The adenovirus E1B 55 kD protein influences mRNA transport via an intranuclear effect on RNA metabolism

The EMBO Journal vol.8 no.8 pp.2329-2336, 1989

The adenovirus El B 55 kd protein influences mRNA transport via an intranuclear effect on RNA metabolism

Keith N.Leppard1 and Thomas Shenk Department of Biology, Princeton University, Princeton, NJ 08544, USA 'Present address: Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK Communicated by L.V.Crawford

The adenovirus type 5 early region 1B encodes a 55 kd polypeptide that functions after transcription and processing to facilitate cytoplasmic accumulation of late viral mRNAs during lytic infection. A virus, d1338, carrying a deletion within the coding region for the 55 kd product, was found to be cold-sensitive for growth. Accumulation of late viral mRNAs was more severely inhibited at 32 than at 37°C in d1338-infected cells. The metabolism of late viral transcripts was analysed within the nucleus of dl338-infected cells at 32°C. Late viral mRNAs failed to accumulate efficiently within a nuclear compartment defimed by specific RNA extraction conditions. Normally, RNA accumulated within this operationally defined compartment after leaving the nuclear matrix and before associating with the nuclear envelope. These results indicate that the 55 kd polypeptide encoded by early region 1B facilitates an intranuclear step in the metabolism of viral mRNAs, resulting in improved transport of these RNAs to the cytoplasm. Key words: adenovirus type 5/EIB 55 kd protein/coldsensitivity/nuclear substructure/RNA transport

tionally, either at the level of RNA transport or by stabilization of RNAs immediately upon entry to the cytoplasm. RNA transport embodies a series of events in the pathway of eukaryotic gene expression which lie between transcription and processing in the nucleus and active translation of an mRNA in the cytoplasm. Transcription and processing are thought to occur in association with a nuclear substructure termed the nuclear matrix or scaffold (Jackson et al., 1981; Ciejek et al., 1982; Lewis et al., 1984). RNA destined to reach the cytoplasm then undergoes a transition to a more readily extractable state (Ciejek et al., 1982; Schroder et al., 1987b) following which it crosses the nuclear envelope and associates with the cytoskeleton where translation occurs (Cervera et al., 1981). During this process, the set of proteins associated with the RNA molecule is substantially changed (reviewed by Dreyfuss, 1986). Thus, the transport process can be broken down into multiple steps (Schroder et al., 1987a) which can be discriminated by differential compartmentalization, extractability and protein associations of the RNA molecule. Any of these could be influenced by the E1B 55 kd protein. In this report we have defined more precisely the site of action of the E1B gene product. The adenovirus mutant, d1338, which fails to produce the E1B 55 kd polypeptide, was found to be cold-sensitive for growth. Its defect in RNA metabolism was enhanced at 32 as compared to 37°C. The cold-sensitivity of the mutant was exploited to demonstrate that the defect in d1338-infected cells occurred at an intranuclear step in RNA metabolism, after transcription and prior to translocation across the nuclear envelope.

Introduction Lytic infection by human adenovirus (Ad) involves expression of the viral genome in a complex and highly ordered fashion. Several of the viral genes which are expressed prior to the onset of DNA replication act to facilitate the expression of the late genes and hence to optimize virus yield. The early 1B (EIB) region encodes two major protein products (Bos et al., 1981). One of these, a 55 kd polypeptide, is found in infected cells associated with a 34 kd product of the E4 transcription unit (Sarnow et al., 1984). This complex has been shown by analysis of mutant viruses to be necessary for the normal accumulation of late viral cytoplasmic mRNAs and for inhibition of the accumulation of host cell mRNA during infection (Babiss and Ginsberg, 1984; Babiss et al., 1985; Halbert et al., 1985; Pilder et al., 1986; Williams et al., 1986). In the absence of the EIB 55 kd polypeptide, no primary defect in transcription rate was detected for late viral genes, their RNAs were polyadenylated and spliced within the nucleus, and the half life of late mRNAs that reached the cytoplasm was normal (Pilder et al., 1986). Therefore, it was concluded that this protein regulates gene expression post-transcrip-

