William P. Halford

ICP0 Is Required for Efficient Reactivation of Herpes Simplex Virus Type 1 from Neuronal Latency

ABSTRACT Relative to wild-type herpes simplex virus type 1 (HSV-1), ICP0-null mutant viruses reactivate inefficiently from explanted, latently infected mouse trigeminal ganglia (TG), indicating that ICP0 is not essential for reactivation but plays a central role in enhancing the efficiency of reactivation. The validity of these findings has been questioned, however, because the replication of ICP0-null mutants is impaired in animal models during the establishment of latency, such that fewer mutant genomes than wild-type genomes are present in latently infected mouse TG. Therefore, the reduced number of mutant viral genomes available to reactivate, rather than mutations in the ICP0 gene per se, may be responsible for the reduced reactivation efficiency of ICP0-null mutants. We have recently demonstrated that optimization of the size of the ICP0 mutant virus inoculum and transient immunosuppression of mutant-infected mice with cyclophosphamide can be used to establish wild-type levels of ICP0-null mutant genomes in latently infected TG (W. P. Halford and P. A. Schaffer, J. Virol. 74:5957–5967, 2000). Using this procedure to equalize mutant and wild-type genome numbers, the goal of the present study was to determine if, relative to wild-type virus, the absence of ICP0 function in two ICP0-null mutants, n212 and 7134, affects reactivation efficiency from (i) explants of latently infected TG and (ii) primary cultures of latently infected TG cells. Although equivalent numbers of viral genomes were present in TG of mice latently infected with either wild-type or mutant viruses, reactivation of n212 and 7134 from heat-stressed TG explants was inefficient (31 and 37% reactivation, respectively) relative to reactivation of wild-type virus (KOS) (95%). Similarly, n212 and 7134 reactivated inefficiently from primary cultures of dissociated TG cells plated directly after removal from the mouse (7 and 4% reactivation, respectively), relative to KOS (60% reactivation). The efficiency and kinetics of reactivation of KOS, n212, and 7134 from cultured TG cells (treated with acyclovir to facilitate the establishment of latency) in response to heat stress or superinfection with a nonreplicating HSV-1 ICP4−mutant, n12, were compared. Whereas heat stress induced reactivation of KOS from 69% of latently infected TG cell cultures, reactivation of n212 and 7134 was detected in only 1 and 7% of cultures, respectively. In contrast, superinfection with the ICP4− virus, which expresses high levels of ICP0, resulted in the production of infectious virus in nearly 100% of cultures latently infected with KOS, n212, or 7134 within 72 h. Thus, although latent mutant viral genome loads were equivalent to that of wild-type virus, in the absence of ICP0, n212 and 7134 reactivated inefficiently from latently infected TG cells during culture establishment and following heat stress. Collectively, these findings demonstrate that ICP0 is required to induce efficient reactivation of HSV-1 from neuronal latency.

Alpha/Beta Interferon and Gamma Interferon Synergize To Inhibit the Replication of Herpes Simplex Virus Type 1

ABSTRACT In vivo evidence suggests that T-cell-derived gamma interferon (IFN-γ) can directly inhibit the replication of herpes simplex virus type 1 (HSV-1). However, IFN-γ is a weak inhibitor of HSV-1 replication in vitro. We have found that IFN-γ synergizes with the innate IFNs (IFN-α and -β) to potently inhibit HSV-1 replication in vitro and in vivo. Treatment of Vero cells with either IFN-β or IFN-γ inhibits HSV-1 replication by <20-fold, whereas treatment with both IFN-β and IFN-γ inhibits HSV-1 replication by ∼1,000-fold. Treatment with IFN-β and IFN-γ does not prevent HSV-1 entry into Vero cells, and the inhibitory effect can be overcome by increasing the multiplicity of HSV-1 infection. The capacity of IFN-β and IFN-γ to synergistically inhibit HSV-1 replication is not virus strain specific and has been observed in three different cell types. For two of the three virus strains tested, IFN-β and IFN-γ inhibit HSV-1 replication with a potency that approaches that achieved by a high dose of acyclovir. Pretreatment of mouse eyes with IFN-β and IFN-γ reduces HSV-1 replication to nearly undetectable levels, prevents the development of disease, and reduces the latent HSV-1 genome load per trigeminal ganglion by ∼200-fold. Thus, simultaneous activation of IFN-α/β receptors and IFN-γ receptors appears to render cells highly resistant to the replication of HSV-1. Because IFN-α or IFN-β is produced by most cells as an innate response to virus infection, the results imply that IFN-γ secreted by T cells may provide a critical second signal that potently inhibits HSV-1 replication in vivo.

