Human cytomegalovirus virions differentially incorporate viral and host cell RNA during the assembly process

Abstract

While analyzing human cytomegalovirus (HCMV) gene expression in infected cells by RNA-specific nucleic acid sequence-based amplification (NASBA), positive results were observed for HCMV RNA encoded by several viral genes immediately after the addition of the virus. UV-inactivated virus also gave a positive NASBA result without establishing active infection, suggesting that RNA was associated with the inoculum. Highly purified virions devoid of cellular contamination proved to be positive for viral RNA encoding both immediate-early (UL123) and late (UL65) gene products. Virion-associated RNA might be incorporated specifically or without selection during the virion assembly. In the latter case, cellular RNA would also be present in the virion. A high-abundant cellular RNA encoded by GAPDH and even U1A RNA, which is expressed at low levels, were detected in the virion fraction, whereas cellular DNA was absent. Virion fractionation revealed that cellular RNA was absent in purified de-enveloped capsids. In conclusion, cellular and viral RNA was present between the capsid and envelope of the virion, whereas in the capsid only viral RNA could be detected. The results suggest that virion-associated viral and cellular RNA is incorporated nonspecifically during virion assembly.

FIG. 1

NASBA and PCR analyses of the nucleic acids detected in virions, capsids, viral DNA, and infected cells. Nucleic acids were isolated from gradient-purified virions (lanes 1 to 3), de-enveloped capsids (lanes 4 to 6), viral nucleic acids (lanes 7 to 9), and infected cells (lanes 10 to 12). (A) IE2.3 NASBA assay; (B) DNA amplification by PCR of pp67 (UL65); (C through E) NASBA assay of pp67 (UL65) (C); U1A (D); and GAPDH (E). −, untreated; + DNase 1, DNase 1 treatment; + RNase A, RNase A treatment.