Herpes simplex virus type 1 (HSV-1) UL56 gene is involved in viral intraperitoneal pathogenicity to immunocompetent mice

Summary A comparison of the pathogenicity in mice of the recombinant herpes simplex virus type 1 (HSV-1) strain HSV-1-M- LacZ, in which the U_L56 gene has been deleted, was made with its parental strain F, following infection in different mouse strains. The polymerase chain reaction (PCR) technique was used to study the migration of virus DNA in the mouse model. Tissues from adult mice infected intraperitoneally (IP) with one of three HSV-1 strains (F, HFEM or HSV-1-LacZ) were examined for the presence of viral DNA. DNA of the pathogenic strain F was detected in the adrenal glands, spinal cord, brain, liver and pancreas. DNA of HSV-1-M-LacZ was detected in the same tissues. However, DNA of the apathogenic strain HFEM was detected transiently (on days 2 and 3 p.i., but not days 1, 5 or 7), only in the adrenal glands and no viral DNA was detected in any of the other tissues. HSV-1 pathogenic strains injected intraperitoneally into newborn mice (7 days old) killed most of the mice. In the surviving mice viral DNA of the three virus strains was found in peritoneal exudate cells (PEC), adrenal glands, spinal cord, liver and spleen. It was found that HSV-1-M-LacZ, which lacks the U_L56 gene, resembled in pathogenicity to the newborn mice the pathogenic HSV-1 strains F and KOS. The PCR technique was used to trace viral DNA in tissues of the mice which survived HSV-1 infection at 7 weeks of age. Only HSV-1 (KOS) DNA was detected in the pancreas. The brains of these mice did not contain viral DNA. It is suggested that HSV-1 DNA may reside in surviving HSV-1- infected newborn mice in a latent state in nonneural tissues.     

The herpes simplex virus type 1 immediate-early polypeptide ICP4 is required for expression of globin genes located in the viral genome.

We infected Vero cells with ICP4-deficient herpes simplex virus recombinants bearing the rabbit beta-globin and human alpha 2-globin genes under the control of their own promoters and found that globin gene expression occurred only when ICP4 was provided in trans. These results demonstrate that ICP4 is required for the activity of globin promoters located in the viral genome and support the hypothesis that these cellular promoters are functionally equivalent to HSV early regulatory regions.     

Adeno-associated virus DNA replication is induced by genes that are essential for HSV-1 DNA synthesis

Adeno-associated virus (AAV) DNA replication is not detectable unless cells are coinfected with a helper adenovirus (Ad) or herpesvirus or unless AAV infection is carried out in certain established cell lines that have been treated with various metabolic inhibitors or uv irradiation. In helper-dependent infections, it has been shown that AAV DNA synthesis depends on one or more early Ad genes, whereas little is known concerning any herpesvirus gene that promotes AAV DNA synthesis. In this study we tested the ability of four cloned Xbal fragments of herpes simplex virus type 1 (HSV-1) DNA to induce AAV DNA synthesis in Vero cells. Cotransfections, which were carried out with pAV1 (an infectious AAV2 plasmid), revealed that AAV DNA synthesis could be optimally induced by three of these clones (C,D, and F) plus a clone of the HSV-1 ICP4 (IE 175) gene. ICP4, an immediate early gene, was presumably required to activate expression of other HSV genes. To help identify the additionally needed HSV genes, we tested Xbal C,D, and F subclones that contain genes previously found necessary for origin-dependent HSV DNA synthesis and found that at least five of these genes (UL 5, 8, 9, 29, and 30) contributed to the induction of AAV DNA synthesis. In contrast to their absolute requirement for HSV DNA synthesis, none of these genes were strictly necessary for AAV DNA replication. Because they are all known to specify proteins that are directly involved in HSV DNA synthesis, our results suggest that some or all of their products also may directly participate in the replication of AAV DNA.     

A sequence in HpaI-P fragment of herpes simplex virus-1 DNA determines intraperitoneal virulence in mice

The virulence of herpes simplex virus-1 (HSV-1) strains by the intraperitoneal (ip) route of injection in mice depends on the presence of an intact sequence in the HpaI DNA fragment P within coordinates 0.762 to 0.787. Deletion of the HpaI-P region (e.g., strain HFEM) abrogates the ability of the virus to infect mice by the ip route without affecting pathogenicity by the intracerebral (ic) route. A recombinant virus (M1C1) derived from DNA of the HSV-1 HFEM strain and the MLUIDNA fragment (coordinates 0.761 to 0.796) spanning the HpaI-P sequence of the pathogenic strain F regained pathogenicity for mice by the ip route.     

