Biomedical subjects

David M Knipe

Publications and source records attributed to David M Knipe.

12 recordsLinked to original sources

PubMed2003

Herpes simplex virus replication compartments can form by coalescence of smaller compartments.

Herpes simplex virus (HSV) uses intranuclear compartmentalization to concentrate the viral and cellular factors required for the progression of the viral life cycle. Processes as varied as viral DNA replication, late gene expression, and capsid assembly take place within discrete structures within the nucleus called replication compartments. Replication compartments are hypothesized to mature from a few distinct structures, called prereplicative sites, that form adjacent to cellular nuclear matrix-associated ND10 sites. During productive infection, the HSV single-stranded DNA-binding protein ICP8 localizes to replication compartments. To further the understanding of replication compartment maturation, we have constructed and characterized a recombinant HSV-1 strain that expresses an ICP8 molecule with green fluorescent protein (GFP) fused to its C terminus. In transfected Vero cells that were infected with HSV, the ICP8-GFP protein localized to prereplicative sites in the presence of the viral DNA synthesis inhibitor phosphonoacetic acid (PAA) or to replication compartments in the absence of PAA. A recombinant HSV-1 strain expressing the ICP8-GFP virus replicated in Vero cells, but the yield was increased by 150-fold in an ICP8-complementing cell line. Using the ICP8-GFP protein as a marker for replication compartments, we show here that these structures start as punctate structures early in infection and grow into large, globular structures that eventually fill the nucleus. Large replication compartments were formed by small structures that either moved through the nucleus to merge with adjacent compartments or remained relatively stationary within the nucleus and grew by accretion and fused with neighboring structures.

Animals↗

PubMed2003

C-terminal region of herpes simplex virus ICP8 protein needed for intranuclear localization.

The herpes simplex virus single-stranded DNA-binding protein, ICP8, localizes initially to structures in the nucleus called prereplicative sites. As replication proceeds, these sites mature into large globular structures called replication compartments. The details of what signals or proteins are involved in the redistribution of viral and cellular proteins within the nucleus between prereplicative sites and replication compartments are poorly understood; however, we showed previously that the dominant-negative d105 ICP8 does not localize to prereplicative sites and prevents the localization of other viral proteins to prereplicative sites (J. Virol. 74 (2000) 10122). Within the residues deleted in d105 (1083 to 1168), we identified a region between amino acid residues 1080 and 1135 that was predicted by computer models to contain two alpha-helices, one with considerable amphipathic nature. We used site-specific and random mutagenesis techniques to identify residues or structures within this region that are required for proper ICP8 localization within the nucleus. Proline substitutions in the predicted helix generated ICP8 molecules that did not localize to prereplicative sites and acted as dominant-negative inhibitors. Other substitutions that altered the charged residues in the predicted alpha-helix to alanine or leucine residues had little or no effect on ICP8 intranuclear localization. The predicted alpha-helix was dispensable for the interaction of ICP8 with the U(L)9 origin-binding protein. We propose that this C-terminal alpha-helix is required for localization of ICP8 to prereplicative sites by binding viral or cellular factors that target or retain ICP8 at specific intranuclear sites.

Amino Acid Sequence↗

PubMed2003

Vaginal submucosal dendritic cells, but not Langerhans cells, induce protective Th1 responses to herpes simplex virus-2.

Herpes simplex virus (HSV) type 2 infection occurs primarily at the genital mucosal surfaces and is a leading cause of ulcerative lesions. Despite the availability of animal models for HSV-2 infection, little is known regarding the mechanism of immune induction within the vaginal mucosa. Here, we examined the cell types responsible for the initiation of protective Th1 immunity to HSV-2. Intravaginal inoculation of HSV-2 led to a rapid recruitment of submucosal dendritic cells (DCs) to the infected epithelium. Subsequently, CD11c(+) DCs harboring viral peptides in the context of MHC class II molecules emerged in the draining lymph nodes and were found to be responsible for the stimulation of IFNgamma secretion from HSV-specific CD4(+) T cells. Other antigen-presenting cells including B cells and macrophages did not present viral peptides to T cells in the draining lymph nodes. Next, we assessed the relative contribution to immune generation by the Langerhans cells in the vaginal epithelium, the submucosal CD11b(+) DCs, and the CD8alpha(+) lymph node DCs. Analysis of these DC populations from the draining lymph nodes revealed that only the CD11b(+) submucosal DCs, but not Langerhans cell-derived or CD8alpha(+) DCs, presented viral antigens to CD4(+) T cells and induced IFNgamma secretion. These results demonstrate a previously unanticipated role for submucosal DCs in the generation of protective Th1 immune responses to HSV-2 in the vaginal mucosa, and suggest their importance in immunity to other sexually transmitted diseases.

