Eculizumab-induced reversal of dialysis-dependent kidney failure from C3 glomerulonephritis.

C3 glomerulopathy (C3G) is characterized by C3 deposits with minimal immunoglobulin deposition caused by alternative complement pathway dysregulation. Unfortunately, no therapeutic intervention has consistently improved outcomes for patients with C3G. Eculizumab, a monoclonal antibody to C5, is currently the only approved complement-specific agent with some efficacy in the treatment of C3 glomerulonephritis (C3GN). Here, we describe a patient with acute crescentic C3GN with no identified complement mutation or family history of renal disease who required dialysis for 6 months. Five months after initiation of eculizumab, she became dialysis independent, showing improvement is possible after adequate time on eculizumab.

A protein encoded by the bovine herpesvirus 1 open reading frame E gene induces neurite-like morphological changes in mouse neuroblastoma cells and is expressed in trigeminal ganglionic neurons.

Bovine herpes virus 1 (BHV-1), like other alpha-herpesvirinae subfamily members, establishes latency in sensory neurons. Periodically BHV-1 reactivates from latency, resulting in virus shedding and spread to uninfected cattle. Although reactivation from latency does not usually lead to recurrent disease, the latency-reactivation cycle is crucial for virus transmission. The latency-related (LR) RNA is abundantly expressed during latency, and expression of a LR encoded protein is necessary for dexamethasone-induced reactivation from latency in cattle. Within LR promoter sequences, a small open reading frame (ORF) was identified (ORF-E) that is antisense to the LR-RNA, and downstream of the bICP0 gene. ORF-E transcription is consistently detected in trigeminal ganglia (TG) of latently infected calves, suggesting ORF-E expression plays a role in the latency-reactivation cycle. Polyclonal antiserum directed against an ORF-E peptide or the entire ORF-E protein specifically recognizes the nucleus of sensory neurons in TG of latently infected calves. The ORF-E peptide-specific antiserum also recognizes a protein when mouse neuroblastoma cells (neuro-2A) are transfected with an ORF-E expression construct. In contrast to the growth inhibiting properties of the LR gene, stably transfected ORF-E-expressing cells were obtained. Neuro-2A cells stably transfected with a plasmid expressing ORF-E induced morphological changes that resembled neurite-like projections. In contrast, neurite-like projections were not observed following transfection of neuro-2A cells with an empty vector. These studies suggest that a protein encoded by ORF-E has the potential to alter the physiology or metabolism of neuronal cell types, which may be important for long-term latency.

A protein encoded by the bovine herpesvirus 1 latency-related gene interacts with specific cellular regulatory proteins, including CCAAT enhancer binding protein alpha.

Following acute infection, bovine herpesvirus 1 establishes latency in sensory neurons of trigeminal ganglia (TG). Reactivation from latency occurs periodically, resulting in the shedding of infectious virus. The latency-related (LR) RNA is abundantly expressed in TG of latently infected calves, and the expression of LR proteins is necessary for dexamethasone-induced reactivation from latency. Previously published studies also identified an alternatively spliced LR transcript which is abundantly expressed in TG at 7 days after infection and has the potential to encode a novel LR fusion protein. Seven days after infection is when extensive viral gene expression is extinguished in TG and latency is established, suggesting that LR gene products influence the establishment of latency. In this study, we used a bacterial two-hybrid assay to identify cellular proteins that interact with the novel LR fusion protein. The LR fusion protein interacts with two proteins that can induce apoptosis (Bid and Cdc42) and with CCAAT enhancer binding protein alpha (C/EBP-alpha). Additional studies confirmed that the LR fusion protein interacts with human or insect C/EBP-alpha. C/EBP-alpha protein expression is induced in TG neurons of infected calves and after dexamethasone-induced reactivation from latency. Wild-type C/EBP-alpha, but not a DNA binding mutant of C/EBP-alpha, enhances plaque formation in bovine cells. We hypothesize that interactions between the LR fusion protein and C/EBP-alpha promote the establishment of latency.

