Varicella-zoster virus (VZV) transcription during latency in human ganglia: construction of a cDNA library from latently infected human trigeminal ganglia and detection of a VZV transcript.
The entire varicella-zoster virus (VZV) genome appears to be present in latently infected human ganglia, but the extent of virus DNA transcription is unknown. Conventional methods to study virus gene transcripts by Northern (RNA) blotting are not feasible, since ganglia are small and VZV DNA is not abundant. To circumvent this problem, we prepared radiolabeled cDNA from ganglionic RNA, hybridized it to Southern blots containing VZV DNA, and demonstrated the presence of a transcript within the SalI C fragment of the virus genome (R. Cohrs, R. Mahalingam, A. N. Dueland, W. Wolf, M. Wellish, and D. H. Gilden, J. Infect. Dis. 166:S24-S29, 1992). To further map VZV transcripts, in the work described here we constructed a cDNA library from poly(A)+ RNA obtained from latently infected human ganglia. Phage DNA isolated from the library was used in PCR amplifications to detect VZV-specific inserts. The specificity of the PCRs was provided by selection of a primer specific for VZV gene 17, 18, 19, 20, or 21 and a second vector-specific primer. VZV gene 21-specific sequences were identified by PCR amplification. The PCR product contained the XhoI cloning site and poly(A)+ sequences between vector and VZV gene 21 sequences. The sequence motif at the 3' end of VZV gene 21, determined by cloning and sequencing of the PCR product, consisted of 49 to 51 nucleotide bases of 3'-untranslated DNA, the termination codon for the VZV gene 21 open reading frame, and DNA sequences reading into the VZV gene 21 open reading frame.
Cohrs
R J
RJ
Department of Neurology, University of Colorado Health Sciences Center, Denver 80262.
Srock
K
K
Barbour
M B
MB
Owens
G
G
Mahalingam
R
R
Devlin
M E
ME
Wellish
M
M
Gilden
D H
DH
D000368:Aged; D000369:Aged, 80 and over; D000818:Animals; D000914:Antibodies, Viral / Q000097:blood; D001483:Base Sequence; D002460:Cell Line; D002522:Chlorocebus aethiops; D017931:DNA Primers; D018076:DNA, Complementary; D004279:DNA, Viral / Q000096:biosynthesis / Q000302:isolation & purification; D005260:Female; D015870:Gene Expression; D015723:Gene Library; D005814:Genes, Viral; D005822:Genetic Vectors; D014645:Herpesvirus 3, Human / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology*; D006801:Humans; D066298:In Vitro Techniques; D007668:Kidney; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D014158:Transcription, Genetic; D012668:Trigeminal Ganglion / Q000821:virology*; D017735:Virus Latency
D000914:Antibodies, Viral; D017931:DNA Primers; D018076:DNA, Complementary; D004279:DNA, Viral