Identification and characterization of the herpes simplex virus type 2 gene encoding the essential capsid protein ICP32/VP19c.

We describe the characterization of the herpes simplex virus type 2 (HSV-2) gene encoding infected cell protein 32 (ICP32) and virion protein 19c (VP19c). We also demonstrate that the HSV-1 UL38/ORF.553 open reading frame (ORF), which has been shown to specify a viral protein essential for capsid formation (B. Pertuiset, M. Boccara, J. Cebrian, N. Berthelot, S. Chousterman, F. Puvian-Dutilleul, J. Sisman, and P. Sheldrick, J. Virol. 63: 2169-2179, 1989), must encode the cognate HSV type 1 (HSV-1) ICP32/VP19c protein. The region of the HSV-2 genome deduced to contain the gene specifying ICP32/VP19c was isolated and subcloned, and the nucleotide sequence of 2,158 base pairs of HSV-2 DNA mapping immediately upstream of the gene encoding the large subunit of the viral ribonucleotide reductase was determined. This region of the HSV-2 genome contains a large ORF capable of encoding two related 50,538- and 49,472-molecular-weight polypeptides. Direct evidence that this ORF encodes HSV-2 ICP32/VP19c was provided by immunoblotting experiments that utilized antisera directed against synthetic oligopeptides corresponding to internal portions of the predicted polypeptides encoded by the HSV-2 ORF or antisera directed against a TrpE/HSV-2 ORF fusion protein. The type-common immunoreactivity of the two antisera and comparison of the primary amino acid sequences of the predicted products of the HSV-2 ORF and the equivalent genomic region of HSV-1 provided evidence that the HSV-1 UL38 ORF encodes the HSV-1 ICP32/VP19c. Analysis of the expression of the HSV-1 and HSV-2 ICP32/VP19c cognate proteins indicated that there may be differences in their modes of synthesis. Comparison of the predicted structure of the HSV-2 ICP32/VP19c protein with the structures of related proteins encoded by other herpes viruses suggested that the internal capsid architecture of the herpes family of viruses varies substantially.

Genetic and hormonal factors modulate spreading depression and transient hemiparesis in mouse models of familial hemiplegic migraine type 1.

Familial hemiplegic migraine type 1 (FHM1) is an autosomal dominant subtype of migraine with aura that is associated with hemiparesis. As with other types of migraine, it affects women more frequently than men. FHM1 is caused by mutations in the CACNA1A gene, which encodes the alpha1A subunit of Cav2.1 channels; the R192Q mutation in CACNA1A causes a mild form of FHM1, whereas the S218L mutation causes a severe, often lethal phenotype. Spreading depression (SD), a slowly propagating neuronal and glial cell depolarization that leads to depression of neuronal activity, is the most likely cause of migraine aura. Here, we have shown that transgenic mice expressing R192Q or S218L FHM1 mutations have increased SD frequency and propagation speed; enhanced corticostriatal propagation; and, similar to the human FHM1 phenotype, more severe and prolonged post-SD neurological deficits. The susceptibility to SD and neurological deficits is affected by allele dosage and is higher in S218L than R192Q mutants. Further, female S218L and R192Q mutant mice were more susceptible to SD and neurological deficits than males. This sex difference was abrogated by ovariectomy and senescence and was partially restored by estrogen replacement, implicating ovarian hormones in the observed sex differences in humans with FHM1. These findings demonstrate that genetic and hormonal factors modulate susceptibility to SD and neurological deficits in FHM1 mutant mice, providing a potential mechanism for the phenotypic diversity of human migraine and aura.

Prevalence and risk factors for human immunodefiency virus and hepatitis C virus co-infection among drug users in innercity neighborhoods in Houston, Texas

Background. There are 200,000 HIV/HCV co-infected people in the US and IDUs are at highest risk of exposure. Between 52-92% of HIV infected IDUs are chronically infected with HCV. African Americans and Hispanics bear the largest burden of co-infections. Furthermore HIV/HCV co-infection is associated with high morbidity and mortality if not treated. The present study investigates the demographic, sexual and drug related risk factors for HIV/HCV co-infection among predominantly African American injecting and non-injecting drug users living in two innercity neighborhoods in Houston, Texas. ^ Methods. This secondary analysis used data collected between February 2004 and June 2005 from 1,889 drug users. Three case-comparison analyses were conducted to investigate the risk factors for HIV/HCV co-infection. HIV mono-infection, HCV mono-infection and non-infection were compared to HIV/HCV co-infection to build multivariate logistic regression models. Race/ethnicity and age were forced into each model regardless of significance in the univariate analysis. ^ Results. The overall prevalence of HIV/HCV co-infection was 3.9% while 39.8% of HIV infected drug users were co-infected with HCV and 10.7% of HCV infected drug users were co-infected with HIV. Among HIV infected IDUs the prevalence of HCV was 71.7% and among HIV infected NIDUs the prevalence of HCV was 24%. In the multivariate analysis, HIV/HCV co-infection was associated with injecting drug use when compared to HIV mono-infection, with MSM when compared to HCV mono-infection and with injecting drug use as well as MSM when compared to non-infection. ^ Conclusion. HIV/HCV co-infection was associated with a combination of sexual and risky injecting practices. More data on the prevalence and risk factors for co-infection among minority populations is urgently needed to support the development of targeted interventions and treatment options. Additionally there should be a focus on promoting safer sex and injecting practices among drug users as well as the expansion of routine testing for HIV and HCV infections in this high risk population.^

