<p>During Herpes Simplex Virus type 1 (HSV-1) infections, many viral proteins are synthesized and several have proven or suspected roles in regulating viral gene expression. To facilitate the study of the individual activity of one such protein, ICP4, the ICP4 gene was cloned in a plasmid vector, and expresser cell lines containing 5-30% of infected cell levels of ICP4 were established. The ICP4 is functional, correctly processed, and located in the cell nuclei. The endogenous ICP4 gene retained its capacity to respond to viral trans-acting factors, since its expression after superinfection with HSV-2 mimicked that of the viral gene. Although cells infected with lCP4 mutant viruses overproduce ICP4 and other immediate-early proteins, cell lines synthesizing a mutant form of ICP4 did not overproduce this protein, suggesting that autoregulation of the lCP4 gene requires more than 30% of the infected cell level of ICP4 or, alternatively, requires the presence of other viral proteins. After superinfection in the presence of an inhibitor of protein synthesis, the endogenous ICP4 is capable of transactivating viral early genes encoding thymidine kinase, lCP6, ICP8, gB, gD, and gE. In contrast, the early gene for the viral alkaline exonuclease, the early-late gene for VP5 and the late genes for p40 and gC, respond poorly or not at all. This demonstrates that most early genes can be induced by ICP4 in the absence of other viral immediate-early proteins, but that early-late and late genes require supplementary factors.</p> / Doctor of Philosophy (PhD)

Herpes simplex viruses 1 and 2 (HSV-1 and HSV-2) infect and establish latency in peripheral neurons, from which they can reactivate to cause recurrent disease throughout the life of the host. Stress is associated with exacerbation of clinical symptoms and induction of recurrences in humans and animal models. The viruses preferentially replicate and establish latency in different subtypes of sensory neurons, as well as in neurons of the autonomic nervous system that are highly responsive to stress hormones. To determine if stress-related hormones modulate productive and latent HSV-1 and HSV-2 infection within sensory and autonomic neurons, we analyzed viral DNA after treatment of primary adult murine neuronal cultures with the stress hormones epinephrine and corticosterone. Both sensory trigeminal (TG) and sympathetic superior cervical ganglia (SCG) neurons expressed adrenergic receptors and glucocorticoid receptor. In productively infected neuronal cultures, epinephrine treatment significantly increased HSV-1 DNA replication and production of viral progeny in SCG neurons, but no significant differences were found in TG neurons. In contrast, corticosterone significantly decreased HSV-2 DNA replication and production of viral progeny in SCG neurons, but not in TG neurons. In quiescently infected neuronal cultures, epinephrine and corticosterone significantly increased HSV-1 reactivation from sympathetic SCG neurons, but not sensory TG neurons. In contrast, corticosterone increased HSV-2 reactivation from both SCG and TG neurons, but epinephrine had no effect. Adrenergic or epinephrine-induced reactivation of HSV-1 in SCG neurons involved activation of several adrenergic receptors, the cyclic AMP response element binding protein (CREB), the transcription factor β-catenin, and the c-Jun N-terminal kinase (JNK). Corticosterone-induced reactivation of HSV-1 in SCG neurons required activation of glucocorticoid receptor (GCR) and transcription factors CREB and JNK. In contrast, corticosterone-induced reactivation of HSV-2 in TG and SCG neurons could utilize either the GCR or mineralocorticoid receptor (MCR) and most likely involves the chromatin remodeling properties of those receptors. Thus, stress-related hormones, epinephrine and corticosterone, selectively modulate productive and quiescent HSV-1 and HSV-2 infections primarily in sympathetic, but not sensory, neurons through different mechanisms. These results have implications for describing a mechanism by which stress-induced reactivation may occur in humans. / Ph. D. / Herpes simplex virus type 1 and 2 (HSV-1 and HSV-2) are major human pathogens, which establish latency in neurons of the peripheral nervous system and reactivate to cause recurrent disease in humans. Physiological stress, which includes the secretion of the stress hormones epinephrine and cortisol, has been associated with increases in severity of clinical signs and increased recurrent disease in humans and animal models of herpetic disease. The mechanism by which physiological stress induces HSV reactivation has been assumed to be through suppression of the immune system. In addition, it has been assumed that sensory neurons harboring latent HSV are the primary source of reactivating virus for recurrent HSV disease. However, my dissertation provides evidence that the stress hormones epinephrine and corticosterone (the rodent equivalent of cortisol) can act on peripheral neurons in which the virus is latent, rather than through immune system suppression. In addition, my dissertation provides evidence that the autonomic nervous system, which modulates the physiological stress response, is an important source of reactivating virus to cause recurrent disease. The molecular pathway by which epinephrine and corticosterone induce HSV reactivation in primary adult murine neurons involves specific receptors, transcription factors, and protein kinases that could potentially be targeted in humans for inhibition of HSV reactivation and prevention of herpetic recurrent disease.