Functional role and sequence analysis of a lymphocyte orphan opioid receptor

Pharmacological evidence indicates that lymphocytes express opioid receptors, but this finding has been questioned. By DNA sequencing of reverse transcription-polymerase chain reaction products, we have found that mouse lymphocytes express mRNA encoding an orphan opioid receptor. These mRNA transcripts were detected in the CD4+, CD8+, and CD4- CD8- lymphocyte subpopulations. Northern blot analysis confirmed that splenic lymphocytes express a 1.5-kb orphan opioid receptor mRNA. Fifteen bases encoding Tyr71-Arg75 in the first intracellular loop are alternatively spliced, suggesting that orphan opioid receptor mRNA encodes two receptor subtypes. Treatment of lipopolysaccharide-stimulated lymphocytes with orphan opioid receptor antisense oligonucleotides suppressed polyclonal IgG and IgM production by 50%. Our results provide direct evidence that lymphocytes express an opioid-like receptor gene, and suggest that this receptor plays a functional role in immunocompetence.

Green fluorescent protein is a quantitative reporter of gene expression in individual eukaryotic cells

Green fluorescent protein (GFP) has gained widespread use as a tool to visualize spatial and temporal patterns of gene expression in vivo. However, it is not generally accepted that GFP can also be used as a quantitative reporter of gene expression. We report that GFP is a reliable reporter of gene expression in individual eukaryotic cells when fluorescence is measured by flow cytometry. Two pieces of evidence support this conclusion: GFP fluorescence increases in direct proportion to the GFP gene copy number delivered to cells by a replication‐defective adenovirus vector, Ad.CMV‐GFP, and the intensity of GFP fluorescence is directly proportional to GFP mRNA abundance in cells. This conclusion is further supported by the fact that the induction of GFP gene expression from two inducible promoters (i.e., the TRE and ICP0 promoters) is readily detected by flow cytometric measurement of GFP fluorescent intensity. Collectively, the results presented herein indicate that GFP fluorescence is a reliable and quantitative reporter of underlying differences in gene expression.

ICP0, ICP4, or VP16 Expressed from Adenovirus Vectors Induces Reactivation of Latent Herpes Simplex Virus Type 1 in Primary Cultures of Latently Infected Trigeminal Ganglion Cells

ABSTRACT In a previous study, we demonstrated that infected-cell polypeptide 0 (ICP0) is necessary for the efficient reactivation of herpes simplex virus type 1 (HSV-1) in primary cultures of latently infected trigeminal ganglion (TG) cells (W. P. Halford and P. A. Schaffer, J. Virol. 75:3240–3249, 2001). The present study was undertaken to determine whether ICP0 is sufficient to trigger HSV-1 reactivation in latently infected TG cells. To test this hypothesis, replication-defective adenovirus vectors that express wild-type and mutant forms of ICP0 under the control of a tetracycline response element (TRE) promoter were constructed. Similar adenovirus vectors encoding wild-type ICP4, wild-type and mutant forms of the HSV-1 origin-binding protein (OBP), and wild-type and mutant forms of VP16 were also constructed. The TRE promoter was induced by coinfection of Vero cells with the test vector and an adenovirus vector that expresses the reverse tetracycline-regulated transactivator in the presence of doxycycline. Northern blot analysis demonstrated that transcription of the OBP gene in the adenovirus expression vector increased as a function of doxycycline concentration over a range of 0.1 to 10 μM. Likewise, Western blot analysis demonstrated that addition of 3 μM doxycycline to adenovirus vector-infected Vero cells resulted in a 100-fold increase in OBP expression. Wild-type forms of ICP0, ICP4, OBP, and VP16 expressed from adenovirus vectors were functional based on their ability to complement plaque formation in Vero cells by replication-defective HSV-1 strains with mutations in these genes. Adenovirus vectors that express wild-type forms of ICP0, ICP4, or VP16 induced reactivation of HSV-1 in 86% ± 5%, 86% ± 5%, and 97% ± 5% of TG cell cultures, respectively (means ± standard deviations). In contrast, vectors that express wild-type OBP or mutant forms of ICP0, OBP, or VP16 induced reactivation in 5% ± 5%, 8% ± 0%, 0% ± 0%, and 13% ± 6% of TG cell cultures, respectively. In control infections, an adenovirus vector expressed green fluorescent protein efficiently in TG neurons but did not induce HSV-1 reactivation. Therefore, expression of ICP0, ICP4, or VP16 is sufficient to induce HSV-1 reactivation in latently infected TG cell cultures. We conclude that this system provides a powerful tool for determining which cellular and viral proteins are sufficient to induce HSV-1 reactivation from neuronal latency.