The herpes simplex virus type 1 locus that encodes the latency-associated transcript enhances the frequency of encephalitis in male BALB/c mice.

Herpes simplex virus type 1 (HSV-1) is the leading cause of virus-induced encephalitis; however, the viral genes that regulate encephalitis have not been well characterized. In this study, we tested whether the LAT (latency-associated transcript) locus regulates the frequency of encephalitis in male or female mice. Male BALB/c mice are more susceptible to HSV-1-induced encephalitis than age-matched female BALB/c mice. Deletion of LAT coding sequences reduced the frequency of encephalitis. A recombinant virus containing the first 1.5 kb of the LAT coding sequence induces levels of encephalitis in male BALB/c mice similar to those induced by wild-type HSV-1.

The locus encompassing the latency-associated transcript of herpes simplex virus type 1 interferes with and delays interferon expression in productively infected neuroblastoma cells and trigeminal Ganglia of acutely infected mice.

The herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) is the only abundant viral transcript expressed in latently infected neurons. LAT inhibits apoptosis, suggesting that it regulates latency by promoting the survival of infected neurons. The LAT locus also contains a newly described gene (AL), which is antisense to LAT and partially overlaps LAT encoding sequences. When human (SK-N-SH) or mouse (neuro-2A) neuroblastoma cells were infected with a virus that does not express LAT or AL gene products (dLAT2903), beta interferon (IFN-beta) and IFN-alpha RNA expression was detected earlier relative to the same cells infected with HSV-1 strains that express LAT and AL. Infection of neuro-2A cells with dLAT2903 also led to higher levels of IFN-beta promoter activity than in cells infected with wild-type (wt) HSV-1. In contrast, IFN RNA expression was the same when human lung fibroblasts were infected with dLAT2903 or wt HSV-1. When BALB/c mice were infected with dLAT2903, IFN-alpha and IFN-beta RNA expression was readily detected in trigeminal ganglia (TG) 4 days after infection. These transcripts were not detected in TG of mice infected with wt HSV-1 or dLAT2903R (marker-rescued dLAT2903) until 6 days postinfection. When TG single-cell suspensions from infected BALB/c mice were prepared and incubated in vitro with wt HSV-1 as a source of antigen, TG cultures prepared from mice infected with dLAT2903 produced and secreted higher levels of IFN protein than wt HSV-1 or dLAT2903R. Collectively, these studies suggest that the LAT locus interferes with and delays IFN expression.

Latency-related gene encoded by bovine herpesvirus 1 promotes virus growth and reactivation from latency in tonsils of infected calves.

Infection of calves with bovine herpesvirus 1 (BHV-1) results in transient immunosuppression that may lead to bacterium-induced pneumonia and, occasionally, death. Although sensory neurons in the trigeminal ganglia (TG) are the primary site of BHV-1 latency, viral genomes are detected in the tonsils of latently infected calves. Dexamethasone (DEX) consistently induces reactivation from latency, and viral gene expression is detected in TG and tonsils. In sensory neurons of latently infected calves, the latency-related (LR) gene is abundantly expressed and is required for reactivation from latency. In the present study, we compared the abilities of wild-type (wt) BHV-1 and a strain with a mutation in the LR gene (the LR mutant strain) to grow in the tonsils of infected calves and reactivate from latency. Lower levels of the LR mutant virus were detected in the tonsils of acutely infected calves. LR mutant viral DNA was consistently detected by PCR in the tonsils of latently infected calves, suggesting that the establishment of a latent or persistent infection occurred. Although the LR mutant did not reactivate from latency in vivo after DEX treatment, explantation of tonsil tissue from calves latently infected with the LR mutant yielded infectious virus. Relative to wt BHV-1, the LR mutant did not induce explant-induced reactivation as efficiently. These studies indicate that the LR gene promotes virus shedding from tonsil tissue during acute infection and reactivation from latency in tonsil tissue in vivo. We suggest that incorporation of the LR gene mutation into existing modified live vaccines would prevent reactivation from latency in neural and nonneural sites and would thus prevent transmission to other animals.

