School of Biological Sciences, University of Nebraska, Lincoln NE 68588, USA1
Department of Veterinary and Biomedical Sciences, University of Nebraska, Lincoln, Fair Street at East Campus Loop, Lincoln, NE 68583-0905, USA2
Author for correspondence: Clinton Jones. Fax +1 402 472 9690. e-mail cjones{at}unlnotes.unl.edu
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Abstract |
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Main text |
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Infection in cattle is initiated by productive infection of mucosal epithelium. During productive infection, 7080 viral genes are expressed temporally. Like other members of the Alphaherpesvirinae subfamily, BHV-1 establishes and maintains a lifelong latent infection in the sensory ganglionic neurons of its host. The only known viral gene that is expressed abundantly in latently infected neurons is the latency-related (LR) transcript (Rock et al., 1987 ). LR RNA is antisense to bICP0, suggesting that bICP0 expression can be regulated by the antisense nature of the LR RNA. bICP0 is a promiscuous transactivator that can activate all classes of viral genes and is believed to play an important role in the reactivation from latency.
A fraction of LR RNA is polyadenylated and alternatively spliced in bovine trigeminal ganglia, indicating that a subset of LR transcripts is translated into a protein (Devireddy & Jones, 1998 ; Hossain et al., 1995
). A LR protein has been identified (Hossain et al., 1995
) that associates with cyclin-dependent kinase 2 (cdk2)cyclin complexes (Jiang et al., 1998
). LR gene products inhibit S phase entry (Schang et al., 1996
) and interfere with chemically induced apoptosis in transiently transfected cells (Ciacci-Zanella et al., 1999
). A mutation in the LR gene interferes with virus shedding from the eye during acute infection of calves and inhibits dexamethasone-induced reactivation from latency (Inman et al., 2001a
, 2002
). Taken together, these studies indicate that LR gene products play an important role during the infection process of cattle. Our long-term goals are to understand the mechanisms by which LR gene products regulate the latency reactivation cycle in cattle.
Herpes simplex virus type 1 (HSV-1) encodes a latency-associated transcript (LAT), which is transcribed abundantly during latency and is antisense to bICP0 (Jones, 1998 ; Wagner & Bloom, 1997
). LAT expression promotes establishment and reactivation from latency in mouse and rabbit models (Maggioncalda et al., 1996
; Perng et al., 1994
, 1996
, 2000a
; Sawtell, 1997
; Sawtell & Thompson, 1992
; Thompson & Sawtell, 1997
, 2001
). LAT inhibits apoptosis (Ahmed et al., 2002
; Inman et al., 2001b
; Perng et al., 2000a
) and productive infection (Mador et al., 1998
), suggesting that LAT and LR gene products have similar functions. In keeping with this observation, a recent study demonstrated that LR gene products can restore spontaneous reactivation to a HSV-1 LAT- mutant (Perng et al., 2002
).
Since the LR gene and LAT appear to have certain functional similarities, we hypothesized that cells expressing LR gene products would inhibit productive BHV-1 infection. To test this hypothesis, increasing concentrations of a plasmid containing the entire LR gene (LRTwt) (Fig. 1A) were cotransfected with bICP0 and BHV-1 DNA into bovine epidermal cells (9.1.3). A BHV-1 recombinant that contains the
-galactosidase (
-Gal) gene inserted downstream of the gC promoter in place of the gC ORF (BHV-1 blue virus) was used for this study. BHV-1 blue virus grows to similar titres as wild-type BHV-1.
-Gal expression correlates directly with virus replication because the gC promoter is a late promoter and its expression is low prior to viral DNA replication. A time-point of 24 h after transfection was used to count cells expressing
-Gal to minimize the number of virus-infected cells that resulted from virus spread (data not shown). As reported previously (Inman et al., 2001c
), the bICP0 gene alone strongly induced productive infection when cotransfected with genomic DNA (Fig. 1B
). The ability of bICP0 to activate gene expression correlates with stimulating productive infection.
