The Kinetics of VP5 mRNA Expression Is Not Critical for Viral Replication in Cultured Cells

Lieu, Pauline T.; Wagner, Edward K.

doi:10.1128/jvi.74.6.2770-2776.2000

Cited by 13 publications

(10 citation statements)

References 34 publications

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“…The in vivo replication and rescue data seem to indicate that early expression of VP5 confers a replication advantage in the nervous system. With the exception of replication in primary neural bulb cells, this finding is in contrast to in vitro replication data for all cell lines tested (10).…”

Section: Discussioncontrasting

confidence: 54%

“…These studies examined the multistep growth kinetics of VP5 promoter replacement recombinants in mouse embryo fibroblasts, rabbit skin cells (RSC), Vero cells, and serum-starved or quiescent human lung fibroblasts. The results indicated that recombinants and their rescued versions replicated to wild-type titers on these cell lines, and it was concluded that the expression of VP5 is not critical for viral replication in cultured cells (10).…”

mentioning

confidence: 94%

“…The construction and characterization of the promoter-substitution viruses used in this study are described in detail elsewhere (10). All viral recombinants were constructed in the background of the parent strain HSV-1 KOS.…”

Section: Methodsmentioning

confidence: 99%

“…To evaluate whether there is a requirement for VP5 expression at early times during an infection, recombinant viruses were generated in which the native VP5 promoter (␥1) was replaced with either a strict-late (U L 38, another capsid protein), a strong early (U L 50 or dUTPase), or an equal-strength leaky-late (VP16) promoter (10). These studies examined the multistep growth kinetics of VP5 promoter replacement recombinants in mouse embryo fibroblasts, rabbit skin cells (RSC), Vero cells, and serum-starved or quiescent human lung fibroblasts.…”

mentioning

confidence: 99%

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Altering the Expression Kinetics of VP5 Results in Altered Virulence and Pathogenesis of Herpes Simplex Virus Type 1 in Mice

Tran

Lieu

Aguilar

et al. 2002

J Virol

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While many herpes simplex virus (HSV) structural proteins are expressed with strict-late kinetics, the HSV virion protein 5 (VP5) is expressed as a "leaky-late" protein, such that appreciable amounts of VP5 are made prior to DNA replication. Our goal has been to determine if leaky-late expression of VP5 is a requirement for a normal HSV infection. It had been shown previously that recombinant viruses in which the VP5 promoter was replaced with promoters of other kinetic classes (including a strict late promoter) exhibited no alterations in replication kinetics or virus yields in vitro. In contrast, here we report that alterations in pathogenesis were observed when these recombinants were analyzed by experimental infection of mice. Following intracranial inoculation, a recombinant expressing VP5 from a strict-late promoter (U L 38) exhibited an increased 50% lethal dose and a 10-fold decrease in virus yields in the central nervous system, while a recombinant expressing VP5 from an early (dUTPase) or another leaky-late (VP16) promoter exhibited wild-type neurovirulence. Moreover, following infection of the footpad, changing the expression kinetics of VP5 from leaky-late to strict-late resulted in 100-fold-less virus in the spinal ganglia during the acute infection than produced by either the parent virus or the rescued virus. These data indicate that the precise timing of appearance of the major capsid protein plays a role in the pathogenesis of HSV infections and that changing the expression kinetics has different effects in different cell types and tissues.Lytic gene expression of herpes simplex virus type 1 (HSV-1) can be divided into three rough classes of viral transcripts: immediate-early (␣), early (␤), and late (␥) (for a review, see reference 18). The temporal regulation of these classes occurs at the level of transcription and is dictated primarily by differences in promoter structure (6,7,9,20). ␣ transcripts are expressed in the absence of de novo viral protein synthesis and encode proteins important for the transactivation and regulation of viral transcription (14, 16). The products of the ␤ transcripts are primarily proteins involved in the replication of viral genomes. As replication of viral DNA ensues, ␣ and ␤ transcription decreases and gives way to increasing ␥ transcription. The ␥ transcripts encode the majority of proteins involved in the structure, maturation, and morphogenesis of virions. The ␥ transcripts are further divided into two subclasses, leakylate (␥1) and strict-late (␥2), distinguished by their sensitivity to inhibitors of viral DNA replication (1, 17).The sequential and coordinated timing of transcription has undoubtedly been selected for and fine tuned over time to balance limited cellular resources with efficient viral replication and spread. Therefore, since HSV-1 ␥1 genes are expressed at both early and late times, it was postulated that a biological requirement for their expression both prior to and after DNA replication might exist. The HSV major capsid protein VP5 (a produ...