E1 protein of bovine papillomavirus 1 is not required for the maintenance of viral plasmid DNA replication

Derivatives of bovine papillomavirus 1 (BPV1) with temperature-sensitive and dominant-negative mutation the E1 gene were used to determine the requirement for E1 in the maintenance of viral plasmid DNA replication. The abilities of these mutant BPV1 genomes to replicate as nuclear plasmids were monitored at permissive (32 degrees C) and nonpermissive (37 degrees C) temperatures in mouse C127 cells. We found that the temperature-sensitive E1 mutant BPV1 genomes replicate as nuclear plasmids as efficiently as does wild-type BPV1 in C127 cells after shifting to the nonpermissive temperature. These findings indicate that BPV1 does not require E1 for the maintenance of viral plasmids.     

Neurovirulence and latency in inbred mice of two HSV-1 intrastrain variants of divergent pathogenicity

The pathogenicity pattern of the HSV-1 strain ANG which is nonencephalitogenic in mice is compared with that of a selected neurovirulent variant of this strain in DBA-2 mice. After i.p. inoculation both variants replicate to high titers in the mouse peritoneum and build up a virus reservoir in the spleen. Both viruses have no effect on visceral mouse organs other than the spleen; both viruses lead to an inefficient and masked viraemia and both replicate efficiently in CNS tissue after direct intracranial injection. Only the pathogenic variant, however, spreads to the CNS and leads to lethal encephalitis upon intraperitoneal infection. The assumption that infection of the CNS would be mediated by hematogenous transport is not supported by the data obtained from transfer and cocultivation experiments with lymphocytes or experiments involving artificial viraemia. In a model to analyse the capacity of the viruses to invade nerve axons and to induce a latent infection both viruses were found to be latency positive in dorsal root ganglia. It is clear that non-neurovirulent HSV-1 strains are subjected to a postganglionic block of virus spread from the periphery to the CNS. The experiments led to the hypothesis that axonal transport even beyond the dorsal root ganglia to the CNS proceeds unrestricted, whereas lethal CNS invasion is prevented by a restriction of viral replication of HSV-1 ANG in the CNS by a virus-induced host defence mechanism.     

Nucleotide sequence of the bacteriophage P22 genes required for DNA packaging.

The mechanism of DNA packaging by dsDNA viruses is not well understood in any system. In bacteriophage P22 only five genes are required for successful condensation of DNA within the capsid. The products of three of these genes, the portal, scaffolding, and coat proteins, are structural components of the precursor particle, and two, the products of genes 2 and 3, are not. The scaffolding protein is lost from the structure during packaging, and only the portal and coat proteins are present in the mature virus particle. These five genes map in a contiguous cluster at the left end of the P22 genetic map. Three additional genes, 4, 10, and 26, are required for stabilizing of the condensed DNA within the capsid. In this report we present the nucleotide sequence of 7461 bp of P22 DNA that contains the five genes required for DNA condensation, as well as a nonessential open reading frame (ORF109), gene 4, and a portion of gene 10. N-terminal amino acid sequencing of the encoded proteins accurately located the translation starts of six genes in the sequence. Despite the fact that most of these proteins have striking analogs in the other dsDNA bacteriophage groups, which perform highly analogous functions, no amino acid sequence similarity between these analogous proteins has been found, indicating either that they diverged a very long time ago or that they are the products of spectacular convergent evolution.     

The herpes simplex virus type 1 immediate-early protein ICP27 is obligately required for the accumulation of a cellular protein during viral infection

Lytic infection with herpes virus type 1 (HSV-1) causes the accumulation of a 40-kDa cellular protein (p40) which is also overexpressed in cultured cells transformed by HSV or other agents and in human cervical tumors. Accumulation of p40 is dependent upon viral protein synthesis but not viral DNA replication in the infected cell and occurs in the HSV-1 mutants tsK and tsLB2 in which only a defective ICP4 protein and the four other immediate-early proteins are synthesized. By using a panel of HSV-1 strains, each defective in one of these four proteins, we show that only a mutation in the gene encoding ICP27 abolishes p40 accumulation. The defect in this mutant virus can be rescued by a plasmid encoding ICP27 alone indicating that ICP27 is obligately required for p40 accumulation. The significance of this effect as one aspect of the interaction of viral control proteins with cellular genes is discussed.     

