The latency-related gene of bovine herpesvirus-1 can inhibit the ability of bICP0 to activate productive infection

Vicki Geiser1, Melissa Inman2, Yange Zhang2 and Clinton Jones2

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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Transfection of bovine cells with bovine herpesvirus-1 genomic DNA yields low levels of infectious virus. Cotransfection with the bICP0 gene enhances productive infection and virus yield because bICP0 can activate viral gene expression. Since the latency-related (LR) gene overlaps and is antisense to bICP0, the effects of LR gene products on productive infection were tested. The intact LR gene inhibited productive infection in a dose-dependent fashion but LR protein expression was not required. Further studies indicated that LR gene sequences near the 3' terminus of the LR RNA are necessary for inhibiting productive infection. When cotransfected with the bICP0 gene, the LR gene inhibited bICP0 RNA and protein expression in transiently transfected cells. Taken together, these results suggest that abundant LR RNA expression in sensory neurons is one factor that has the potential to inhibit productive infection and consequently promote the establishment and maintenance of latency.


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Bovine herpesvirus-1 (BHV-1) infections cause a significant economic loss to the cattle industry. In addition to clinical symptoms associated with acute infection, abortions, encephalitis, conjunctivitis, respiratory disease, genital disease, pneumonia and immunosuppression can result from infection (Bowland & Shewen, 2000 ; Jones, 1998 ; Tikoo et al., 1995 ). BHV-1-induced immunosuppression leads frequently to secondary bacterial infections, resulting in bronchopneumonia and even death. Increased susceptibility to secondary infection correlates with depressed cell-mediated immunity after infection (Carter et al., 1989 ; Griebel et al., 1987a , b , 1990 ). CD8+ T cell recognition of infected cells is impaired by downregulation of major histocompatibility complex class I expression and the transporter associated with antigen presentation (Hariharan et al., 1993 ; Hinkley et al., 1998 ; Nataraj et al., 1997 ). CD4+ T cell function is impaired during acute infection of calves because BHV-1 has the ability to infect these cells and induces apoptosis (Winkler et al., 1999 , 2000 ). Thus, BHV-1 infections have a profound effect on the health of cattle.

Infection in cattle is initiated by productive infection of mucosal epithelium. During productive infection, 70–80 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 {beta}-galactosidase ({beta}-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. {beta}-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 {beta}-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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Fig. 1. The LR gene inhibits productive infection in a dose-dependent manner. (A) Partial restriction map of the 2 kb LR gene. The position of bICP0 (Wirth et al., 1989 ) and the LR RNA that is transcribed during productive and latent infection is indicated (Bratanich et al., 1992 ; Delhon & Jones, 1997 ; Schang et al., 1996 ). The construction and characteristics of pCMV-bICP0 (bICP0), pCMV-LRT (LRTwt) (Inman et al., 2001c ), pCMV-LRTstop (LRTstop), pCMV-LRT{Delta}SmaI (LRT{Delta}SmaI), pCMV-LRT{Delta}HX (LRT{Delta}HX) (Ciacci-Zanella et al., 1999 ), pCMV-LRT{Delta}PstI (LRT{Delta}PstI) (Jones et al., 1990 ) and pCMV-LRT{Delta}SphI (LRT{Delta}SphI) (Schang et al., 1996 ) were described previously. LRTstop is a plasmid that has a deletion between nt 778 and 809 (SphI sites), an oligonucleotide containing three stop codons and a unique EcoRI site inserted between the two SphI sites (the position of the stop codons are denoted by the arrowhead). pCMV-LRTSacI (LRTSacI) was constructed by cloning the 3' terminal 564 bp region of the LR gene (1373–1937 bp) into the pcDNA3.1- vector (Invitrogen) by standard molecular cloning techniques. All plasmids were purified using caesium chloride purification. (B) Bovine cells (9.1.3) were cotransfected using Superfect (Qiagen), the designated amount of LRTwt, viral DNA prepared from BHV-1 blue virus (1 µg DNA) and the bICP0 plasmid (15·6 ng DNA). Because of the size difference in the plasmid and the BHV-1 genome, this ratio is an approximate 1:1 molecular ratio between BHV-1 DNA and the bICP0 plasmid. This ratio of viral DNA to bICP0 plasmid was optimal for increasing plaque formation and the number cells expressing {beta}-Gal (Inman et al., 2001c ). Increasing concentrations of LR plasmids (4:1, 16:1 and 64:1 concentrations of LR plasmid to bICP0) were added to test the effects of LR gene products on bICP0-induced transactivation. A blank expression vector (pcDNA3.1-) was used to maintain equivalent amounts of DNA. At 24 h after transfection, cells were fixed, stained and the number of blue cells counted. The number of cells expressing {beta}-Gal in cultures cotransfected with bICP0 and BHV-1 DNA was set at 100 per cent productive infection efficiency (black bar). This minimized the differences in cell confluence, Superfect lot variation and transfection efficiency. The results are the average of at least three independent experiments.

 
The intact LR gene (LRTwt) inhibited the ability of bICP0 to stimulate productive infection in a dose-dependent fashion and at the highest concentration of LRTwt, a threefold reduction in productive infection was observed (Fig. 1B). Insertion of three in-frame stop codons at the amino terminus of the first ORF (LRTstop) or deletion of the SmaI site that encompasses splicing sites within the LR gene (LRT{Delta}SmaI) did not relieve repression (Fig. 2A), indicating that expression of a LR protein was not required for repression.