ICP0 antagonizes Stat 1-dependent repression of herpes simplex virus: implications for the regulation of viral latency

BackgroundThe herpes simplex virus type 1 (HSV-1) ICP0 protein is an E3 ubiquitin ligase, which is encoded within the HSV-1 latency-associated locus. When ICP0 is not synthesized, the HSV-1 genome is acutely susceptible to cellular repression. Reciprocally, when ICP0 is synthesized, viral replication is efficiently initiated from virions or latent HSV-1 genomes. The current study was initiated to determine if ICP0s putative role as a viral interferon (IFN) antagonist may be relevant to the process by which ICP0 influences the balance between productive replication versus cellular repression of HSV-1.ResultsWild-type (ICP0+) strains of HSV-1 produced lethal infections in scid or rag2-/- mice. The replication of ICP0- null viruses was rapidly repressed by the innate host response of scid or rag2-/- mice, and the infected animals remained healthy for months. In contrast, rag2-/- mice that lacked the IFN-α/β receptor (rag2-/-ifnar-/-) or Stat 1 (rag2-/-stat1-/-) failed to repress ICP0- viral replication, resulting in uncontrolled viral spread and death. Thus, the replication of ICP0- viruses is potently repressed in vivo by an innate immune response that is dependent on the IFN-α/β receptor and the downstream transcription factor, Stat 1.ConclusionICP0s function as a viral IFN antagonist is necessary in vivo to prevent an innate, Stat 1-dependent host response from rapidly repressing productive HSV-1 replication. This antagonistic relationship between ICP0 and the host IFN response may be relevant in regulating whether the HSV-1 genome is expressed, or silenced, in virus-infected cells in vivo. These results may also be clinically relevant. IFN-sensitive ICP0- viruses are avirulent, establish long-term latent infections, and induce an adaptive immune response that is highly protective against lethal challenge with HSV-1. Therefore, ICP0- viruses appear to possess the desired safety and efficacy profile of a live vaccine against herpetic disease.

Re-Evaluating Natural Resistance to Herpes Simplex Virus Type 1

ABSTRACT It is often stated that individuals of a species can differ significantly in their innate resistance to infection with herpes simplex virus type 1 (HSV-1). Three decades ago Lopez reported that C57BL/6 mice could survive a 5,000-fold-higher inoculum of HSV-1 given intraperitoneally than mice of the A or BALB/c strain (Nature 258:152-153, 1975). Susceptible strains of mice died of encephalitis-like symptoms, suggesting that viral spread to the central nervous system was the cause of death. Although Lopezs study documented that C57BL/6 mice were resistant to the development of HSV-1 encephalitis and mortality, the resistance of C57BL/6 mice to other steps of the HSV-1 infection process was not assessed. The results of the present study extend these observations to clarify the difference between resistance to (i) HSV-1 pathogenesis, (ii) HSV-1 replication, (iii) HSV-1 spread, and (iv) the establishment of latent HSV-1 infection. Although C57BL/6 mice are more resistant to HSV-1 pathogenesis than BALB/c mice, the results of the present study establish that HSV-1 enters, replicates, spreads, and establishes latent infections with virtually identical efficiencies in C57BL/6 and BALB/c mice. These observations raise questions about the validity of the inference that differences in natural resistance are relevant in explaining what differentiates humans with recurrent herpetic disease from the vast majority of asymptomatic carriers of HSV-1 and HSV-2.

Optimized Viral Dose and Transient Immunosuppression Enable Herpes Simplex Virus ICP0-Null Mutants To Establish Wild-Type Levels of Latency In Vivo

ABSTRACT The reduced efficiency with which herpes simplex virus type 1 (HSV-1) mutants establish latent infections in vivo has been a fundamental obstacle in efforts to determine the roles of individual viral genes in HSV-1 reactivation. For example, in the absence of the “nonessential” viral immediate-early protein, ICP0, HSV-1 is severely impaired in its ability to (i) replicate at the site of inoculation and (ii) establish latency in neurons of the peripheral nervous system. The mouse ocular model of HSV latency was used in the present study to determine if the conditions of infection can be manipulated such that replication-impaired, ICP0-null mutants establish wild-type levels of latency, as measured by viral genome loads in latently infected trigeminal ganglia (TG). To this end, the effects of inoculum size and transient immunosuppression on the levels of acute replication in mouse eyes and of viral DNA in latently infected TG were examined. Following inoculation of mice with 2 × 103, 2 × 104, 2 × 105, or 2 × 106 PFU/eye, wild-type virus replicated in mouse eyes and established latency in TG with similar efficiencies at all four doses. In contrast, increasing the inoculum size of the ICP0-null mutants n212 and 7134 from 2 × 105 to 2 × 106PFU/eye significantly decreased the levels of infectious virus detected in the tear films of mice from days 4 to 9 postinfection. In an attempt to establish the biological basis for this finding, the effect of viral dose on the induction of the host proinflammatory response was examined. Quantitative reverse transcription-PCR demonstrated that increasing the inoculum of 7134 from 2 × 104 to 2 × 106 PFU/eye significantly increased the expression of proinflammatory (interleukin 6), cell adhesion (intercellular adhesion molecule 1), and phagocyte-associated (CD11b) genes in mouse eyes 24 h postinfection. Furthermore, transient immunosuppression of mice with cyclophosphamide, but not cyclosporin A, significantly enhanced both the levels of acute n212 and 7134 replication in the eye and the levels of mutant viral genomes present in latently infected TG in a dose-dependent manner. Thus, the results of this study demonstrate that acute replication in the eye and the number of ICP0-null mutant genomes in latently infected TG can be increased to wild-type levels for both n212 and 7134 by (i) optimization of inoculum size and (ii) transient immunosuppression with cyclophosphamide.