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Section: Discussioncontrasting

confidence: 54%

mentioning

confidence: 94%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

Altering the Expression Kinetics of VP5 Results in Altered Virulence and Pathogenesis of Herpes Simplex Virus Type 1 in Mice

Tran

Lieu

Aguilar

et al. 2002

J Virol

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“…Answers may lead to the improvement of already promising CMV-based vac- cine vectors (46). Provocatively, herpesviral promoters seem, in general, to maintain their kinetic properties when moved to or reproduced at a different location within the extant genome (47,48), perhaps making this type of expression system the most predictable and preferable.…”

Section: Discussionmentioning

confidence: 99%

Heterologous Viral Promoters Incorporated into the Human Cytomegalovirus Genome Are Silenced during Experimental Latency

Qin

Penkert

Kalejta

2013

J Virol

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Human cytomegalovirus (HCMV) lytic phase gene expression is repressed upon entry into myeloid lineage cells where the virus establishes latency. Lytic infection is not initiated because the tegument-delivered transactivator protein pp71 fails to enter the nucleus and inactivate the Daxx-mediated cellular intrinsic defense that silences the viral genome. When pp71 is expressed de novo in THP-1 monocytes, it localizes to the nucleus, inactivates the Daxx defense, and initiates lytic infection. We speculated that replacing the native viral promoter that drives pp71 expression with one that is highly and constitutively active in myeloid cells would permit pp71 de novo expression upon infection and that this newly expressed pp71 would accumulate in the nucleus, inactivate the intrinsic defense, and initiate the cascade of lytic gene expression. Surprisingly, we found that this promoter was still subject to normal silencing mechanisms in THP-1 monocytes and primary CD34 ؉ cells, two independent myeloid lineage cells. A second constitutively active heterologous viral promoter located in a different region of the HCMV genome was also silenced in THP-1 and CD34؉ cells. Furthermore, these two independent heterologous viral promoters inserted into three different regions of the HCMV genome in three different viral strains all required prior expression of the viral immediate early proteins for activation in fibroblasts. From this, we conclude that incorporation within the HCMV genome impacts the proclivity of heterologous viral promoters to initiate transcription. These observations have mechanistic implications for the expression of viral genes and transgenes during both lytic infection and latency.

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Two Leaky-Late HSV-1 Promoters Differ Significantly in Structural Architecture

Lieu

Wagner

2000

Virology

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The HSV-1 VP5 and VP16 transcripts are expressed with leaky-late (gamma1) kinetics and reach maximal levels after viral DNA replication. While the minimal VP5 promoter includes only an Sp1 site at -48, a TATA box at -30, and an initiator (Inr) element at the cap site, here we show that elements upstream of -48 can functionally compensate for the mutational loss of the critical Sp1 site at -48. To determine whether this is a general feature of leaky-late promoters, we have carried out a detailed analysis of the VP16 promoter in the context of the viral genome at the gC locus. Sequence analysis suggests a great deal of similarity between the two. Despite this, however, mutational analysis revealed that the 5' boundary of the VP16 promoter extends to ca. -90. This region includes an Sp1 binding site at -46, CAAT box homology at -77, and "E box" (CACGTG) at -85. Mutational and deletional analyses demonstrate that the proximal Sp1 site plays little or no role in promoter strength; despite this it can be shown to bind Sp1 protein using DNA mobility shift assays. Like the VP5 promoter, the VP16 promoter also requires an initiator element at the cap site. The VP16 Inr element differs in sequence from that of the VP5 promoter, and its deletion or mutation has a significantly smaller effect on promoter strength. The difference between these two Inr elements was confirmed by our finding that the VP16 initiator element binds to the 65-kDa YY1 transcription factor, and the VP5 Inr element competes poorly for the binding between the VP16 element and infected cell proteins in comparative bandshift assays. While the VP16 Inr sequence is identical to that of several murine TATA-less promoters, the VP16 Inr requires a TATA box for measurable activity.

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The Kinetics of VP5 mRNA Expression Is Not Critical for Viral Replication in Cultured Cells

Cited by 13 publications

References 34 publications

Altering the Expression Kinetics of VP5 Results in Altered Virulence and Pathogenesis of Herpes Simplex Virus Type 1 in Mice

Altering the Expression Kinetics of VP5 Results in Altered Virulence and Pathogenesis of Herpes Simplex Virus Type 1 in Mice

Heterologous Viral Promoters Incorporated into the Human Cytomegalovirus Genome Are Silenced during Experimental Latency

Two Leaky-Late HSV-1 Promoters Differ Significantly in Structural Architecture

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