Herpes simplex virus latency-associated transcript encodes a protein which greatly enhances virus growth, can compensate for deficiencies in immediate-early gene expression, and is likely to function during reactivation from virus latency - PubMed (original) (raw)

Herpes simplex virus latency-associated transcript encodes a protein which greatly enhances virus growth, can compensate for deficiencies in immediate-early gene expression, and is likely to function during reactivation from virus latency

S K Thomas et al. J Virol. 1999 Aug.

Abstract

Herpes simplex virus types 1 and 2 (HSV1 and HSV2) enter and reactivate from latency in sensory neurons, although the events governing these processes are little understood. During latency, only the latency-associated transcripts (LATs) are produced. However, although the LAT RNAs were described approximately 10 years ago, their function remains ambiguous. Mutations affecting the LATs have minimal effects other than a small reduction in establishment of and reactivation from latency in some cases. Mutations in putative LAT-contained open reading frames (ORFs) have so far shown no effect. The LATs consist of a large species from which smaller (approximately 2 kb), nuclear, nonlinear LATs which are abundant during latency are spliced. Thus, translation of ORFs in these smaller LATs would not usually be expected to be possible, and if expressed at all, their expression might be tightly regulated. Here we show that deregulated expression of the largest HSV1 2-kb LAT-contained ORF in various cells of neuronal and nonneuronal origin greatly enhances virus growth in a manner specific to HSV1-the HSV1 LAT ORF has no effect on the growth of HSV2. Similar results of enhanced growth were found when the HSV1 LAT ORF was constitutively expressed from within the HSV1 genome. The mechanism of LAT ORF action was strongly suggested to be by substituting for deficiencies in immediate-early (IE) gene expression (particularly ICP0), because deregulated LAT ORF expression, as well as enhancing wild-type virus growth, was also found to allow efficient growth of viruses with mutations in ICP0 or VMW65. Such viruses otherwise exhibit considerable growth defects. IE gene expression deficiencies are often the block to productive infection in nonpermissive cells and are also evident during latency. These results, which we show to be protein- rather than RNA-mediated effects, strongly suggest a function of the tightly regulated expression of a LAT ORF-encoded protein in the reactivation from HSV latency.

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Figures

FIG. 1

The HSV DNA genome, the LAT region, and constructs used in this study. (a) The LATs are transcribed from the long terminal repeat regions of the genome (larger rectangles), resulting in production of a large primary transcript, antisense to the IE1 transcript, which is spliced to release smaller LATs that are abundant during latency. Potential 2-kb LAT-contained ORFs in HSV1 and HSV2 are shown, as are regions with promoter activity (LAP1 and LAP2). LAP1 is predominantly responsible for 2-kb LAT production in latency (7). The HSV1 LAT ORF studied here is shaded. (b) Constructs and viruses used.

FIG. 2

Deregulated expression of the HSV1 LAT ORF enhances virus growth. (a) HSV1 growth curves on six randomly picked ND7 and BHK clones stably transfected with pcDNA3LAT compared to that of pcDNA3-transfected control cells. (b) Growth curves of three separately plaque-purified 17+/pR20.5/5/LAT virus stocks compared to that of a 17+/pR20.5/5 control virus on unmanipulated BHK or ND7 cells or ND7 cells stably transfected with pcDNA3LAT (Lat N5 in panel a). MOI = 0.1 and 0.01 for ND7 and BHK cells, respectively. (c) Growth curves of HSV1 and HSV2 (strain HG52) on unmanipulated ND7 cells and ND7 cells stably transfected with pcDNA3/LAT (Lat N5). MOI = 0.01. (d) Growth of HSV1 expressing the LAT ORF in vivo. Twenty 3-week-old female BALB/c mice were footpad inoculated with 105 PFU of either 17+/pR20.5/5 or 17+/pR20.5/5/LAT. Pairs of mice from each group were killed on days 1 to 10 after inoculation, DRG L1 to L6 were harvested and manually homogenized, and titers of samples were determined for virus content on BHK cells. The points represent results from individual mice. (e) Northern blot showing LAT ORF encoding RNA expressed from the CMV promoter in 17+/pR20.5/5/LAT at the indicated times after infection of BHK cells (MOI = 1) and that of 17+/pR20.5 control virus (see Materials and Methods). p.i., postinfection.

FIG. 2

FIG. 3

Effect of deregulated LAT ORF expression on HSV1 ICP0 mutants. Growth curves of an HSV1 strain 17+ ICP0 deletion mutant (_dl_1403) (28) and VMW65 insertional mutant (_in_1814) (1) on ND7 (Lat N3, N5, and Neo in Fig. 2a) and BHK (Lat B2, B5, and Neo in Fig. 2a) cells stably transfected with pcDNA3/LAT or pcDNA3 and to those of wild-type virus (strain 17+) on pcDNA3-transfected BHK and ND7 cells are shown. MOI = 0.01 in each case.

FIG. 4

The HSV1 LAT ORF functions as a protein. (a) Viruses and cell lines were produced as described in Fig. 1, except where a frameshift mutation was inserted into the LAT ORF. Growth curves for wild-type HSV1 are shown for four representative ND7 cell lines (ND7LATmut) compared to that of a representative unaltered LAT ORF-expressing line (Lat N5 in Fig. 2a). A Northern blot (b) showing LAT ORF- and mutant LAT ORF-specific RNA expression in these cells is also shown (LAT ORF cells N2, N3, N5, and N6 in Fig. 2, and four representative mutant LAT ORF cell lines [see Materials and Methods]). con, control pcDNA3 transfected cells. (c) LAT ORF protein expression in vitro. [35S]Met-labelled in vitro transcription/translation reactions (TnT; Promega) were performed with pcDNA3LAT, pcDNA3LATmut, and a control luciferase-encoding plasmid. Reactions were run on a 15% polyacrylamide gel before autoradiographic detection of protein products.

FIG. 5

Model of LAT function during HSV latency and reactivation.

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Herpes simplex virus latency-associated transcript encodes a protein which greatly enhances virus growth, can compensate for deficiencies in immediate-early gene expression, and is likely to function during reactivation from virus latency - PubMed (original) (raw)