No description available.

Purpose: To investigate the immune responses in cornea, ocular draining lymph nodes and spleen as well as the priming site of HSV-specific lymphocytes and their possible role in HSK development. To explore the possible treatment of HSK by transplanting corneal allograft and collagen artificial cornea. Methods: BALB/c mice corneas were infected with RE strain HSV-1. Immunohistochemistry of eye sections was performed and flow cytometry was carried out for cell suspension of cornea, TG, submandibular ocular draining lymph node (SMDLN),a non-ocular related lymph node and spleen at various times post HSV-1 eye inoculation. Results: There were strong immune responses in ocular draining lymph nodes post HSV-1 ocular infection, with a significant increasing number of innate cells as well as B cells and T cells. These changes were not observed in non-ocular related lymph nodes or spleen. An antigen specific response to HSV-1 antigen stimulation was observed <i>in vitro</i> for crude cells from ocular draining lymph node and to a lesser extent for spleen, but no changes were observed for the cells from non-ocular related lymph node. Interestingly, removal of ocular draining lymph nodes or spleen prior to HSV inoculation did not prevent HSK, but adversely impaired the control of viral replication, which was indicated by severe blepharitis and encephalitis. Both corneal allograft and collagen artificial cornea failed to survive in HSK eye, and retro-corneal membrane and degradation were the main obstacles for artificial cornea. Conclusions: HSV-specific lymphocytes are primed mainly in ocular draining lymph nodes, however, these cells might not be necessarily required for HSK development. Prevention of retro-corneal membrane seems to be important for survival of both corneal allograft and artificial cornea in HSK eyes.

Herpes simplex virus type 1 (HSV-1) is a virus commonly causing cold sores in humans, however, virulent infections are known to produce debilitating encephalitis and death. HSV-1 transcription is carried out by the host cell RNA polymerase II in a tightly regulated temporal cascade. The first genes transcribed, the a genes, are activated in the absence of viral DNA synthesis. Transcription of the other temporal classes, the β, βγ, and γ genes is dependent upon the protein products of the a genes for activation. The purpose of this study was to investigate the factors that contribute to this rigid regulation of HSV-1 transcription. This investigation sought to identify some of the cellular and viral transcription factors that activate transcription of genes of the later kinetic classes. Two separate approaches were utilized in these investigations. 1) In vitro transcription using a soluble, cell free system to study the transcriptional regulation of the VP5 gene, and 2) DNA competition binding assays to identify and characterize the protein-DNA complexes resulting from interaction between the cisacting DNA sequences of the VP5 gene, other viral genes, and the proteins that bind to them. Attempts at in vitro transcription of β, βγ, and γ genes were unsuccessful. Because these genes require a products for activation, it was necessary to prepare nuclear extracts from infected cells. However, HSV-1 contains endogenous RNase activities which are components of the biochemical machinery by which the virus directs host transcription to the synthesis of viral molecules. The uses of virus deficient in the host shut-off function and various drugs were unsuccessful. Previous work in the Millette laboratory demonstrated a sequence in the VP5 promoter that played a significant role in the up regulation of expression of that gene. DNA binding competition studies using a number of HSV-1 sequences exhibiting partial homology to this sequence demonstrated that these sequences all compete for the binding of the same protein factor. Similarly, a piece of the human immunodeficiency virus (HIV) exhibiting a seven base pair homology also exhibited weak competition.