Infected cell protein 0 encoded by bovine herpesvirus 1 can activate caspase 3 when overexpressed in transfected cells.

Infection of cattle or bovine cells with bovine herpesvirus 1 (BHV-1) leads to increased apoptosis. Previous studies indicated that BHV-1 infected cell protein 0 (bICP0), the major transcriptional regulatory protein of BHV-1, is toxic in transiently transfected cells. Point mutations within the zinc RING finger of bICP0 reduced toxicity and eliminated the ability of bICP0 to activate viral gene expression. In mouse neuroblastoma cells (neuro-2A) and bovine turbinate cells, bICP0 activated caspase 3, a key regulatory protein in the apoptotic pathway. A pro-apoptotic gene (Bax), but not bICP0, induced caspase 3 cleavage and activation by 8 h after transfection of neuro-2A cells. Conversely, bICP0 or the N-terminal 356 aa of bICP0 did not induce caspase 3 cleavage in neuro-2A cells until 30 h after transfection, suggesting that bICP0 stimulates caspase 3 cleavage by an indirect mechanism. These studies indicate that the toxic functions of bICP0 correlate with caspase 3 cleavage and activation.

Identification of a novel bovine herpesvirus 1 transcript containing a small open reading frame that is expressed in trigeminal ganglia of latently infected cattle.

Bovine herpesvirus 1 (BHV-1), like other Alphaherpesvirinae subfamily members, establishes latency in sensory neurons. The latency-related (LR) RNA is abundantly expressed during latency, and expression of an LR protein is required for the latency reactivation cycle in cattle. Within LR promoter sequences, a 135-amino-acid open reading frame (ORF) was identified, ORF-E, that is antisense to the LR RNA. ORF-E is also downstream of the gene encoding the major viral transcriptional activator, bICP0. Strand-specific reverse transcription-PCR demonstrated that a transcript containing ORF-E was consistently expressed in trigeminal ganglia (TG) of latently infected calves, productively infected cultured cells, and acutely infected calves. As expected, a late transcript encoding glycoprotein C was not detected in TG of latently infected calves. The ORF-E transcript is polyadenylated and is expressed early when cultured bovine cells are productively infected. Protein coding sequences containing ORF-E were fused to green fluorescent protein (GFP) to examine the cellular localization of the putative protein. In transiently transfected mouse neuroblastoma (neuro-2A) and human neuroblastoma (SK-N-SH) cells, the ORF-E/GFP fusion protein was detected in discreet domains within the nucleus. In contrast, the ORF-E/GFP fusion protein was detected in the cytoplasm and nucleus of rabbit skin cells and bovine kidney cells. As expected, the GFP protein was expressed in the cytoplasm and nucleus of transfected cells. These studies indicate that the ORF-E transcript is consistently expressed during latency. We suggest that the ORF-E gene regulates some aspect of the latency reactivation cycle.

A mutation in the latency-related gene of bovine herpesvirus 1 inhibits protein expression from open reading frame 2 and an adjacent reading frame during productive infection.

The latency-related (LR) gene of bovine herpesvirus 1 (BHV-1) is abundantly expressed during latency. A mutant BHV-1 strain that contains three stop codons at the 5' terminus of the LR gene (LR mutant) does not reactivate from latency. This study demonstrates that the LR mutant does not express open reading frame 2 or an adjacent reading frame that lacks an initiating ATG (reading frame C). Since the LR mutant and wild-type BHV-1 express similar levels of LR RNA, we conclude that LR protein expression plays an important role in regulating the latency reactivation cycle in cattle.

The bovine herpesvirus-1 LR ORF2 is critical for this gene's ability to restore the high wild-type reactivation phenotype to a herpes simplex virus-1 LAT null mutant.