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The 9.1.3 bovine cell line is an immortalized bovine epidermal cell line that expresses the simian virus 40 large T antigen (Hegde et al., 1998 ), a gene that can regulate the cell cycle and apoptosis (Hardwick, 1998
). To rule out the possibility that the T antigen influenced the conclusions from Fig. 2(A)
, the ability of the LR gene to interfere with productive infection was examined in bovine foetal lung cells, a low passage cell type. As observed in 9.1.3 cells, LRwt, LRT
SphI and LRTSacI repressed bICP0-induced activation of productive infection (Fig. 2B
). In contrast, LRT
PstI did not repress bICP0-induced productive infection.
Additional experiments were performed to test whether the LR gene could interfere with bICP0 expression. Cotransfection of 9.1.3 cells with bICP0 and BHV-1 DNA clearly led to higher levels of bICP0 RNA (Fig. 3A, lane 2) compared to cultures that were cotransfected with BHV-1 DNA and LRT
PstI (Fig. 3A
, lane 1). When a plasmid expressing LR gene products (LRTwt) was cotransfected with the bICP0 plasmid and BHV-1 DNA, lower levels of bICP0 RNA were detected at 24 h after transfection (Fig. 3A
, lane 3). As expected, abundant bICP0 RNA was detected in 9.1.3 cells infected with BHV-1 (Fig. 3A
, lane 4) but not in mock-infected cells (Fig. 3A
, lane 5).
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The effects of LRTwt on bICP0 protein expression were examined in transiently transfected cells, in the absence of other viral genes. Human 293 cells were used for this study because they can be transfected very efficiently and high levels of bICP0 are expressed in transiently transfected 293 cells (Fig. 3C). A plasmid expressing LRTwt cotransfected with a bICP0 plasmid expressed lower levels of the bICP0 protein when compared to cultures cotransfected with the bICP0 plasmid and LRT
PstI (Fig. 3C
). However, LRTwt had no effect on the steady-state levels of cdk2. In summary, the studies in Fig. 3
indicated that the LR gene has the potential to inhibit bICP0 RNA and protein expression.
Taken together, these studies indicated that LR RNA repressed productive infection by reducing bICP0 RNA and protein steady-state levels. Furthermore, it was also clear that LR gene products did not completely block productive infection in cultured bovine cells. Since proteins encoded by the LR gene interfere with apoptosis in transiently transfected cells (Ciacci-Zanella et al., 1999 ) and the latency reactivation cycle in cattle (Inman et al., 2001a
, 2002
), the LR gene appears to have more than one function. We propose that in the context of neuronal latency, LR gene products and neuronal-specific cellular factors inhibit productive infection and promote neuronal survival by inhibiting apoptosis.
LR RNA and bICP0 have complementary nucleic acid sequences; therefore, the two mRNAs could hybridize causing a reduction in bICP0 expression. Double-stranded RNA (dsRNA) formed from the hybridized mRNAs may be cleaved and degraded through the stimulation of interferon-regulated, dsRNA-activated pathways. The first pathway involves a protein kinase (PKR), which phosphorylates and inactivates the translation factor eIF2, leading to a generalized suppression of protein synthesis and cell death (reviewed by Clemens & Elia, 1997 ). A second dsRNA-response pathway involves the synthesis of 2-5' polyadenylic acid followed by activation of a sequence-non-specific RNase (RNaseL) (Player & Torrence, 1998
). Additional antisense effects may result in the reduction in translation initiation or premature termination of transcription. The 163 nt SphISalI fragment contains sequences (5' GAAAC 3') that resemble consensus ribozyme sites [5' GAA(G/A)C 3'] (Bratty et al., 1993
). Interestingly, a previous study also identified ribozyme-like sequences in HSV-1 LAT (Hui & Lo, 1998
). A growing number of studies have also demonstrated that a variety of plants, animals and viruses encode RNA-silencing systems that are RNA based (Storz, 2002
), which, in this case, would be LR RNA-silencing bICP0 expression. Future studies will attempt to identify how LR RNA interferes with bICP0 RNA and protein expression.
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Acknowledgments |
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Received 22 February 2002;
accepted 10 August 2002.