HSV-2 may have adverse consequences in HIV. I evaluated the impact of HSV-2 co-infection on (highly active antiretroviral therapy)-untreated HIV infection in a systematic review of observational studies (study 1) and a retrospective cohort (study 2). I further evaluated whether HSV reactivation rates in co-infected persons differ by use of suppressive cART (study 3). Study 1 found modest evidence that HSV-2 seropositivity may be associated with accelerated progression to opportunistic infection or clinical AIDS, but not with increased HIV viral load. Some evidence suggests that HSV-2 disease activity is associated with increased HIV viral load and decreased CD4 counts. Study 2 compared rates of CD4 count change by HSV-2 status (Focus HerpeSelect ELISA) among 218 patients with a past period of ART-untreated follow-up using mixed linear regression models. No significant difference in the rate of CD4 count change was observed in HSV-2 seropositives at +13.6 cells/mm3/year (p=0.12) in univariate analysis, and -4.5 cells/mm3/year (p=0.68) in analysis adjusted for sex, HSV-1, oral and genital HSV symptoms, immigrant status, and immigrant*time interaction. These findings support the need for carefully designed and executed studies of HSV-2 suppression as an adjunctive management strategy for HIV disease, but raise questions regarding the exact mechanism of negative synergy between these viruses and the relative importance of HSV-2 latency and replication in driving these effects. In Study 3, 44 cART-naïve and 41 treated (HIV RNA<50 copies/mL) HIV+ adults with HSV-1 and/or 2 co-infection collected oral, genital and anal swabs daily for 28 days. Negative binomial models were used to quantify the relationship between cART and HSV shedding (Roche LightCycler HSV1/2). Overall HSV shedding was low, at a median (IQR) of 3.6% (0, 14.3%) of days. No relationship was seen between cART and HSV-1 or 2 shedding in univariate (RR=1.55, 95%CI=0.83,2.87) or multivariate analysis adjusted for sex, baseline CD4, recent immigrant status, and time since HIV diagnosis (aRR=1.05, 95%CI=0.43,2.58). Null results were also observed for HSV-1 and HSV-2 considered separately. That HSV shedding persists despite cART suggests that trials of anti-HSV drugs for improving HIV outcomes may be warranted in such patients.

Thesis (Ph. D.)--University of Chicago, Committee on Virology, 2001. / Includes bibliographical references. Also available on the Internet.

Thesis (Ph. D.)--University of Chicago, Committee on Virology, June 2001. / Includes bibliographical references. Also available on the Internet.

HSV-2 may have adverse consequences in HIV. I evaluated the impact of HSV-2 co-infection on (highly active antiretroviral therapy)-untreated HIV infection in a systematic review of observational studies (study 1) and a retrospective cohort (study 2). I further evaluated whether HSV reactivation rates in co-infected persons differ by use of suppressive cART (study 3). Study 1 found modest evidence that HSV-2 seropositivity may be associated with accelerated progression to opportunistic infection or clinical AIDS, but not with increased HIV viral load. Some evidence suggests that HSV-2 disease activity is associated with increased HIV viral load and decreased CD4 counts. Study 2 compared rates of CD4 count change by HSV-2 status (Focus HerpeSelect ELISA) among 218 patients with a past period of ART-untreated follow-up using mixed linear regression models. No significant difference in the rate of CD4 count change was observed in HSV-2 seropositives at +13.6 cells/mm3/year (p=0.12) in univariate analysis, and -4.5 cells/mm3/year (p=0.68) in analysis adjusted for sex, HSV-1, oral and genital HSV symptoms, immigrant status, and immigrant*time interaction. These findings support the need for carefully designed and executed studies of HSV-2 suppression as an adjunctive management strategy for HIV disease, but raise questions regarding the exact mechanism of negative synergy between these viruses and the relative importance of HSV-2 latency and replication in driving these effects. In Study 3, 44 cART-naïve and 41 treated (HIV RNA<50 copies/mL) HIV+ adults with HSV-1 and/or 2 co-infection collected oral, genital and anal swabs daily for 28 days. Negative binomial models were used to quantify the relationship between cART and HSV shedding (Roche LightCycler HSV1/2). Overall HSV shedding was low, at a median (IQR) of 3.6% (0, 14.3%) of days. No relationship was seen between cART and HSV-1 or 2 shedding in univariate (RR=1.55, 95%CI=0.83,2.87) or multivariate analysis adjusted for sex, baseline CD4, recent immigrant status, and time since HIV diagnosis (aRR=1.05, 95%CI=0.43,2.58). Null results were also observed for HSV-1 and HSV-2 considered separately. That HSV shedding persists despite cART suggests that trials of anti-HSV drugs for improving HIV outcomes may be warranted in such patients.

To investigate the localization and targeting of sodium channels in neurons, an efficient means of gene delivery will need to be established. Two amplicon-based viral approaches and a recombinant whole virus approach were attempted in order to package and express RIIA sodium channel tagged with a c-myc epitope (RIIA-myc) with the ultimate purpose of developing a Herpes virus-based model system for targeting studies. / Immunofluorescent staining of transfected epithelial cells was carried out to demonstrate that constructs created for use in these HSV-based approaches were capable of a high level of expression of RIIA-myc. Measurements of $ beta$-galactosidase reporter gene expression observed in cultured cells infected with RIIA amplicon virus suggested successful packaging of amplicon DNA. However, RIIA-myc expression from amplicon virus was not apparent, which may suggest recombination events occurred upon packaging of constructs. Difficulties in selection for recombinants with acyclovir prevented the recombinant whole virus approach from being pursued.