One Functional Copy of the Long Terminal Repeat Gene Specifying the Immediate-early Polypeptide IE 110 Suffices for a Productive Infection of Human Foetal Lung Cells by Herpes Simplex Virus

SUMMARYin the process of generating restriction endonuclease site deletion mutants, we have isolated and characterized three mutants of herpes simplex virus type 2 (HSV-2), strain HG52, with large genomic deletions in Us and TRs. The deleted sequences (7.5 kb) extend from 0-94 map coordinates (m.c.) to 0-99 m.c. and are presumed to contain the HSV-2 gene equivalents of US10, 11 and 12, one copy of immediate early (IE) gene 3 and one copy of an origin of replication (ORls). One of the mutants (HG52X163X12) has a simple deletion whereas in the two others (HG52X 163X14 and HG52X163X21) the deleted sequences have been replaced by inverted duplications of Us/IRs sequences between 0.83 and 0-91 m.c. such that the molecules have short region inverted repeats extended by 6 kb on either side. All three are viable, stable and grow in tissue culture indicating that the polypeptides coded by the HSV-2 genes equivalent to US10, 11 and 12 are n0n-essential for lytic growth in BHK21/C13 cells. In addition the lack of one copy of the HSV-2 equivalent of IE gene 3 and ORIs in HG52X163X12 shows that only one copy of each suffices for viability. However the temperature restriction of the mutants at 38-5 °C suggests that one or more of the polypeptides coded by the deleted sequences may be required in conjunction with another polypeptide(s) for viral growth or stability at the higher temperature. INTRODUCTIONDeletions in one copy of the long repeat sequences of herpes simplex virus type 2 (HSV-2) strain HG52 occur at a frequency of 24~o by passage eight from the original field isolation (Harland & Brown, 1985). The described deletions range in size with the maximum being 9 kb and encompass the entire long repeat with the exception of the 'a' sequences. Other strains of HSV-2 exhibit simdar deletions showing that the HG52 strain is not exceptional. Deletions in the long repeat had been observed previously in intertypic reeombinants when Davison et al. (1981) suggested that they probably arose as a consequence of heterotypic repetitive regions. Our observations show that this could not be the explanation but that deletions within one copy of the long repeats of the HG52 genome are relatively common. These deletion variants do not have a selective disadvantage (Harland & Brown, 1985); thus only one copy of immediate early (IE) gene 1 and the other coding information within the long repeats is sufficient for viable growth in tissue culture. The product of IE gene 1, i.e. Vm,~ IE 110, in conjunction with V mw IE175 has an enhancing effect on early polypeptide transcription (Everett, 1984) but it has not been shown whether V~wlE110 is essential for lytic growth.The high frequency of spontaneous deletions within the long repetitive sequences raises the question of whether similar deletions could occur in the short repetitive sequences of the genome. The repeat (TRs) and unique short (Us) regions of the HSV-l genome have been fully sequenced (Murchie & McGeoch, 1982;McGeoch et al., 1985), The IE3 gene coding for

Section: Discussionmentioning

confidence: 78%

mentioning

confidence: 78%

Section: Growth Of Virusmentioning

confidence: 99%

See 1 more Smart Citation

Three Mutants of Herpes Simplex Virus Type 2: One Lacking the Genes US10, US11 and US12 and Two in which RS Has Been Extended by 6 kb to 0{middle dot}91 Map Units with Loss of US Sequences between 0{middle dot}94 and the US/TRS Junction

Brown¹,

Harland²

1987

“…There is no published information on the extent of unique-sequence transcription during productive infections with HVS. The possibility that all the functions required for a productive infection with HVS may be encoded within a small fraction of the unique genome sequences cannot be dismissed a priori, given such familiar precedents as bacteriophage 2 and examples of duplicated and dispensable functions in the genome of herpes simplex virus (Davison et al, 1981 ;Post & Roizman, 1981). However, such a situation would be unexpected and the persistence of host protein synthesis may have prevented the detection of a significant number of virus-specified early gene products.…”

Section: Demonstration O F a N Electrophoretically Distinct Nuclear Pmentioning

confidence: 99%

Proteins Specified by Herpesvirus Saimiri: Identification and Properties of Virus-specific Polypeptides in Productively Infected Cells

Randall¹,

Honess²,

O’Hare³

1983

SUMMARYThe synthesis and accumulation of more than 30 virus-induced polypeptides was detected after infection of permissive primate cells with high multiplicities of herpesvirus saimiri. These virus-induced polypeptides had apparent mol. wt. of from 12000 (12K) to 250K and differed in molar abundance by up to two orders of magnitude. The majority of virus-induced polypeptides satisfied multiple criteria for their virus specificity. Polypeptides of ll0K, 76K, 51K and 31K to 29K were synthesized at maximum rates early in infection, but the majority of proteins were made at high rates late in infection. Virus-specific polypeptides were substrates for a number of post-synthetic modifications. The l17K, 85K, 76K, 31K and 30K polypeptides were each processed to forms with altered electrophoretic mobility. The 117K and 85K polypeptides were among the virus-specified substrates for glycosylation and virus-induced polypeptides of 59K, 51K, 30K and 26K were phosphorylated. The early 51K phosphoprotein and the 30K to 31K polypeptides were rapidly translocated to the nucleus of infected cells. The 31K polypeptide was processed to a 29K product which remained stably associated with the nuclear fraction. The 30K nuclear protein was shown to be the precursor of a 28K polypeptide which was released in a soluble form into the cytoplasmic fraction and the culture medium of cells at late times in the virus growth cycle. Many other polypeptides accumulated slowly in nuclear (e.g. 250K, 150K, 130K, 110K and 38K) or in cytoplasmic (e.g. 117K, 85K and 28K) fractions Of infected cells in tbrms which could be differentiated by the use of detergents or differences in stability to salt extraction. The mol. wt., relative molarities and some features of the post-synthetic processing of herpesvirus saimiri polypeptides more closely resembled the properties of gene products of Epstein-Barr virus than those of herpes simplex virus.

“…Banks of tandem repeats are believed to be responsible for the variation in size of certain restriction fragments, which is seen between strains and between single plaque isolates within a strain (Lonsdale et al, 1980). Variation is also found in the size of the terminal and joint fragments which contain the 'a' sequence but this is due to variability in the number of 'a' sequences present (Davison & Wilkie, 1981).…”

Section: Introductionmentioning

confidence: 99%

Deletion and Duplication Variants around the Long Repeats of Herpes Simplex Virus Type 1 Strain 17

Maclean

Brown

1987

SUMMARYThree variants of herpes simplex virus type 1 strain 17 were isolated. One variant had a deletion of 2-5 x 106 M r in IRL/UL and 0.8 × 106 Mr in TRL such that sequences were deleted from both long repeats. The deletion in UL removed the 20K and 22K open reading frames. The second variant had a deletion of 3.5 × 106Mr in IRL/UL which again removed the 20K and 22K open reading frames. The third variant had a similar deletion in UL, but in this case the deleted sequences were replaced by sequences from the left end Of UL, such that the long repeat was extended by 3 x 106 M r and the overall genome size by 2 × 106 M r. All three variants grew almost normally in vitro, The analysis of 11 isolates with extensive variation in the long repeats outside the 'a' sequence is also reported.