Correlation between pathogenicity of Shigella and intraperitoneal survival in mice

Phagocytosis of virulent and avirulent strains of Shigella and Escherichia coli in the mouse peritoneum was studied. A direct correlation between bacterial virulence and resistance to phagocytosis by peritoneal phagocytes was demonstrated. Virulent strains were less readily cleared and were able to multiply to a limited extent within the peritoneal cavity. An epimerase-deficient, rough mutant of S. flexneri 2a was highly susceptible to phagocytosis. Restoration of the cell wall structure in these mutants resulted in a significant increase in their resistance to phagocytosis. Susceptibility to phagocytosis in smooth S. flexneri was age-dependent. Cells from 16-hr cultures were more resistant to removal from the peritoneum than were cells from 48- and 72-hr cultures.     

Characterization of factors determining Rickettsia tsutsugamushi pathogenicity for mice.

Pathogenicity of Rickettsia tsutsugamushi for laboratory mice is known to be influenced by at least three factors: (i) route of inoculation, (ii) antigenic strain, and (iii) natural resistance of the host. By using Karp, Gilliam, and Kato strains of R. tsutsugamushi, we examined the effect of these three pathogenicity factors on the kinetics of infection and the development of immunity in BALB/cDub and C3H/HeDub mice. The appearance of rickettsemia in the pathogenic infections generally preceded infections of reduced pathogenicity by 1 to 2 days in both magnitude and time of onset. Mice infected by the subcutaneous route with normally pathogenic rickettsiae, i.e., Gilliam-infected C3H/HeDub mice and Karp-infected BALB/cDub mice, consistently maintained a detectable rickettsemia over a 1-year period. Rickettsiae were recovered from the spleens of 95% (19 of 20) of these mice 52 weeks postinfection. In contrast, mice with infections of reduced pathogenicity, i.e., BALB/cDub mice infected by intraperitoneal and subcutaneous inoculation with Gilliam, did not have detectable rickettsemia from week 20 through week 52 postinfection except for a single mouse on week 44 postinfection. Rickettsiae were detected in the spleens of only 40% (8 of 20) of these mice after 1 year. In both Gilliam-infected mouse strains, protection against heterologous challenge with Karp or Kato rickettsial strains was incomplete up to 7 days postimmunization. Infections of reduced pathogenicity did not result from an enhanced systemic immune response by the host. The onset of the humoral response was not different for the pathogenic and reduced-pathogenicity infections. Pathogenicity differences seemed to result from the more rapid growth of the rickettsiae in the pathogenic infections.     

c-Jun N-terminal kinase 1 is required for Toll-like receptor 1 gene expression in macrophages

The regulation of innate immune responses to pathogens occurs through the interaction of Toll-like receptors (TLRs) with pathogen-associated molecular patterns and the activation of several signaling pathways whose contribution to the overall innate immune response to pathogens is poorly understood. We demonstrate a mechanism of control of murine macrophage responses mediated by TLR1/2 heterodimers through c-Jun N-terminal kinase 1 (JNK1) activity. JNK controls tumor necrosis factor alpha production and TLR-mediated macrophage responses to Borrelia burgdorferi, the causative agent of Lyme disease, and the TLR1/TLR2-specific agonist PAM(3)CSK(4). JNK1, but not JNK2, activity regulates the expression of the tlr1 gene in the macrophage cell line RAW264.7, as well as in primary CD11b(+) cells. We also show that the proximal promoter region of the human tlr1 gene contains an AP-1 binding site that is subjected to regulation by the kinase and binds two complexes that involve the JNK substrates c-Jun, JunD, and ATF-2. These results demonstrate that JNK1 regulates the response to TLR1/2 ligands and suggest a positive feedback loop that may serve to increase the innate immune response to the spirochete.     

