Ada Yukht

The Latency-Associated Transcript Gene Enhances Establishment of Herpes Simplex Virus Type 1 Latency in Rabbits

ABSTRACT The latency-associated transcript (LAT) gene the only herpes simplex virus type 1 (HSV-1) gene abundantly transcribed during neuronal latency, is essential for efficient in vivo reactivation. Whether LAT increases reactivation by a direct effect on the reactivation process or whether it does so by increasing the establishment of latency, thereby making more latently infected neurons available for reactivation, is unclear. In mice, LAT-negative mutants appear to establish latency in fewer neurons than does wild-type HSV-1. However, this has not been confirmed in the rabbit, and the role of LAT in the establishment of latency remains controversial. To pursue this question, we inserted the gene for the enhanced green fluorescent protein (EGFP) under control of the LAT promoter in a LAT-negative virus (ΔLAT-EGFP) and in a LAT-positive virus (LAT-EGFP). Sixty days after ocular infection, trigeminal ganglia (TG) were removed from the latently infected rabbits, sectioned, and examined by fluorescence microscopy. EGFP was detected in significantly more LAT-EGFP-infected neurons than ΔLAT-EGFP-infected neurons (4.9% versus 2%, P < 0.0001). The percentages of EGFP-positive neurons per TG ranged from 0 to 4.6 for ΔLAT-EGFP and from 2.5 to 11.1 for LAT-EGFP (P = 0.003). Thus, LAT appeared to increase neuronal latency in rabbit TG by an average of two- to threefold. These results suggest that LAT enhances the establishment of latency in rabbits and that this may be one of the mechanisms by which LAT enhances spontaneous reactivation. These results do not rule out additional LAT functions that may be involved in maintenance of latency and/or reactivation from latency.

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

ABSTRACT 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.

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

ABSTRACT 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.

Three Herpes Simplex Virus Type 1 Latency-Associated Transcript Mutants with Distinct and Asymmetric Effects on Virulence in Mice Compared with Rabbits

ABSTRACT Herpes simplex virus type 1 latency-associated transcript (LAT)-null mutants have decreased reactivation but normal virulence in rabbits and mice. We report here on dLAT1.5, a mutant with LAT nucleotides 76 to 1667 deleted. Following ocular infection of rabbits, dLAT1.5 reactivated at a lower rate than its wild-type parent McKrae (6.1 versus 11.8%; P = 0.0025 [chi-square test]). Reactivation was restored in the marker-rescued virus dLAT1.5R (12.6%;P = 0.53 versus wild type), confirming the importance of the deleted region in spontaneous reactivation. Compared with wild-type or marker-rescued virus, dLAT1.5 had similar or slightly reduced virulence in rabbits (based on survival following ocular infection). In contrast, in mice,dLAT1.5 had increased virulence (P< 0.0001). Thus, deletion of LAT nucleotides 76 to 1667 increased viral virulence in mice but not in rabbits. In contrast, we also report here that LAT2.9A, a LAT mutant that we previously reported to have increased virulence in rabbits (G. C. Perng, S. M. Slanina, A. Yuhkt, B. S. Drolet, W. J. Keleher, H. Ghiasi, A. B. Nesburn, and S. L. Wechsler, J. Virol. 73:920–929, 1999), had decreased virulence in mice (P = 0.03). In addition, we also found that dLAT371, a LAT mutant that we previously reported to have wild-type virulence in rabbits (G. C. Perng, S. M. Slanina, H. Ghiasi, A. B. Nesburn, and S. L. Wechsler, J. Virol. 70:2014–2018, 1996), had decreased virulence in mice (P < 0.05). Thus, these three mutants, each of which encodes a different LAT RNA, have different virulence phenotypes. dLAT1.5 had wild-type virulence in rabbits but increased virulence in mice. In contrast, LAT2.9A had increased virulence in rabbits but decreased virulence in mice, anddLAT371 had wild-type virulence in rabbits but decreased virulence in mice. Taken together, these results suggest that (i) the 5′ end of LAT and/or a gene that overlaps part of this region is involved in viral virulence, (ii) this virulence appears to have species-specific effects, and (iii) regulation of this virulence may be complex.

Oxidized LDL activated eosinophil polarize macrophage phenotype from M2 to M1 through activation of CD36 scavenger receptor

BACKGROUND AND AIMS Inflammation, particularly innate immunity, plays an important role in cardiovascular diseases. The aim of this study was to investigate whether atherogenic determinants such as oxidized LDL modulate the phenotype of eosinophils. METHODS Cultured eosinophils were treated with oxidized LDL and the expression of selective inflammatory and anti-inflammatory cytokines was determined. In addition, the eosinophil receptor and signaling that mediate these events were identified. RESULTS Treatment of cultured eosinophils with oxidized LDL (Ox-LDL) specifically induced the expression of IFNα and IFNβ without affecting expression of other proinflammatory cytokines, such as TNFα, IL-1β, and IL-6. In macrophages, Ox-LDL downregulated expression of both IFNα and IFNβ, suggesting that the effect of Ox-LDL on the expression of type I interferons is specific to eosinophils. Furthermore, we noted that eosinophils constitutively expressed IL-4 and IL-13, and Ox-LDL markedly downregulated their expression. Analysis of Ox-LDL signaling revealed that eosinophils constitutively expressed SRB2, CD36, and CD68 scavenger receptors, and Ox-LDL markedly induced the expression of CD36. Further analysis of CD36 signaling by siRNA and neutralizing antibodies showed that the induction of type I IFN by Ox-LDL is mediated by CD36 signaling whereas downregulation of IL-4 is independent of CD36 activation. We further showed that peritoneal macrophages treated with condition medium collected from Ox-LDL treated eosinophils markedly induced the expression of M1 markers such as iNOS, IL6, SOSC3 and TNFα whereas the condition medium from non-treated eosinophils significantly induced expression of M2 markers like ARG1 and CCL24. CONCLUSIONS Our data suggest that an atherogenic condition could activate eosinophils and modulate the phenotype of macrophages (from M2 to M1 phenotype), in part, through the CD36 receptor signaling.