optimized for labeling and neurochemical characterization of neural circuitry.
In this study we have modified the neuroinvasiveness of pseudorabies virus strain Bartha, a commonly utilized trans-synaptic tract-tracer. In addition, we sought to facilitate detection of cellular mRNAs in neurons infected with the virus. In order to modify spreading characteristics, we inserted the lacZ or the GFP (green fluorescent protein) genes into the genomic loci containing the putative latency-associated transcript promoter (P(LAT2)), resulting in the disruption of the promoter function. Following rat kidney injection, mutant viruses labeled central autonomic neurons in a slower and much more restricted manner than the parent Bartha strain. Since both reporter genes were controlled by the human cytomegalovirus immediate early (IE) 1 promoter, they exhibited IE expression kinetics. This property proved to be important for the co-detection of reporter proteins with neuronal mRNAs, readily detected at early but not at late stage of infection, as shown in tyrosine-hydroxylase expressing A5 catecholaminergic neurons and in serotonin transporter expressing raphe magnus neurons.
Boldogköi
Zsolt
Z
Laboratory of Neuromorphology, Department of Anatomy, Semmelweis University, Budapest, Hungary.
Reichart
Aniko
A
Tóth
Ida E
IE
Sik
Attila
A
Erdélyi
Ferenc
F
Medveczky
Istvan
I
Llorens-Cortes
Catherine
C
Palkovits
Miklos
M
Lenkei
Zsolt
Z
D000818:Animals; D048429:Cell Size; D004274:DNA, Recombinant; D004279:DNA, Viral; D015967:Gene Expression Regulation, Viral; D017781:Genes, Immediate-Early; D017930:Genes, Reporter; D011558:Herpesvirus 1, Suid / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D017403:In Situ Hybridization; D007668:Kidney / Q000166:cytology / Q000502:physiology; D009415:Nerve Net; D009474:Neurons / Q000502:physiology* / Q000648:ultrastructure; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D051381:Rats; D017207:Rats, Sprague-Dawley; D013194:Staining and Labeling / Q000379:methods*; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism; D014779:Virus Replication
D004274:DNA, Recombinant; D004279:DNA, Viral; D014764:Viral Proteins