Animals↗

PubMed2003

Conformational changes in the herpes simplex virus ICP8 DNA-binding protein coincident with assembly in viral replication structures.

The herpes simplex virus (HSV) single-stranded DNA-binding protein, ICP8, is required for viral DNA synthesis. Before viral DNA replication, ICP8 colocalizes with other replication proteins at small punctate foci called prereplicative sites. With the onset of viral genome amplification, these proteins become redistributed into large globular replication compartments. Here we present the results of immunocytochemical and biochemical analysis of ICP8 showing that various antibodies recognize distinct forms of ICP8. Using these ICP8-specific antibodies as probes for ICP8 structure, we detected a time-dependent appearance and disappearance of ICP8 epitopes in immunoprecipitation assays. Immunofluorescence staining of ICP8 in cells infected with different HSV mutant viruses as well as cells transfected with a limited number of viral genes demonstrated that these and other antigenic changes occur coincident with ICP8 assembly at intranuclear replication structures. Genetic analysis has revealed a correlation between the ability of various ICP8 mutant proteins to form the 39S epitope and their ability to bind to DNA. These results support the hypothesis that ICP8 undergoes a conformational change upon binding to other HSV proteins and/or to DNA coincident with assembly into viral DNA replication structures.

Cell Line↗

PubMed2003

Latent herpes simplex virus infection of sensory neurons alters neuronal gene expression.

The persistence of herpes simplex virus (HSV) and the diseases that it causes in the human population can be attributed to the maintenance of a latent infection within neurons in sensory ganglia. Little is known about the effects of latent infection on the host neuron. We have addressed the question of whether latent HSV infection affects neuronal gene expression by using microarray transcript profiling of host gene expression in ganglia from latently infected versus mock-infected mouse trigeminal ganglia. (33)P-labeled cDNA probes from pooled ganglia harvested at 30 days postinfection or post-mock infection were hybridized to nylon arrays printed with 2,556 mouse genes. Signal intensities were acquired by phosphorimager. Mean intensities (n = 4 replicates in each of three independent experiments) of signals from mock-infected versus latently infected ganglia were compared by using a variant of Student's t test. We identified significant changes in the expression of mouse neuronal genes, including several with roles in gene expression, such as the Clk2 gene, and neurotransmission, such as genes encoding potassium voltage-gated channels and a muscarinic acetylcholine receptor. We confirmed the neuronal localization of some of these transcripts by using in situ hybridization. To validate the microarray results, we performed real-time reverse transcriptase PCR analyses for a selection of the genes. These studies demonstrate that latent HSV infection can alter neuronal gene expression and might provide a new mechanism for how persistent viral infection can cause chronic disease.

Animals↗

PubMed2002

Type 3 reovirus neuroinvasion after intramuscular inoculation: viral genetic determinants of lethality and spinal cord infection.

To better understand the mechanisms by which neurotropic viruses invade peripheral nerve pathways and produce CNS disease, we defined the type 3 (T3) reovirus genes that are determinants of the capacity of reovirus T3 strain Dearing (T3D) and T3 clone 9 (C9) to infect the spinal cord and kill mice after hindlimb injection. T3D and C9 viruses are both highly virulent (LD(50) < 10(1) PFU) after intracranial injection of neonatal mice. However, C9 is significantly more lethal than T3D after either intramuscular injection (LD(50) < 10(1) vs LD(50) 10(4) PFU) or peroral injection (LD(50) 10(3.4) vs LD(50) > 10(8.3) PFU). Using reassortant viruses containing different combinations of genes derived from T3D and C9, we found that the S1 gene, encoding the cell attachment protein sigma 1 and the nonstructural protein sigma 1s, and the L3 gene, encoding the core shell protein lambda 1 were the primary determinants of lethality after intramuscular injection. The L3 gene and the L2 gene encoding spike protein, lambda 2, determined differences in spinal cord titer after intramuscular injection. A C9 x T3D mono-reassortant containing all T3D genes except for the C9-derived L3 was lethal after peroral injection. These studies indicate that the S1, L2, and L3 genes all play a potential role in neuroinvasiveness and provide the first identification of a role in pathogenesis for the L3 gene.

