The herpes simplex virus type 1 U(L)17 gene encodes virion tegument proteins that are required for cleavage and packaging of viral DNA

Abstract

Previous studies have suggested that the U(L)17 gene of herpes simplex virus type 1 (HSV-1) is essential for virus replication. In this study, viral mutants incorporating either a lacZ expression cassette in place of 1,490 bp of the 2,109-bp U(L)17 open reading frame [HSV-1(deltaU(L)17)] or a DNA oligomer containing an in-frame stop codon inserted 778 bp from the 5' end of the U(L)17 open reading frame [HSV-1(U(L)17-stop)] were plaque purified on engineered cell lines containing the U(L)17 gene. A virus derived from HSV-1(U(L)17-stop) but containing a restored U(L)17 gene was also constructed and was designated HSV-1(U(L)17-restored). The latter virus formed plaques and cleaved genomic viral DNA in a manner indistinguishable from wild-type virus. Neither HSV-1(deltaU(L)17) nor HSV-1(U(L)17-stop) formed plaques or produced infectious progeny when propagated on noncomplementing Vero cells. Furthermore, genomic end-specific restriction fragments were not detected in DNA purified from noncomplementing cells infected with HSV-1(deltaU(L)17) or HSV-1(U(L)17-stop), whereas end-specific fragments were readily detected when the viruses were propagated on complementing cells. Electron micrographs of thin sections of cells infected with HSV-1(deltaU(L)17) or HSV-1(U(L)17-stop) illustrated that empty capsids accumulated in the nuclei of Vero cells, whereas DNA-containing capsids accumulated in the nuclei of complementing cells and enveloped virions were found in the cytoplasm and extracellular space. Additionally, protein profiles of capsids purified from cells infected with HSV-1(deltaU(L)17) compared to wild-type virus show no detectable differences. These data indicate that the U(L)17 gene is essential for virus replication and is required for cleavage and packaging of viral DNA. To characterize the U(L)17 gene product, an anti-U(L)17 rabbit polyclonal antiserum was produced. The antiserum reacted strongly with a major protein of apparent Mr 77,000 and weakly with a protein of apparent Mr 72,000 in wild-type infected cell lysates and in virions. Bands of similar sizes were also detected in electrophoretically separated tegument fractions of virions and light particles and yielded tryptic peptides of masses characteristic of the predicted U(L)17 protein. We therefore conclude that the U(L)17 gene products are associated with the virion tegument and note that they are the first tegument-associated proteins shown to be required for cleavage and packaging of viral DNA.

FIG. 1

Schematic representation of collinear HSV sequences relevant to the production and documentation of U_L17 insertion and deletion viruses. (A) Line 1, representation of the HSV-1 genome (open rectangles represent inverted repeat regions flanking the U_L and U_S components); line 2, schematic representation of sequences within the genome of recombinant virus R7224 relevant to these studies; lines 3 and 4, schematic representation of the construction of a plasmid containing the lacZ gene driven by an SV40 promoter in the reverse orientation from U_L17 and the relevant sequences in the resulting recombinant HSV genome (filled rectangles, SV40 promoter; open rectangles, lacZ sequences); line 5, schematic representation of the BglII O and P fragments in HSV-1(R7224) and HSV-1(ΔU_L17) DNAs, as shown in Fig. 2; line 6, schematic collinear diagram of the U_L17 probe used in the experiment in Fig. 2. The probe contains both U_L17 and U_L15 exon II-specific sequences and hybridizes to both the BglII O fragment, which contains U_L15 exon II-specific sequences, and the BglII P fragment, which contains both U_L17-specific sequences and U_L15 exon II-specific sequences. (B) Line 1, representation of the HSV-1(17) genome (open rectangles represent inverted repeat regions flanking the U_L and U_S components); line 2, collinear representation of a set of cosmid DNAs cotransfected into cells for production of the HSV-1(U_L17-stop) recombinant virus (the position of the oligomer inserted into U_L17 encoding stop codons in all three potential open reading frames is indicated); line 3, schematic representation of sequences in the BglII P fragment of HSV-1 DNA (the arrows represent the direction and approximate lengths of the indicated open reading frames, and the position of the XbaI site within the DNA oligomer inserted within U_L17 is indicated); line 4, collinear representation of relevant DNA sequences within the probe used in the experiment illustrated in Fig. 2; line 5, collinear representation of the BglII P fragment with the XbaI and BglII restriction enzyme sites which were used to generate the hybridizing band shown in Fig. 2; line 6, collinear representation of the HSV-1 DNA sequences used to restore the oligomer insertion to wild-type sequences.