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Fig. 2. Localization of sequences in the LR gene that inhibit productive infection. (A) As described in Fig. 1(B), bovine cells (9.1.3) were cotransfected using Superfect with the viral DNA prepared from BHV-1 blue virus (1 µg DNA), bICP0 plasmid (15·6 ng DNA) and increasing concentrations of the designated plasmids containing various regions of the LR gene. (B) Bovine foetal lung cells were cotransfected using Superfect with the designated amounts of BHV-1 blue virus genome (1 µg DNA), bICP0 plasmid (15·6 ng DNA) and the specified ratios of LR plasmids, as described in Fig. 1(B). A blank expression vector (pcDNA3.1-) was used to maintain equivalent amounts of DNA. At 24 after transfection, cells were fixed, stained and the number of blue cells counted as described in Fig. 1(B). The results are the average of at least three independent experiments.

 
Deletion of the first 523 bp of the LR promoter (LRT{Delta}HX) only reduced the level of repression slightly (Fig. 2A). A LR promoter construct that contained approximately 200 nt of LR-coding RNA sequences (Bratanich et al., 1992 ) (LRT{Delta}PstI) did not interfere with bICP0-activated productive infection. Deletion of the SphI fragment (986–1777 bp) (LRT{Delta}SphI) repressed productive infection, but not as efficiently as LRTwt, LRTstop or LRT{Delta}SmaI. This suggested that the 163 bp SphI–SalI fragment played a role in repressing productive infection. Attempts to clone the 163 bp fragment failed, indicating that the fragment was unstable in Escherichia coli (data not shown). A larger construct containing 564 bp of the 3' terminus of LR-coding RNA sequences was cloned (LRTSacI) and this construct interfered consistently with productive infection, but not as efficiently as LRTwt (Fig. 2A). In summary, these studies demonstrated that LR RNA sequences located near the 3' terminus of the LR gene played an important role in inhibiting productive BHV-1 infection.

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{Delta}SphI and LRTSacI repressed bICP0-induced activation of productive infection (Fig. 2B). In contrast, LRT{Delta}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{Delta}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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Fig. 3. LR gene products inhibit expression of bICP0 RNA and protein expression. (A) Lanes: 1, bovine cells (9.1.3) cotransfected with BHV-1 DNA (1 µg DNA) and LRT{Delta}PstI (1 µg DNA); 2, BHV-1 DNA (1 µg DNA), the bICP0 plasmid (15·6 ng DNA) and LRT{Delta}PstI (1 µg DNA) (64:1 ratio between LRT{Delta}PstI and bICP0) cotransfected into 9.1.3 cells; 3, BHV-1 DNA (1 µg DNA), the bICP0 plasmid (15·6 ng DNA) and LRTwt (1 µg DNA) (64:1 ratio between LRTwt and bICP0) cotransfected into 9.1.3 cells; 4, 9.1.3 cells infected with BHV-1 (m.o.i. of 4) for 6 h; 5, mock-infected cells. Total RNA was prepared at 24 h after transfection (lanes 1–3) or at the designated time after infection. Equivalent amounts of RNA (15 µg) were electrophoresed in a 1% formaldehyde–agarose gel and the RNA transferred to a nylon filter. (B) Bovine cells (9.1.3) cotransfected with BHV-1 DNA (1 µg DNA), bICP0 (15·6 ng DNA) and increasing concentrations of the designated LR plasmids, as described in Fig. 1(B). At 24 h after transfection, total RNA was prepared, equivalent amounts of RNA (15 µg) were electrophoresed on a 1% formaldehyde–agarose gel and the RNA transferred to a nylon filter. For the Northern blots in (A, B), a 32P-labelled probe (approximately 1100 bp) that encompassed the amino-terminal region of the bICP0 protein was prepared by random priming. This fragment was a SalI–HindII fragment derived from pCMV-bICP0 and is downstream of LR gene sequences shown in Fig. 1(A). Consequently, this probe does not hybridize specifically to LR RNA. The arrows denote the position of the bICP0 transcript. (C) Human 293 cells cotransfected with 3 µg of plasmid expressing a Flag-tagged, full-length bICP0 protein (pCMV-bICP0) and 9 µg (lanes 1 and 3) or 18 µg DNA (lanes 2 and 4) of LRT{Delta}PstI or LRTwt. The bICP0 expression vector contains a FLAG epitope and was described previously (Inman et al., 2001c ; Zhang & Jones, 2001 ). At 48 h after transfection, cells were lysed in 150 µl SDS–PAGE buffer and equivalent amounts of proteins electrophoresed on a 8% SDS polyacrylamide gel. The bICP0 protein in the Western blot was detected with a monoclonal antibody directed against the FLAG epitope (Stratagene). The cdk2 protein was detected using an antibody available commercially from Santa Cruz Biotechnology. Arrows denote the position of bICP0 (98 kDa) and cdk2 (35 kDa).

 
Increasing levels of LRTwt were cotransfected with BHV-1 DNA to test whether there was a dose-dependent reduction in bICP0 expression (Fig. 3B). At all concentrations of the LRT{Delta}PstI plasmid, higher levels of the bICP0 transcript were observed relative to cultures that were cotransfected with LRTwt and BHV-1 DNA. As expected, cells transfected with the bICP0 plasmid but not LRTwt expressed a transcript that hybridized with the bICP0 probe.

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{Delta}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 SphI–SalI 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.


   Acknowledgments
 
A Public Health Service grant (1P20RR15635) and USDA grants (2000-02060 and 2002-02450) supported this study. We thank S. Chowdhury (Kansas State University, Kansas, USA) for the BHV-1 gC blue virus.


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Received 22 February 2002; accepted 10 August 2002.