Quantitative competitive NASBA for measuring mRNA expression levels of the immediate early 1, late pp67, and immune evasion genes US3, US6 and US11 in cells infected with human cytomegalovirus.
Different cell types were infected with human cytomegalovirus (HCMV) and RNA expression dynamics were analyzed by quantitative NASBA assays for IE1 (UL123), pp67 (UL65) and the immune evasion genes (US3, US6 and US11). The quantitative NASBA assays gave reproducible quantification in the range of 10(3)-10(6) RNA copies for IE1 and pp67 RNA, from 3x10(3) to 1x10(6) RNA copies for US6 and US11 RNA, and from 1x10(4) to 1x10(6) RNA copies for US3 RNA. SMC, HAEC and HUVEC cells infected with an, in endothelial cells, propagated HCMV strain (VHL/E) showed similar RNA expression dynamics for the analyzed genes. Expression of all genes studied was observed within the first 4 h post-infection. The first gene for which expression could be detected was IE1, followed by US3, US11, pp67 and US6. Fibroblasts infected with HCMV strain AD169 showed a different RNA expression pattern for US3. Translation of the mRNA studied was demonstrated by detection of the proteins 48 h post-infection by immunofluorescence.
Greijer
A E
AE
Organon Teknika, Boxtel, The Netherlands.
Adriaanse
H M
HM
Kahl
M
M
Tacken
N M
NM
Oldenburg
N
N
Sijlmans
A
A
van de Crommert
J M
JM
Dekkers
C A
CA
Sillekens
P T
PT
Middeldorp
J M
JM
D002478:Cells, Cultured / Q000821:virology; D003587:Cytomegalovirus / Q000235:genetics* / Q000276:immunology / Q000378:metabolism*; D003586:Cytomegalovirus Infections / Q000821:virology; D006023:Glycoproteins; D006801:Humans; D017874:Immediate-Early Proteins / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D021161:Self-Sustained Sequence Replication / Q000379:methods*; D012680:Sensitivity and Specificity; D014759:Viral Envelope Proteins / Q000235:genetics / Q000378:metabolism; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
D006023:Glycoproteins; C088645:IE1 protein, cytomegalovirus; D017874:Immediate-Early Proteins; D008565:Membrane Proteins; D012333:RNA, Messenger; D012367:RNA, Viral; D016601:RNA-Binding Proteins; C074546:US11 protein, herpesvirus; C095111:US3 protein, cytomegalovirus; D014759:Viral Envelope Proteins; D014764:Viral Proteins; C039447:glycoprotein D, Human herpesvirus 1