Viral interference of HSV-1: Properties of the intracellular viral progeny DNA

Intracellular progeny DNA was isolated and characterized from cells infected with standard herpes simplex virus or from cells coinfected with standard virus and with a virus stock obtained by serial passages at high multiplicity of infection (HP virus). The latter was shown to contain an excess of variant virus particles interfering with the production of infectious progeny virus. The ratio of plaque-forming to interfering virus in the HP virus stock used in this study was determined to be $\text{1}\text{10}$. In both infections similar amounts of unit length viral DNA were synthesized. Restriction endonuclease digestion of intracellular viral progeny DNA yielded fragment patterns showing that the majority of the DNA molecules present in standard and HP HSV DNA yield the same restriction fragments. That the typical end fragments could be demonstrated suggests a correct processing of concatemeric precursor DNA into HSV unit length DNA. Despite the obvious similarity of the two DNA species the infectivity in transfection assays of progeny DNA formed in HP virus-infected cells was by three log lower than that of standard DNA. As shown by controls involving cotransfections with standard viral DNA and heterologous DNA, this difference can be attributed to the presence in HP DNA of HSV DNA molecules that interfere with the plaque formation by standard DNA. An alteration in various steps of DNA processing such as DNA methylation and incorporation of ribonucleotides into the DNA was demonstrated not to correlate with interference. Both in HP and standard virus DNA preparations methylation was below the level of detection of 10 5-methylcytosine residues per unit length of viral DNA. Low values were also obtained for the uridine content of the DNA. Almost 100% of the radioactivity incorporated could be recovered as deoxyribonucleosides and less than 0.07% as uridine.     

Analysis of the transcription pattern of HSV-1 UL52 and UL53 genes

The UL52 and UL53 genes of herpes simplex virus type-1 are both located in theBamHI-L DNA fragment, with an overlap of 14 amino acids. An RNase protection experiment was designed to determine the 5′ termini of both the UL52 and UL53 mRNAs. The 5′ end of the UL52 mRNA was found to be located 100 bp upstream of its ATG initiation codon. Surprisingly, the 5′ terminus of the UL53 gene was found to be downstream of its putative initiation codon. Therefore, it was suggested that the translation of the UL53 open reading frame (ORF) starts at an internal initiation codon that is located 55 codons downstream of the putative one. A hybrid selection experiment was performed in which the UL53-specific mRNA was selected from BSC-1 cells infected with HSV-1 KOS and translated in vitro. The translation product of the UL53 message was found to be 32 kD (shorter than the original 37.5 kD ORF). The size of the protein obtained corresponds with the expected translation product starting at the downstream initiation codon. Analysis of the sequence upstream of this initiation codon reveals the presence of a promotor sequence. Therefore, we suggest that the UL53 protein is 54 amino acids shorter than was previously suggested and is located at coordinates 112,341–113,193.     

The HSV-1 UL45 gene product is not required for growth in Vero cells.

We have constructed a HSV-1 UL45 null mutant (UL45 delta) by inserting a TK-lacZ cassette into a BclI site near the 5' end of the UL45 gene. A polyclonal antiserum produced to an Escherichia coli trpE:UL45 fusion protein was used to show that an 18-kDa polypeptide corresponding to the predicted UL45 gene product was produced in HSV-1 strain KOS-infected Vero cells but was not detected in UL45 delta-infected Vero cells. The absence of the 18-kDa protein had only a slight effect on viral growth in cell culture, indicating that the UL45 gene product is not essential for growth in Vero cells. However, the burst size of UL45 delta was smaller than HSV-1 KOS in Vero and HeLa cells. UL45 delta also had a smaller plaque size and an altered plaque morphology.     

Identification of an orphan response regulator required for the virulence of Francisella spp. and transcription of pathogenicity island genes

Francisella tularensis is a category A agent of biowarfare/biodefense. Little is known about the regulation of virulence gene expression in Francisella spp. Comparatively few regulatory factors exist in Francisella, including those belonging to two-component systems (TCS). However, orphan members of typical TCS can be identified. To determine if orphan TCS members affect Francisella gene expression, a gene encoding a product with high similarity to the Salmonella PmrA response regulator (FTT1557c/FNU0663.2) was deleted in Francisella novicida (a model organism for F. tularensis). The F. novicida pmrA mutant was defective in survival/growth within human and murine macrophage cell lines and was 100% defective in virulence in mice at a dose of up to 10(8) CFU. In addition, the mutant strain demonstrated increased susceptibility to antimicrobial peptide killing, but no differences were observed between the lipid A of the mutant and the parental strain, as has been observed with pmrA mutants of other microbes. The F. novicida pmrA mutant was 100% protective as a single-dose vaccine when challenge was with 10(6) CFU of F. novicida but did not protect against type A Schu S4 wild-type challenge. DNA microarray analysis identified 65 genes regulated by PmrA. The majority of these genes were located in the region surrounding pmrA or within the Francisella pathogenicity island (FPI). These FPI genes are also regulated by MglA, but MglA does not regulate pmrA, nor does PmrA regulate MglA. Thus, the orphan response regulator PmrA is an important factor in controlling virulence in F. novicida, and a pmrA mutant strain is an effective vaccine against homologous challenge.