Cytokine and chemokine production in HSV-1 latently infected trigeminal ganglion cell cultures: Effects of hyperthermic stress

The establishment of a primary trigeminal ganglion (TG) cell culture latently infected with herpes simplex virus type 1 (HSV-1) has been useful in studying stress-induced reactivation of the latent virus. However, the immune profile of this culture system prior to and after stress has never been established. In the present manuscript, cytokine and chemokine production were measured in primary cultures of TG cells obtained from uninfected and HSV-1 latently infected mice. Supernates from TG cell cultures contained detectable interleukin (IL)-6 but not IL-1beta, IL-2, IL-10, interferon (IFN)-gamma or tumor necrosis factor (TNF)-alpha as determined by ELISA. The basal level of IL-6 in uninfected TG cell cultures was 20.5 +/- 2.3 ng/ml, whereas latently infected TG cells produced significantly less IL-6 (12.1 +/- 1.9 ng/ml). Supernates from TG cell cultures also contained detectable levels of C-10, MCP-1 and eotaxin but little to no MIP-1alpha, MIP-1beta, or MIP-2. While there were no differences in the basal level of MCP-1 and eotaxin in TG cell cultures from HSV-1-infected and uninfected mice, C10 levels were significantly higher in TG cultures originating from infected mice compared to uninfected ones (5.86 +/- 0.61 ng/ml compared to 1.18 +/- 0.16 ng/ml). Hyperthermic stress (43 degrees C, 180 min), which induces reactivation of latent HSV-1 from TG cell cultures, significantly reduced IL-6 and C-10 levels from both uninfected and latently infected TG cell cultures. However, there was no correlation between cytokine/chemokine levels and HSV-1 reactivation. Immunofluorescent studies showed TG cell cultures contained 10% MAC-3+ staining cells (macrophage specific) but no dendritic cells. By comparison, cells from freshly isolated TG contained 6% positive dendritic cells but < 1% MAC-3 + cells. Both in vivo and in vitro TG consisted of a low percentage of CD3+ and CD8+ cells. Hyperthermic stress (43 degrees C for 3 h) eliminated the lymphocyte population as determined by RT-PCR. Whereas no spontaneous reactivation has been reported in mice, spontaneous reactivation occurred in 4.5% (10/220) of TG cell cultures surveyed over a 20 day period. Collectively, the dichotomy between HSV-1 replication and reactivation comparing the in vitro and in vivo HSV-1 latency models may reside, in part, to the differences in the levels of cytokines, chemokines and immune cell populations within the microenvironment of the in vitro and in vivo TG.

Evidence that spontaneous reactivation of herpes virus does not occur in mice

BackgroundSome species, including humans and rabbits, exhibit periodic viral reactivation and shed infectious virus at the infected end organ. Mice may be an exception, because spontaneous shedding of infectious virus rarely, if ever, occurs. However, spontaneous molecular reactivation, i.e., the expression of a few viral genes and the synthesis of the viral glycoproteins coded for by these genes, has been reported. This finding has prompted the assumption that molecular reactivation is an indicator of reactivation and the production of infectious virus. The goal of this study was to differentiate between viral gene expression during latency and the episodic production of infectious virus in mice.ResultsViral reactivation and infection were not seen in herpes simplex virus type 1 (HSV-1) latent ganglion graft recipient BALB/c scid or immunocompetent BALB/c mice, which survived the 65-day observation period with no evidence of viral infection although the immunocompetent mice developed cellular and humoral immunity to HSV-1. In contrast, BALB/c scid recipients of ganglia containing reactivating virus invariably developed a local and, subsequently, systemic viral infection and died within 14 days. Immunocompetent BALB/c mice that received ganglion grafts containing reactivating virus survived the infection and became immune to the virus. Trigeminal ganglia removed from scid and immunocompetent recipient graft sites 5, 14, and 28 days after transplantation contained latent virus and viable neurons.ConclusionThe results suggest that, within the limits of detection of the experiments, spontaneous episodic production of immunogenic viral antigens but not of infectious virus occurs in mouse neural ganglia during latency.