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J. Virol., Jan 1998, 366-379, Vol 72, No. 1
Copyright © 1998, American Society for Microbiology

Defective growth correlates with reduced accumulation of a viral DNA replication protein after low-multiplicity infection by a human cytomegalovirus ie1 mutant

RF Greaves and ES Mocarski
Department of Medicine, Cambridge University Clinical School, Addenbrookes Hospital, United Kingdom. rfg@mole.bio.cam.ac.uk

To investigate the importance of the IE1 p72 regulatory protein during human cytomegalovirus replication, a recombinant virus unable to synthesize IE1 p72 was constructed. The Towne strain mutant CR208 lacked exon 4 of the major immediate-early gene and was isolated and complemented in an IE1-expressing immortalized human fibroblast line (ihfie1.3). Replication of CR208 in primary human fibroblasts was completed after an input multiplicity of 10 PFU/cell but was severely- impaired at 0.1 PFU/cell. CR208 formed plaques with lower efficiency on primary fibroblasts than on ihfiel.3 cells, and the relationship between the CR208 inoculum size and the resulting number of undersized plaques was nonlinear, indicating that multiple particles of CR208 were required to initiate lytic replication in a single primary fibroblast. After infection of primary fibroblasts with CR208 at 5 PFU/cell, a normal pattern of viral antigens was detected, although IE1 p72 was absent. During lower-multiplicity infections, IE2 protein was consistently detected at similar levels in a similar proportion of CR208-infected cells relative to the case for a Towne infection, but many fewer CR208-infected cells contained the ppUL44 polymerase accessory protein when evaluated at 24 or 48 h after infection. Furthermore, fibroblasts infected with CR208 at a low multiplicity failed to form viral DNA replication compartments, despite having expressed IE2 p86. These low-multiplicity growth and expression defects were corrected in two rescued derivatives of CR208 able to synthesize IE1 p72. One rescued virus (CR249) carried a deletion removing the large intron between exons 1 and 2 of the ie1-ie2 locus, revealing that this intron was dispensable for growth in cell culture.


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