FIG. 2

Scanned digital images of the autoradiographs of the electrophoretically separated viral DNAs. Lanes contain viral DNA purified from cells infected with the indicated viruses digested with BglII (lanes 1 to 3) and with BglII and XbaI (lanes 4 to 6) and probed with radiolabeled U_L17 sequences (shown schematically in Fig. 1A, line 6, and Fig. 1B, line 4, respectively). The sizes (in kilobase pairs) of the DNA fragments are indicated to the right of each panel.

FIG. 3

Digitally scanned images of autoradiographs of electrophoretically separated viral DNA probed with end-specific sequences. Vero cells (or G5 cells where specified) were infected with the indicated viruses. Viral DNAs were purified, digested with BamHI, transferred to nitrocellulose, and hybridized with radiolabeled BamHI S DNA. The positions of the BamHI S fragments representing the termini of the long components in linear viral genomes and the S-P fragment derived from the junction of the long and short components in linear and concatemeric viral genomes are indicated.

FIG. 4

Scanned digital images of electron micrographs. Vero (top) and G5 (bottom) cells were fixed 14 h after infection with HSV-1(ΔU_L17). Thin sections were prepared and viewed with a Phillips EM 201 electron microscope. For size comparisons, HSV capsids are 120 nm in diameter. The left inset contains an image of the extracellular space of G5 cells infected with HSV-1(ΔU_L17). RM delineates the reticular meshwork referred to in the text; NM delineates the nuclear membrane; A, B, and C indicate A, B, and C capsids, respectively.

FIG. 5

Scanned digital images of electron micrographs. Vero (top) and G5 (bottom) cells were fixed 14 h after infection with HSV-1(U_L17-stop). The inset shows an image of the cytoplasm and extracellular space of G5 cells infected with HSV-1(U_L17-stop). NM delineates the nuclear membrane; B and C indicate B and C capsids, respectively.

FIG. 6

Scanned digital image of a denaturing polyacrylamide gel containing silver-stained capsid-associated proteins. Capsids were purified from cells infected with HSV-1(ΔU_L17) or HSV-1(F). Positions of the major capsid proteins are indicated. Relative molecular weights are indicated in thousands.

FIG. 7

Scanned digital image of an immunoblot probed with U_L17-specific antibody. Lanes 1 to 3, HEp-2 cells mock infected or infected with the indicated virus; lane 4, virions purified from cells infected with HSV-1(F). The proteins in lysates were electrophoretically separated, transferred to nitrocellulose, and probed with the antibody directed against U_L17. The apparent M_r of the proteins reacting with the antibody are indicated.

FIG. 8

Digitally scanned image of a denaturing polyacrylamide gel containing Coomassie blue-stained virion- and light particle-associated proteins. Virions (V) and light particles (L) were purified from cells infected with HSV-1(17) (wild type) or a virus lacking the U_L47 gene (U_L47⁻). Electrophoretic profiles of proteins associated with envelope (E), capsid-tegument (CT), or tegument (T) fractions are also shown. The position of the U_L19 major capsid protein is indicated to show its presence in greatly reduced amounts in light particles which lack capsids; the small amount present likely originated from contaminating virions. Positions of the U_L46 and U_L47 proteins and bands a and b (containing products of the U_L17 gene, as discussed in the text) are also indicated.