Administration, Oral↗

PubMed2002

Type 3 reovirus neuroinvasion after intramuscular inoculation: direct invasion of nerve terminals and age-dependent pathogenesis.

Neonatal but not adult mice are vulnerable to reovirus invasion of the central nervous system after peripheral inoculation. After hindlimb injection, type 3 reovirus travels via the sciatic nerve to replicate in spinal cord motor neurons before spread to the brain and development of lethal encephalitis. Here we provide ultrastructural evidence for direct reovirus invasion of unmyelinated neonatal motor nerve terminals within 2 h and replication in spinal cord motor neurons within 14 h after hindlimb injection of 1-day-old mice. In adult mice, resistance to reovirus lethality after intracranial (IC) injection correlates with the restriction of virus growth in cortical neurons. We found that neuroinvasion also is age dependent after intramuscular injection. Virus lethality and CNS infection decreased sharply during the first postnatal week, while lethality after IC injection continued for 2 additional weeks. Mice inoculated at 7 days of age with high virus doses suffered paralysis of the injected limb, but significant brain infection was not lethal. These results suggest that reovirus invasion of the neonatal CNS is restricted by several progressive age-dependent mechanisms.

Age Factors↗

PubMed2002

Macrophage-derived complement component C4 can restore humoral immunity in C4-deficient mice.

Mice with a disrupted C4 locus (C4(-/-)) have an impaired immune response to thymus-dependent Ags. To test the role of bone marrow-derived C4 in humoral immunity, we reconstituted deficient animals with wild-type bone marrow or an enriched fraction of bone marrow-derived macrophages. C4 chimeras were immunized with 4-hydroxy-3-nitrophenyl(5) conjugated to keyhole limpet hemocyanin (NP(5)- KLH) or infected with HSV-1, and the Ab response was evaluated. Wild-type bone marrow rescued the humoral immune response to both Ags, i.e., the soluble Ag and HSV-1, demonstrating that local C4 production is sufficient for humoral responses. Although the C4 chimeric animals lacked detectable C4 in their sera, C4 mRNA was identified in splenic sections by in situ hybridization, and C4 protein deposits were identified in the germinal center areas of splenic follicles by immunofluorescence staining. Macrophages derived from bone marrow produced sufficient C4 protein to restore the humoral response to NP(5)-KLH in C4-deficient animals when administered along with Ag. Cell-sorting experiments, followed by C4-specific RT-PCR, identified splenic macrophages (CD11b(+), CD11c(-)) as a cellular source for C4 synthesis within the spleen.

Adoptive Transfer↗

PubMed2002

Herpes simplex virus.

Herpes simplex virus (HSV) commonly causes human infections in the orofacial region (HSV-1) and in the genital region (HSV-2). Productive viral infection in mucosal epithelial cells may result in clinical symptoms and is followed by a latent infection within sensory neurons. During productive infection a large number of viral gene products are expressed while during latent infection few or no viral proteins are expressed. Reactivation from latency results in recurrent infections and disease at or near the primary site of infection. Understanding the details of the two stages of the HSV life cycle is a particular focus of current research on HSV. The virus interacts with and modifies numerous host cell functions in both epithelial and neuronal cells, and studies of HSV have enhanced our knowledge of many fundamental processes in eukaryotic cells. Ongoing research continues to uncover novel effects of HSV on cells, and a complete understanding of HSV infection during both productive and latent infection should allow the design of new antiviral agents and vaccines and increased knowledge of basic cell and molecular biology. This review article is designed to provide an introduction to HSV biology and key aspects of the infection cycle.

Animals↗

PubMed2002

Neither LAT nor open reading frame P mutations increase expression of spliced or intron-containing ICP0 transcripts in mouse ganglia latently infected with herpes simplex virus.

