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The human cytomegalovirus (HCMV) basic phosphoprotein pp 150, encoded by the UL32 gene, together with the two other major phosphoproteins, pp65 (ppUL83) and pp71 (ppUL82) and several minor structural proteins, form the tegument around the viral nucleocapsid. Experiments were undertaken to locate the area of assembly of tegument proteins ppl50 and pp65 and nucleocapsids in fibroblasts, in order to assess the functional role of these two structural proteins in HCMV morphogenesis. Whereas ppl50 expression starts during the cytoplasmic maturation of HCMV, pp65 is expressed in the early and late phases of HCMV gene transcription. Western blot analysis of isolated cell fractions showed that ppl50 is initially (48 h postinfection) localized in the nucleus, associated either with the nuclear membrane or with viral assembly regions, and later (72 h post-infection) in the cytoplasm. By indirect immunofluorescence, ppl50 and pp65 could be detected in nuclear subcompartments and were strongly associated with the nuclear membrane. Using immunogold analysis by electron microscopy, pp65 was exclusively detected within the matrix of cytoplasmic and extracellular dense bodies and of dense body-like structures in the nucleoplasm. These were localized in close contact with hypertrophic nucleoli, in the proximity of developing nucleocapsids and in special patches at the inner nuclear membrane L Positive immunostaining of ppl50 was observed at the surface of developing nucleocapsids concentrated within viral assembly regions in the nucleoplasm. Additionally, the tegument of cytoplasmic and extracellular virions was stained, whereas dense bodies or nuclear dense body-like structures did not react. Thus, the acquisition of the tegument protein pp 150 seems to start in special nuclear subcompartments of the HCMV-infected fibroblasts.

Human cytomegalovirus (HCMV) open reading frame UL32 codes for the basic phosphoprotein pp150 (ppUL32), an abundant constituent of the virion tegument. In order to study its potential role in the assembly and/or transport of progeny particles, astrocytoma cell lines (U373MG) were generated, stably expressing a 2n1 kb 5h fragment of UL32 in antisense orientation under the control of the HCMV major immediate early promoter. The steady-state level of the UL32 sense mRNA and pp150 synthesis were strongly reduced in infected antisense cell lines. Neither immediate early and early gene expression, nor viral DNA replication, was inhibited ; the expression of the late gene product gB (gpUL55) was also reduced, but mainly at the level of translation. Control experiments indicated that this dif-

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