During neuronal latency of herpes simplex virus (HSV)-1, the latency-associated transcript (LAT) is the only viral gene readily detectable. LAT is required for the high-level reactivation phenotype in animal models. LAT's anti-apoptotic activity was recently demonstrated by our group and it was proposed that LAT's anti-apoptotic function is involved in enhancing the reactivation phenotype. Recently, using chimeric virus CJLAT, it was shown that the reactivation phenotype of LAT(-) mutant dLAT2903 can be restored to wild-type levels by inserting the bovine herpes virus (BHV)-1 latency-related (LR) gene into the LAT locus of this HSV-1 LAT deletion mutant. Although transcription of the LR gene, like LAT, inhibits apoptosis, LR appears to be multifunctional. To investigate whether the LR gene's anti-apoptotic function was responsible for restoring the high-reactivation phenotype, a mutated BHV-1 LR gene was inserted into the LAT locus of HSV-1 generating the chimeric virus CJLATmut. This mutation consists of three stop codons inserted just after the ATG of the first LR open reading frame (ORF2). In plasmids and in a BHV-1 mutant, this mutation eliminated the LR gene's anti-apoptotic activity, strongly suggesting that ORF2 encodes a protein responsible for LR's anti-apoptotic activity. Reactivation of the CJLATmut virus, in both rabbits and mice, was significantly lower than in wild-type McKrae virus (P=0.0001 and P=0.0003, respectively) and CJLAT virus, containing wild-type LR in place of LAT (P<0.0001) and was similar to LAT(-) dLAT2903 (P=0.8 and P=0.7, respectively). Thus, disruption of BHV-1 LR ORF2 eliminated the high-reactivation phenotype.

Infection of cattle with a bovine herpesvirus 1 strain that contains a mutation in the latency-related gene leads to increased apoptosis in trigeminal ganglia during the transition from acute infection to latency.

Bovine herpesvirus 1 (BHV-1) is an important pathogen of cattle and infection is usually initiated via the ocular or nasal cavity. After acute infection, the primary site for BHV-1 latency is sensory neurons in the trigeminal ganglia (TG). Reactivation from latency occurs sporadically, resulting in virus shedding and transmission to uninfected cattle. The only abundant viral transcript expressed during latency is the latency-related (LR) RNA. An LR mutant was constructed by inserting three stop codons near the beginning of the LR RNA. This mutant grows to wild-type (wt) efficiency in bovine kidney cells and in the nasal cavity of acutely infected calves. However, shedding of infectious virus from the eye and TG was dramatically reduced in calves infected with the LR mutant. Calves latently infected with the LR mutant do not reactivate after dexamethasone treatment. In contrast, all calves latently infected with wt BHV-1 or the LR rescued mutant reactivate from latency after dexamethasone treatment. In the present study, we compared the frequency of apoptosis in calves infected with the LR mutant to calves infected with wt BHV-1 because LR gene products inhibit apoptosis in transiently transfected cells. A sensitive TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) assay and an antibody that detects cleaved caspase-3 were used to identify apoptotic cells in TG. Both assays demonstrated that calves infected with the LR mutant for 14 days had higher levels of apoptosis in TG compared to calves infected with wt BHV-1 or to mock-infected calves. Viral gene expression, except for the LR gene, is extinguished by 14 days after infection, and thus this time frame is operationally defined as the establishment of latency. Real-time PCR analysis indicated that lower levels of viral DNA were present in the TG of calves infected with the LR mutant throughout acute infection. Taken together, these results suggest that the antiapoptotic properties of the LR gene play an important role during the establishment of latency.

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

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.

A novel herpes simplex virus type 1 transcript (AL-RNA) antisense to the 5' end of the latency-associated transcript produces a protein in infected rabbits.

