The analysis of gene expression and regulation has been hindered thus far, however, by the limited number of mapped genes. Only a few proteins of HCMV-infected cells can at present be assigned to defined regions on the genome \cite{Cranage_1986, Heilbronn_1987, Jahn_1987, Kouzarides_1987, Mach_1986, Mocarski_1985, Ruger_1987}; from such studies it appears that E and late genes of HCMV are scattered over the entire genome. In contrast, the IE genes are clustered in a restricted area \cite{Jahn_1984a, Stinski_1983}. The major IE gene of HCMV (IE1) and its gene product, a nuclear phosphoprotein of $M_r$ 68K to 72K, as well as two other genes from the IE region (IE2 and IE3) have been extensively characterized \cite{Boshart_1985, Henninghausen_Fleckenstein_1986, Jahn_1984a, Staprans_Spector_1986, Stenberg_1984, Wilkinson_1984}. Here we report on the structure of a fourth gene from the IE region of HCMV that appears to be different from all other IE genes thus far described in herpesviruses. The transcript from this gene, an RNA of about 5 kb, appears to be present in RNA preparations from infected cells purified at IE, E and late times after infection \cite{DeMarchi_1983, Jahn_1984a, Wathen_Stinski_1982}. It spans a DNA fragment for which \textit{in vitro} transforming activity has been reported \cite{Nelson_1984}. Completed sequence data, fine mapping and kinetics of the 5 kb RNA in the infected permissive cell are provided.

\section{Methods}

\subsection{Viruses and cell culture}
Strains AD169 and Towne as well as wild-type HCMV were propagated in primary human foreskin fibroblast cell cultures according to standard procedures. Total cell IE and late RNA and RNA from uninfected cells (mock infection (MI) RNA) was purified as described before \cite{Jahn_1984a}. Total E RNA from HCMV-infected cells was prepared in the presence of either phosphonoacetic acid (PAA) or phosphonoformic acid (PFA), two potent inhibitors of the virus-encoded DNA polymerase, as described by \cite{Wathen_Stinski_1982}.

\subsection{RNA purification}
Total cellular RNA, as well as poly(A)$^{+}$ and poly(A)$^{-}$ RNA, was purified according to published procedures \cite{Chirgwin_1979, Jahn_1984a} with the exceptions that guanidine thiocyanate buffer (5.7 M guanidine thiocyanate, 50 mM-lithium citrate, 0.1\% lithium laurylsulphate, 0.1\% 2-mercaptoethanol) was used instead of guanidine hydrochloride buffer for cell lysis and that the final treatment of the RNA probes with proteinase K was omitted. Cytoplasmic and nuclear fractions of RNA were isolated as described by \cite{Ortin_Doerfler_1975}. After separation and centrifugation at 3000 r.p.m. for 10 min at 4$^\circ$C, the supernatant was mixed with 3 volumes of guanidine thiocyanate buffer and centrifuged through a CsCl cushion for 20 to 24 h at 28000 r.p.m. in an SW41 rotor to give the cytoplasmic fraction. Similarly, the pellet (nuclear fraction) was resuspended in guanidine thiocyanate buffer and centrifuged as just described. The resulting pellets were resuspended and RNA was ethanol-precipitated.

\subsection{Northern blot hybridization}
Northern blot analyses were carried out with 0.5 to 1.0\% denaturing agarose gels as described \cite{Jahn_1984a}. Double-stranded DNA probes were labelled either by nick translation or, in order to increase sensitivity, by replacement synthesis using T4 DNA polymerase. Hybridization conditions and washings were as described earlier. For hybridization of synthetic oligonucleotides to Northern blots, the DNA was labelled at the 5$'$ end with [$\gamma$-$^{32}$P]dATP to a specific activity of $10^7$ to $10^8$ c.p.m./$\mu$g. Hybridization was carried out in a buffer containing 25\% deionized formamide, 5$\times$ SSC, 0.1\% SDS, 1$\times$ Denhardt's solution, 20 mM-sodium phosphate pH 7.0 and 100 $\mu$g of yeast RNA per ml at 25$^\circ$C to 30$^\circ$C for at least 48 h. Washing was done three times for 15 min each in a buffer containing 2$\times$ SSC, 0.1\% SDS and 20 mM-sodium phosphate, at 40 to 42$^\circ$C.

\subsection{Nuclease protection analysis}
Nuclease protection analyses with dsDNA probes were carried out by a modification of the technique of \cite{Berk_Sharp_1977} using either S1 nuclease or mung bean nuclease. Appropriate DNA fragments were separated on non-denaturing acrylamide gels and electro-eluted according to \cite{Stenberg_1984}. For 3$'$ end mapping, the DNA was end-labelled using T4 DNA polymerase. Twenty to 30 $\mu$g of total late RNA or MI RNA was hybridized to $10^5$ to $5 \times 10^5$ c.p.m. of end-labelled DNA fragments at 56$^\circ$C for 3 h in 10 to 20 $\mu$l buffer containing 80\% deionized formamide, 400 mM-NaCl, 40 mM-PIPES and 1 mM-EDTA. After hybridization the probes were diluted in 300 $\mu$l of ice-cold buffer containing 30 mM-sodium acetate pH 4.6, 50 mM-NaCl, 1 mM-ZnCl$_2$ and 5\% glycerol together with 270 units (U)/ml S1 nuclease or 50 to 100 U/ml mung bean nuclease, respectively. Digestion with S1 nuclease was carried out at 30$^\circ$C for 30 min; mung bean nuclease digestion was carried out at 37$^\circ$C for 30 to 60 min. The resulting fragments were sized on 6 to 8\% denaturing sequencing gels. Nuclease protection analyses with M13 ssDNA probes from genomic regions of high AT content were performed using a modification of the method described by \cite{Burke_1984} using unlabelled M13 ssDNA clones. Five to 20 $\mu$g of total late RNA and 0.5 to 2.5 $\mu$g ssDNA were coprecipitated and resuspended in 10 $\mu$l of hybridization buffer (0.4 M-NaCl, 10 mM-PIPES pH 6.4). Denaturation of the probes at 85$^\circ$C for 5 min was followed by hybridization for at least 3 h at temperatures between 50 and 60$^\circ$C. S1 or mung bean nuclease