Cross -packing among retroviruses and characterization of the feline immunodeficiency virus (FIV) packaging signal: Implications for the development of FIV-based gene transfer systems

Feline immunodeficiency virus (FIV)-based gene transfer systems are being seriously considered for human gene therapy as an alternative to vectors based on primate lentiviruses, a genetically complex group of retroviruses capable of infecting non-dividing cells. The greater phylogenetic distance between the feline and primate lentiviruses is thought to reduce chances of the generation of recombinant viruses. However, safety of FIV-based vector systems has not been tested experimentally. Since primate lentiviruses such as human and simian immunodeficiency viruses (HIV/SIV) can cross-package each other's genomes, we tested this trait with respect to FIV. Unexpectedly, both feline and primate lentiviruses were reciprocally able to both cross-package and propagate each other's RNA genomes. This was largely due to the recognition of viral packaging signals by the heterologous proteins. However, a simple retrovirus such as Mason-Pfizer monkey virus (MPMV) was unable to package FIV RNA. Interestingly, FIV could package MPMV RNA, but not propagate it for further steps of replication. These findings suggest that upon co-infection of the same host, cross-packaging may allow distinct retroviruses to generate chimeric variants with unknown pathogenic potential. ^ In order to understand the packaging determinants in FIV, we conducted a detailed mutational analysis of the region thought to contain FIV packaging signal. We show that the first 90–120 nt of the 5′ untranslated region (UTR) and the first 90 nt of gag were simultaneously required for efficient FIV RNA packaging. These results suggest that the primary FIV packaging signal is multipartite and discontinuous, composed of two core elements separated by 150 nt of the 5 ′UTR. ^ The above studies are being used towards the development of safer FIV-based self-inactivating (SIN) vectors. These vectors are being designed to eliminate the ability of FIV transfer vector RNAs to be mobilized by primate lentiviral proteins that may be present in the target cells. Preliminary test of the first generation of these vectors has revealed that they are incapable of being propagated by feline proteins. The inability of FIV transfer vectors to express packageable vector RNA after integration should greatly increase the safety of FIV vectors for human gene therapy. ^

Ascorbic acid effects on the immune system and type -1 /type -2 cytokine balance in humans

Vitamin C (ascorbic acid--AA) can have a substantial impact on human health by reducing the incidence and/or severity of coryza. Studies also suggest it has immunomodulatory functions in humans. Immune function is controlled by cytokines, such as type-1 cytokines (IFNγ) that promote antiviral immunity and type-2 cytokines (IL-4, IL-10) that promote humoral immunity. Knowing the mechanisms responsible for both antiviral immunity and type-1/type-2 cytokine balance, we sought to identify AA-induced alterations of human peripheral blood mononuclear cells (PBMC) in vivo and in vitro . We hypothesized that AA modulates the immune system, altering both number and function of PBMC. We first described the effect of 14 days of oral (1 gram) AA in healthy subjects. AA increased circulating natural killer (NK) cells, CD25+ and HLA-DR+ T cells, and PMA/ionomycin-stimulated intracellular IFNγ. We subsequently developed models for in vitro use. We determined that AA was toxic in vitro to T cells when used at doses found intracellularly but doses found in plasma from individuals taking 1gm/day AA were nontoxic. The model that most fully reproduced our in vivo intracellular cytokine findings used dehydroascorbic acid and buffers to deliver AA intracellularly. This model generated the largest increase in IFNγ at physiologic plasma concentrations. Previous studies demonstrate that chronic psychological stress is associated with a type-2 cytokine response. We hypothesized that vitamin C could prevent the type-2 cytokine shift associated with stress. In a study of medical students taking 1 g AA or placebo, a significant increase in IFNγ was seen intracellularly in CD4+ and CD8+ cells and in tetanus-stimulated cultures in the AA group only. We also observed increases in IFNγ/IL-4 and IFNγ/IL-10 ratios with AA supplementation, indicating a type-1 shift. Furthermore, we noted increased numbers of NK cells and activated T cells in the peripheral blood in the AA treated group only. Lastly, we investigated the role of the CD40L/CD40 and CD28/B7 costimulatory pathway in these cytokine alterations. AA did not have any effect on either pathway studied. Thus costimulatory pathways are not contributing to AA induced modulation of the type-1/type-2 immune balance. ^