The DR1 and DR6 first exons of human herpesvirus 6A are not required for virus replication in culture and are deleted in virus stocks that replicate well in T-cell lines

Abstract

Human herpesvirus 6A (HHV-6A) and HHV-6B are lymphotropic viruses which replicate in cultured activated cord blood mononuclear cells (CBMCs) and in T-cell lines. Viral genomes are composed of 143-kb unique (U) sequences flanked by approximately 8- to 10-kb left and right direct repeats, DR(L) and DR(R). We have recently cloned HHV-6A (U1102) into bacterial artificial chromosome (BAC) vectors, employing DNA replicative intermediates. Surprisingly, HHV-6A BACs and their parental DNAs were found to contain short approximately 2.7-kb DRs. To test whether DR shortening occurred during passaging in CBMCs or in the SupT1 T-cell line, we compared packaged DNAs from various passages. Restriction enzymes, PCR, and sequencing analyses have shown the following. (i) Early (1992) viral preparations from CBMCs contained approximately 8-kb DRs. (ii) Viruses currently propagated in SupT1 cells contained approximately 2.7-kb DRs. (iii) The deletion spans positions 60 to 5545 in DR(L), including genes encoded by DR1 through the first exon of DR6. The pac-2-pac-1 packaging signals, the DR7 open reading frame (ORF), and the DR6 second exon were not deleted. (iv) The DR(R) sequence was similarly shortened by 5.4 kb. (v) The DR1 through DR6 first exon sequences were deleted from the entire HHV-6A BACs, revealing that they were not translocated into other genome locations. (vi) When virus initially cultured in CBMCs was passaged in SupT1 cells no DR shortening occurred. (vii) Viral stocks possessing short DRs replicated efficiently, revealing the plasticity of herpesvirus genomes. We conclude that the DR deletion occurred once, producing virus with advantageous growth "conquering" the population. The DR1 gene and the first DR6 exon are not required for propagation in culture.

FIG. 1.

Preparation of packaged DNase I-resistant viral DNA. Concentrated samples from infected cell medium without (−) and with DNase I (D) treatment were tested for the presence of cell DNA by PCR employing the hGAPDH (G) primers and for the presence of viral DNA by PCR with the L primers from positions 15000 to 15602 of HHV-6A (U1102) DNA. NC is a negative control without DNA template. PC is a positive control, including primers and total infected cell DNA.

FIG. 2.

Preparations of packaged DNase I-resistant DNA from different HHV-6 preparations. Concentrated samples from infected cell medium without (−) and with DNase I (D) treatment were tested for the presence of cell DNA by PCR employing the hGAPDH (G) primers and for the presence of viral DNA by PCR with the L primers from positions 15000 to 15602 of HHV-6A (U1102) DNA. The sizes of PCR products are marked, based on the known DNA sequences. Lanes 1 and 17 include the 1-kb DNA marker (1 kb). NC, negative control (without template).

FIG. 3.

The DRs of the U1102-N-CB→S laboratory stock are shorter than the DRs from the various packaged viral stocks. DNAs extracted from the virus preparations embedded in agar plugs were digested with the SfiI (lanes 1 to 5) and the HpaI (lanes 6 to 10) enzymes. The PFGE gels were blotted and hybridized with the pac-2-pac-1 probe. Fragment sizes are denoted in kb.

FIG. 4.

Scheme of the pGEM cloning and sequencing of the DR of U1102-N-CB→S. (A) DNA from packaged virus was amplified by PCR, employing the pac-1-pac-2 primers. The ∼2.7-kb product was cloned into pGEM, and the resultant clones were sequenced. (B) Schematic diagram of the deleted DR in U1102-N-CB→S, based on the sequenced pGEM clones, showing an ∼5.4-kb deletion between bp 60 and 5545. The deletion is marked with a large X. The DR7 sequence (positions 5629 to 6720) overlaps the second exon of DR6, as denoted in PubMed. Relative positions of the DR7 start and stop codons are marked by dotted lines. (C) Schematic diagram of the arrangement of sequences within the HHV-6A (U1102) DR based on the PubMed sequence denoting the pac-1 and pac-2 signals, the telomeric repeats (tel), the DR1 and DR6 exons (denoted as e-1 and e-2), and the DR5 ORF.

FIG. 5.

The U1102-N-CB→S virus contains a majority of molecules with deleted DR and a minority of genomes with nondeleted DR. Lanes 3 and 6, EtBr and blot hybridizations of SfiI-digested packaged DNA preparations from the U1102-N-CB→S; lanes 5 and 8, EtBr and blot hybridizations of SfiI-digested packaged DNA preparations from the early stock U1102-CB→S which was propagated 24 passages in SupT1 cells. “1 kb” represents the 1-kb DNA marker, and M represents the midrange DNA marker.

FIG. 6.

PCR analyses of SfiI fragments with the DR1, DR5, and DR6 primers. Analyses of the U1102-N-CB→S and the early U1102-CB→S stock propagated 24 times in SupT1 cells. Viral DNAs from virions in the gel plugs were digested with SfiI and fractionated in the gel to ∼4.1-, ∼9.5-, and 150-kb bands, which were tested for the presence of the DR sequences by PCR. The following primers were employed: DR1 exon 1 (1-1); DR1 exon 2 (1-2); DR5 (5); DR6 exon 1 (6-1); DR6 exon 2 (6-2); and the L primers from positions 15000 to 15602. “1 kb” represents the 1-kb DNA ladder. The sizes of the PCR-amplified fragments are depicted at the left side of the gel. The sizes of some of the 1-kb ladder bands are depicted on the right.

FIG. 7.

PCR analyses of HHV-6A BAC clones for DR1, DR5, and DR6 DNA sequences. The three independently derived HHV-6A BAC clones (BAC-20, BAC-44, and BAC-38) were tested for the presence of the DR sequences by PCR. The following primers were employed: DR1 exon 1 (1-1); DR1 exon 2 (1-2); DR5 (5); DR6 exon 1 (6-1); and DR6 exon 2, 6-2. The R primers were from positions 134263 to 134591 of HHV-6A (U1102). “1 kb” represents the 1-kb DNA size ladder.