Following primary ocular infection, herpes simplex virus type 1 (HSV-1) establishes a lifelong latent infection in sensory neurons of the trigeminal ganglia. Latency-associated transcript (LAT), the only known viral gene abundantly transcribed during HSV-1 neuronal latency, is required for high levels of reactivation. Recently we showed that three different mutants that do not alter the LAT promoter but contain deletions within the 5' end of the primary LAT transcript affect viral virulence (G. C. Perng et al., J. Virol. 75:9018-9028, 2001). In contrast, in LAT-null mutants viral virulence appears unaltered (T. M. Block et al., Virology 192:618-630, 1993; D. C. Bloom et al., J. Virol. 68:1283-1292, 1994; J. M. Hill et al., Virology 174:117-125, 1990; G. C. Perng et al., J. Virol. 68:8045-8055, 1994; F. Sedarati, K. M. Izumi, E. K. Wagner, and J. G. Stevens, J. Virol. 63:4455-4458, 1989). We therefore hypothesized that the 5' end of LAT and/or an as yet unidentified gene that overlaps part of this region is involved in viral virulence. We report here on the discovery and initial characterization of a novel HSV-1 RNA consistent with such a putative gene. The novel RNA was antisense to the 5' end of LAT and was designated AL-RNA (anti-LAT sense RNA). The AL-RNA overlapped the core LAT promoter and the first 158 nucleotides of the 5' end of the primary LAT transcript. AL-RNA was detected in extracts from neuron-like cells (PC-12) infected with wild-type HSV-1 but not in cells infected with a mutant with the AL region deleted. The deletions in each of the above three mutants with altered virulence encompass the 5' end of the AL-RNA, and these mutants cannot transcribe AL. This supports the hypothesis that the AL gene may play a role in viral virulence. Based on comparison to the corresponding genomic sequence, the AL-RNA did not appear to be spliced. The AL-RNA was polyadenylated and contained an open reading frame capable of encoding a protein 56 amino acids in length with a predicted molecular mass of 6.8 kDa. Sera from three of three rabbits infected with wild-type HSV-1 but not sera from any of three rabbits infected with a mutant with the AL-RNA region deleted recognized the Escherichia coli recombinantly expressed AL open reading frame on Western blots. In addition, four of six rabbits infected with wild-type virus developed enzyme-linked immunosorbent assay titers against one or more AL synthetic peptides. These results suggest that an AL protein is produced in vivo.

A mutation in the latency-related gene of bovine herpesvirus 1 disrupts the latency reactivation cycle in calves.

Bovine herpesvirus 1 (BHV-1) is an important pathogen of cattle, and infection is usually initiated via the ocular or nasal cavity. Following acute infection, the primary site for BHV-1 latency is the sensory neuron. Reactivation from latency occurs sporadically, resulting in virus shedding and transmission to uninfected cattle. The only abundant viral transcript expressed during latency is the latency-related (LR) RNA, suggesting that it mediates some aspect of latency. An LR mutant was constructed by inserting three stop codons near the beginning of the LR-RNA, suggesting that expression of LR proteins would be altered. The LR mutant grew with wild-type (wt) efficiency in bovine kidney cells (MDBK). When calves were infected with the LR mutant, a dramatic decrease (3 to 4 logs) in ocular, but not nasal, viral shedding occurred during acute infection relative to the wt or the LR-rescued virus (M. Inman, L. Lovato, A. Doster, and C. Jones, J. Virol. 75:8507-8515, 2001). In this study, we examined the latency reactivation cycle in calves infected with the LR mutant and compared these results to those from calves infected with wt BHV-1 or the LR-rescued virus. During acute infection, lower levels of infectious virus were detected in trigeminal ganglion homogenates from calves infected with the LR mutant. As judged by in situ hybridization, BHV-1-positive neurons were detected in trigeminal ganglia of calves infected with the wt but not the LR mutant. Although LR-RNA was detected by reverse transcription-PCR in calves latently infected with the LR mutant, a semiquantitative PCR analysis revealed that lower levels of viral DNA were present in trigeminal ganglia of calves infected with the LR mutant. Dexamethasone treatment of calves latently infected with wt BHV-1 or the LR-rescued virus, but not the LR mutant, consistently induced reactivation from latency, as judged by shedding of infectious virus from the nose or eyes and increases in BHV-1-specific antibodies. In summary, this study demonstrates that wt expression of LR gene products plays an important role in the latency reactivation cycle of BHV-1 in cattle.