Latent infections by herpes simplex virus are characterized by repression of productive-cycle gene expression. Several hypotheses to explain this repression involve inhibition of expression of the immediate-early gene activator ICP0 during latency. To address these hypotheses, we developed quantitative reverse transcriptase-PCR assays that detected spliced and intron-containing ICP0 transcripts in mouse ganglia latently infected with wild-type virus. In these ganglia, the numbers of spliced ICP0 transcripts correlated better with the numbers of transcripts from the immediate-early gene encoding ICP4 than with those from the early gene encoding thymidine kinase. There were fewer spliced than intron-containing ICP0 transcripts on average, with considerable ganglion-to-ganglion variation. We then investigated whether ICP0 expression in latently infected ganglia is reduced by the latency-associated transcripts (LATs) and whether splicing of ICP0 transcripts is inhibited by the product of open reading frame (ORF) P. A LAT deletion mutation which essentially eliminates expression of the major LATs did not appreciably increase levels of ICP0 transcripts. LAT deletion mutants did, however, appear to express reduced levels of intron-containing ICP0 transcripts. ORF P mutations did not alter levels of ICP0 transcripts in a manner consistent with inhibition of ICP0 splicing by ORF P. Although these results argue against antisense inhibition of ICP0 expression by LATs or inhibition of ICP0 splicing by ORF P, they are consistent with the possibilities of a block between immediate-early and early gene expression and regulation of spliced versus intron-containing ICP0 transcripts in latently infected ganglia.

Animals↗

PubMed2002

Association of herpes simplex virus type 1 ICP8 and ICP27 proteins with cellular RNA polymerase II holoenzyme.

Herpes simplex virus 1 (HSV-1) infection causes the shutoff of host gene transcription and the induction of a transcriptional program of viral gene expression. Cellular RNA polymerase II is responsible for transcription of all the viral genes, but several viral proteins stimulate viral gene transcription. ICP4 is required for all delayed-early and late gene transcription, ICP0 stimulates transcription of viral genes, and ICP27 stimulates expression of some early genes and transcription of at least some late viral genes. The early DNA-binding protein, ICP8, also stimulates late gene transcription. We therefore investigated which HSV proteins interact with RNA polymerase II. Using immunoprecipitation and Western blotting methods, we observed the coprecipitation of ICP27 and ICP8 with RNA polymerase II holoenzyme. The association of ICP27 with RNA polymerase II was detectable as early as 3 h postinfection, while ICP8 association became evident by 5 h postinfection, and the association of both was independent of viral DNA synthesis. Infections with ICP27 gene mutant viruses revealed that ICP27 is required for the association of ICP8 with RNA polymerase II, while studies with ICP8 gene deletion mutants showed no apparent role for ICP8 in the association of ICP27 with RNA polymerase II. The association of ICP27 and ICP8 with RNA polymerase II holoenzyme appeared to be independent of nucleic acids. We hypothesize that the interaction of ICP27 with RNA polymerase II holoenzyme reflects its role in stimulating early and late gene expression and/or its role in inhibiting host transcription and that the interaction of ICP8 with RNA polymerase II holoenzyme reflects its role in stimulating late gene transcription.

Animals↗

PubMed2002

Herpes simplex virus vectors elicit durable immune responses in the presence of preexisting host immunity.

Herpes simplex virus (HSV) recombinants are being developed as vaccine vectors for the expression of heterologous antigens. There is concern, however, that preexisting HSV immunity may decrease their effectiveness. We have addressed this issue in an animal model. Immunized mice were inoculated with a replication-defective HSV-1 vector that expressed the Escherichia coli beta-galactosidase protein as a model antigen. We assessed vector efficacy by analyzing the immunoglobulin G (IgG) antibody response and cellular proliferative response directed against beta-galactosidase. We report that the ability of the vector to induce antibody or proliferative responses was not diminished by preexisting immunity to HSV. Of further note, the anti-HSV and anti-beta-galactosidase IgG responses following vector administration were extremely durable in both immunized and naive mice. These results indicate that the ability of a replication-defective HSV-derived vaccine vector to elicit long-lived immune responses in mice is not impaired by prior HSV exposure.

Animals↗