Analysis of the human cytomegalovirus genomic region from UL146 through UL147A reveals sequence hypervariability, genotypic stability, and overlapping transcripts

Abstract

Background: Although the sequence of the human cytomegalovirus (HCMV) genome is generally conserved among unrelated clinical strains, some open reading frames (ORFs) are highly variable. UL146 and UL147, which encode CXC chemokine homologues are among these variable ORFs.

Results: The region of the HCMV genome from UL146 through UL147A was analyzed in clinical strains for sequence variability, genotypic stability, and transcriptional expression. The UL146 sequences in clinical strains from two geographically distant sites were assigned to 12 sequence groups that differ by over 60% at the amino acid level. The same groups were generated by sequences from the UL146-UL147 intergenic region and the UL147 ORF. In contrast to the high level of sequence variability among unrelated clinical strains, the sequences of UL146 through UL147A from isolates of the same strain were highly stable after repeated passage both in vitro and in vivo. Riboprobes homologous to these ORFs detected multiple overlapping transcripts differing in temporal expression. UL146 sequences are present only on the largest transcript, which also contains all of the downstream ORFs including UL148 and UL132. The sizes and hybridization patterns of the transcripts are consistent with a common 3'-terminus downstream of the UL132 ORF. Early-late expression of the transcripts associated with UL146 and UL147 is compatible with the potential role of CXC chemokines in pathogenesis associated with viral replication.

Conclusion: Clinical isolates from two different geographic sites cluster in the same groups based on the hypervariability of the UL146, UL147, or the intergenic sequences, which provides strong evidence for linkage and no evidence for interstrain recombination within this region. The sequence of individual strains was absolutely stable in vitro and in vivo, which indicates that sequence drift is not a mechanism for the observed sequence hypervariability. There is also no evidence of transcriptional splicing, although multiple overlapping transcripts extending into the adjacent UL148 and UL132 open reading frames were detected using gene-specific probes.

Figure 1

Map of HCMV UL144 through UL132 open reading frames. Upper map shows the general structure of the complete HCMV genome. Lower map is an expansion of the region of interest at the indicated position on the HCMV genome. U_L, unique long; U_S, unique short; IR_L, internal repeat long; IR_S, internal repeat short; TR_L, terminal repeat long; TR_S, terminal repeat short. Numbered arrows indicate riboprobe sequences. Nucleotide numbers below the map are based on the Toledo sequence (GenBank accession number U33331).

Figure 2

UL146 phylogenetic analysis. UL146 amino acid sequences from 48 clinical strains, plus Towne and Toledo. Group designations correspond to those of Dolan et al. [2].

Figure 3

UL146-147 intergenic region phylogenetic analysis. Dendrogram of intergenic nucleotide sequences from 32 strains. Group designations are the same as those in Figure 2.

Figure 4

UL147 phylogenetic analysis. UL147 amino acid sequences from 32 strains. Group designations are the same as those in Figure 2.

Figure 5

RT-PCR amplification. (A) RT-PCR products containing UL146 through UL147A orfs. Lane 1 NW23-1; Lane 2 NW23-1 no RT control. Lane 3 CH15; Lane 4 CH15 no RT control. Lane 5 CH25. Lane 6 CH25 no RT control. Lane 7 CH22; Lane 8 CH 22 no RT control. Lane 9 CH-14. Lane 10 no RT control. (B) RT-PCR products containing portions of region UL146 through UL132 from strains CH21 and CH23. Lane 1 CH21 UL146-UL147A; Lane 2 no RT control. Lane 3 CH23 UL146-UL147A; Lane 4 no RT control. Lane 5 CH21 UL146-UL148; Lane 6 no RT control. Lane 7 CH23 UL146-UL148; Lane 8 no RT control; Lane 9 CH21 UL146-UL132; Lane 10 no RT control; Lane 11 CH23 UL146-132; Lane 12 no RT control.

Figure 6

Northern analysis of total transcripts from UL146 through UL132. Transcriptional pattern associated with UL146 through UL132 from different HCMV clinical strains. (A) Total RNA extracted from cells infected with designated strains and hybridized with UL146 CH22-specific riboprobe number 1 (See Figure 1). (B) Same blot as in (A), but rehybridized with riboprobe number 2 (Figure 1) containing UL146 through UL147A orfs derived from strain CH22. (C) Total RNA extracted from cells infected with designated strains and hybridized with UL147 CH19-specific riboprobe number 3 (Figure 1).

Figure 7

Northern analysis of temporal transcriptional pattern of UL146 through UL132. Total RNA was extracted at the indicated time points post-inoculation from cells infected with strain CH2 and hybridized with riboprobe number 1 derived from strain CH22 in the absence (A) or presence (B) of 400 μM foscarnet. (C) and (D) same as (A) and (B) but hybridized with riboprobe number 2. (E) and (F) same as (C) and (D) but hybridized with riboprobe 4. Lanes labeled U contain RNA from uninfected cells. Ribroprobe 5 data are not shown but are identical to ribroprobe 4 data as indicated by parentheses. Agarose gels showing 28S ribosomal RNA for loading control below (C) through (F).