A gene capable of blocking apoptosis can substitute for the herpes simplex virus type 1 latency-associated transcript gene and restore wild-type reactivation levels.

After ocular herpes simplex virus type 1 (HSV-1) infection, the virus travels up axons and establishes a lifelong latent infection in neurons of the trigeminal ganglia. LAT (latency-associated transcript), the only known viral gene abundantly transcribed during HSV-1 neuronal latency, is required for high levels of reactivation. The LAT function responsible for this reactivation phenotype is not known. Recently, we showed that LAT can block programmed cell death (apoptosis) in neurons of the trigeminal ganglion in vivo and in tissue culture cells in vitro (G.-C. Perng et al., Science 287:1500-1503, 2000; M. Inman et al., J. Virol. 75:3636-3646, 2001). Consequently, we proposed that this antiapoptosis function may be a key to the mechanism by which LAT enhances reactivation. To study this further, we constructed a recombinant HSV-1 virus in which the HSV-1 LAT gene was replaced by an alternate antiapoptosis gene. We used the bovine herpes virus 1 (BHV-1) latency-related (LR) gene, which was previously shown to have antiapoptosis activity, for this purpose. The resulting chimeric virus, designated CJLAT, contains two complete copies of the BHV-1 LR gene (one in each viral long repeat) in place of the normal two copies of the HSV-1 LAT, on an otherwise wild-type HSV-1 strain McKrae genomic background. We report here that in both rabbits and mice reactivation of CJLAT was significantly greater than the LAT null mutant dLAT2903 (P < 0.0004 and P = 0.001, respectively) and was at least as efficient as wild-type McKrae. This strongly suggests that a BHV-1 LR gene function was able to efficiently substitute for an HSV-1 LAT gene function involved in reactivation. Although replication of CJLAT in rabbits and mice was similar to that of wild-type McKrae, CJLAT killed more mice during acute infection and caused more corneal scarring in latently infected rabbits. This suggested that the BHV-1 LR gene and the HSV-1 LAT gene are not functionally identical. However, LR and LAT both have antiapoptosis activity. These studies therefore strongly support the hypothesis that replacing LAT with an antiapoptosis gene restores the wild-type reactivation phenotype to a LAT null mutant of HSV-1 McKrae.

The zinc ring finger in the bICP0 protein encoded by bovine herpesvirus-1 mediates toxicity and activates productive infection.

The bICP0 protein encoded by bovine herpesvirus 1 (BHV-1) is believed to activate transcription and consequently productive infection. Expression of full-length bICP0 protein is toxic in transiently transfected mouse neuroblastoma cells (neuro-2A) in the absence of other viral genes. However, bICP0 does not appear to directly induce apoptosis. Although bICP0 is believed to be functionally similar to the herpes simplex virus type 1-encoded ICP0, the only protein domain that is well conserved is a C3HC4 zinc ring finger located near the N terminus of both proteins. Site-specific mutagenesis of the zinc ring finger of bICP0 demonstrated that it was important for inducing aggregated chromatin structures in transfected cells and toxicity. The zinc ring finger was also required for stimulating productive infection in bovine cells and for trans-activating the thymidine kinase (TK) promoter of herpes simplex virus type 1. Deletion of amino acids spanning 356-677 of bICP0 altered subcellular localization of bICP0 and prevented trans-activation of the TK promoter. However, this deletion did not prevent trans-activation of the viral genome. Taken together, these studies indicated that bICP0 has several functional domains, including the zinc ring finger, which stimulate productive infection and influence cell survival.