Results The E1B 55 kd mutant phenotype at 32°C Several point mutations mapping to the E1B 55 kd protein have been isolated that either were selected for cold-sensitive growth in HeLa cells or were found subsequently to be coldsensitive (Ho et al., 1982). Accordingly, the growth of a mutant carrying a deletion within the E1B gene, d1338, was compared at 32°C and 37°C and the mutant proved to be cold-sensitive. The yield of d1338 was reduced by a factor of 200 over wild-type at 32°C compared to a factor of 50 at 37'C, and the excess length of eclipse in mutant infections was increased from 2 h at 370C to 24 h at 320C (data not

shown). In previous studies carried out at 37°C (Pilder et al., 1986), d1338 expressed its early genes normally, but exhibited a reduced cytoplasmic accumulation of mRNAs encoded by the major late transcription unit. To determine whether the d1338 defect at the lower temperature was qualitatively similar to that observed at 37°C, viral DNA and protein synthesis were monitored as a function of time post-infection at 32 and 370C. The 32°C infections

2329 ©IRL Press2

K.N.Leppard and T.Shenk

Fig. 1. Steady-state levels of early and late RNAs encoded by mutant and wild-type viruses. HeLa cells, infected at a multiplicity of 10 p.f.u./cell with d1309 or dl338 and cultured at 32°C, were harvested at the times indicated and total cytoplasmic and total nuclear RNA prepared. Levels of EIA, L3, L4 and L5 transcripts were determined by quantitative RNase protection assays. The figure shows the amounts of labelled probe protected by either 5 jig of cytoplasmic or 2 jig of nuclear RNA, detected by autoradiography using preflashed film.

proceeded more slowly than the 37°C infections but at each temperature, viral DNA synthesis by wild-type and mutant viruses commenced at the same time and accumulation of DNA occurred with very similar kinetics (data not shown). Similarly, by pulse labelling and immunoprecipitation analysis, early EIA and E2A proteins were synthesized at comparable rates by the two viruses during the early phase of infection at both temperatures, synthesis continuing at high levels into the late phase for the mutant virus only (data not shown). Similar results have been obtained previously for E2A at 37°C (S.Pilder and T.Shenk, unpublished data). When late proteins were analysed, the mutant virus synthesized these at much reduced rates at both temperatures (data not shown). The appearance of major capsid protein from the mutant virus was substantially delayed at 32°C, as expected from the extended eclipse period of the virus seen at this temperature. The d1338 defect at 32°C was next analysed at the RNA level. Accumulation of viral mRNAs in the nucleus and cytoplasm of infected HeLa cells was monitored as a function of time after infection at 32°C (Figure 1). Steady-state levels of the early ElA-coded mRNAs in the nuclear and cytoplasmic compartments of mutant (dl338) and wild-type (d1309) virus-infected cells were similar throughout the time period tested. In contrast, the late L3, L4 and L5 mRNAs exhibited both a delayed appearance and reduced accumulation in the cytoplasm of dl338-infected cells. This appeared to be due in part to a delayed accumulation of the RNAs in the nuclear compartment of infected cells. However, the reduction in cytoplasmic late mRNA levels was clearly greater than could be explained by the reduced levels of these RNAs in the nucleus. At 37°C, transcription rates for both early and late genes were normal in d1338-infected cells until very late after infection (Pilder et al., 1986). Transcription rates were analysed at 32°C (Figure 2), and, as predicted by the steady-

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state levels, early viral genes such as E1A were transcribed at very similar rates in mutant (d1338) and wild-type (dl309) virus-infected cells. This was also true for the L3, L4 and L5 segments of the major late transcription unit until 40 h after infection. Rates observed in mutant and wild-type virusinfected cells differed by 2- to 3-fold or less, a difference too small to account for the substantial differences in steadystate levels of cytoplasmic L3, L4 and L5 mRNAs. After 40 h, however, the transcription rates of late mRNAs encoded by d1338 dropped precipitously as compared to the rates observed for d1309. This has been observed previously at 37'C (Pilder et al., 1986), and may result from the failure to produce adequate quantities of an as yet unidentified late gene product which could further stimulate late transcription. Results of steady-state and transcription rate analyses for the 40 h time point are presented in a quantitative fashion in Table I. Wild-type and mutant viruses exhibited similar rates of transcription and steady-state levels of the early ElA RNA. The steady-state levels of cytoplasmic RNAs derived from the major late unit are reduced by a factor of 5 to 21 in mutant as compared to wild-type virus-infected cells. The reduction in transcription rates (