Katherine R. Spindler

Viral disruption of the blood-brain barrier

The blood-brain barrier (BBB) provides significant protection against microbial invasion of the brain. However, the BBB is not impenetrable, and mechanisms by which viruses breach it are becoming clearer. In vivo and in vitro model systems are enabling identification of host and viral factors contributing to breakdown of the unique BBB tight junctions. Key mechanisms of tight junction damage from inside and outside cells are disruption of the actin cytoskeleton and matrix metalloproteinase activity, respectively. Viral proteins acting in BBB disruption are described for HIV-1, currently the most studied encephalitic virus; other viruses are also discussed.

Mouse Adenovirus Type 1-Induced Breakdown of the Blood-Brain Barrier

ABSTRACT Infection with mouse adenovirus type 1 (MAV-1) results in fatal acute encephalomyelitis in susceptible mouse strains via infection of brain endothelial cells. Wild-type (wt) MAV-1 causes less brain inflammation than an early region 3 (E3) null virus in C57BL/6 mice. A mouse brain microvascular endothelial cell line infected with wt MAV-1 had higher expression of mRNAs for the proinflammatory chemokines CCL2 and CCL5 than mock- and E3 null virus-infected cells. Primary mouse brain endothelial cells infected with wt virus had elevated levels of CCL2 compared to mock- or E3 null virus-infected cells. Infection of C57BL/6 mice with wt MAV-1 or the E3 null virus caused a dose-dependent breakdown of the blood-brain barrier, primarily due to direct effects of virus infection rather than inflammation. The tight junction proteins claudin-5 and occludin showed reduced surface expression on primary mouse brain endothelial cells following infection with either wt MAV-1 or the E3 null virus. mRNAs and protein for claudin-5, occludin, and zona occludens 2 were also reduced in infected cells. MAV-1 infection caused a loss of transendothelial electrical resistance in primary mouse brain endothelial cells that was not dependent on E3 or on MAV-1-induced CCL2 expression. Taken together, these results demonstrate that MAV-1 infection caused breakdown of the blood-brain barrier accompanied by decreased surface expression of tight junction proteins. Furthermore, while the MAV-1-induced pathogenesis and inflammation were dependent on E3, MAV-1-induced breakdown of the blood-brain barrier and alteration of endothelial cell function were not dependent on E3 or CCL2.

Fatal Disseminated Mouse Adenovirus Type 1 Infection in Mice Lacking B Cells or Bruton's Tyrosine Kinase

ABSTRACT Mouse adenovirus type 1 (MAV-1) infection of B-cell-deficient and Brutons tyrosine kinase (Btk)-deficient mice resulted in fatal disseminated disease resembling human adenovirus infections in immunocompromised patients. Mice lacking B cells or Btk were highly susceptible to acute MAV-1 infection, in contrast to controls and mice lacking T cells. To our knowledge, this is the first demonstration that mice with an X-linked immunodeficiency phenotype (Btk deficient) are susceptible to virus-induced disease. Mice lacking B cells or Btk on a C57BL/6 background succumbed with encephalomyelitis, hepatitis, and lymphoid necrosis. Mice lacking B cells on a BALB/c background succumbed with enteritis and hepatitis. Survival of acute MAV-1 infection correlated with early T-cell-independent neutralizing antibody and T-cell-independent antiviral immunoglobulin M. Treatment of MAV-1-infected Btk−/− mice 4 to 9 days postinfection with antiserum harvested 6 to 9 days postinfection from MAV-1-infected Btk+/+ mice was therapeutic. Our findings implicate a critical role for B-cell function in preventing disseminated MAV-1 infection, particularly production of early T-cell-independent antiviral immunoglobulin M.

T Cells Cause Acute Immunopathology and Are Required for Long-Term Survival in Mouse Adenovirus Type 1-Induced Encephalomyelitis

ABSTRACT Infection of adult C57BL/6 (B6) mice with mouse adenovirus type 1 (MAV-1) results in dose-dependent encephalomyelitis. Utilizing immunodeficient mice, we analyzed the roles of T cells, T-cell subsets, and T-cell-related functions in MAV-1-induced encephalomyelitis. T cells, major histocompatibility complex (MHC) class I, and perforin contributed to acute disease signs at 8 days postinfection (p.i.). Acute MAV-1-induced encephalomyelitis was absent in mice lacking T cells and in mice lacking perforin. Mice lacking α/β T cells had higher levels of infectious MAV-1 at 8 days, 21 days, and 12 weeks p.i., and these mice succumbed to MAV-1-induced encephalomyelitis at 9 to 16 weeks p.i. Thus, α/β T cells were required for clearance of MAV-1. MAV-1 was cleared in mice lacking perforin, MHC class I or II, CD4+ T cells, or CD8+ T cells. Our results are consistent with a model in which either CD8+ or CD4+ T cells are sufficient for clearance of MAV-1. Furthermore, perforin contributed to MAV-1 disease but not viral clearance. We have established two critical roles for T cells in MAV-1-induced encephalomyelitis. T cells caused acute immunopathology and were required for long-term host survival of MAV-1 infection.

The role of integration in oncogenic progression of HPV-associated cancers

Persistent infection with a subset of “high oncogenic risk” human papillomaviruses (HPVs) can promote the development of cancer. In these cancers, the extrachromosomal viral genome has often become integrated into the host genome. The integration event is thought to drive oncogenesis by dysregulating expression of the E6 and E7 viral oncogenes, leading to inactivation of critical cell cycle checkpoints and increased genetic instability in the host. This Pearl reviews the evidence that gave rise to the current textbook paradigm of HPV integration events and their consequences and incorporates new findings that demonstrate that stochastic integration events can promote oncogenesis in many ways.

Defensins Potentiate a Neutralizing Antibody Response to Enteric Viral Infection

α-defensins are abundant antimicrobial peptides with broad, potent antibacterial, antifungal, and antiviral activities in vitro. Although their contribution to host defense against bacteria in vivo has been demonstrated, comparable studies of their antiviral activity in vivo are lacking. Using a mouse model deficient in activated α-defensins in the small intestine, we show that Paneth cell α-defensins protect mice from oral infection by a pathogenic virus, mouse adenovirus 1 (MAdV-1). Survival differences between mouse genotypes are lost upon parenteral MAdV-1 infection, strongly implicating a role for intestinal defenses in attenuating pathogenesis. Although differences in α-defensin expression impact the composition of the ileal commensal bacterial population, depletion studies using broad-spectrum antibiotics revealed no effect of the microbiota on α-defensin-dependent viral pathogenesis. Moreover, despite the sensitivity of MAdV-1 infection to α-defensin neutralization in cell culture, we observed no barrier effect due to Paneth cell α-defensin activation on the kinetics and magnitude of MAdV-1 dissemination to the brain. Rather, a protective neutralizing antibody response was delayed in the absence of α-defensins. This effect was specific to oral viral infection, because antibody responses to parenteral or mucosal ovalbumin exposure were not affected by α-defensin deficiency. Thus, α-defensins play an important role as adjuvants in antiviral immunity in vivo that is distinct from their direct antiviral activity observed in cell culture.

Identification of Quantitative Trait Loci for Susceptibility to Mouse Adenovirus Type 1

ABSTRACT Adult SJL/J mice are highly susceptible to mouse adenovirus type 1 (MAV-1) infections, whereas other inbred strains, including BALB/cJ, are resistant (K. R. Spindler, L. Fang, M. L. Moore, C. C. Brown, G. N. Hirsch, and A. K. Kajon, J. Virol. 75:12039-12046, 2001). Using congenic mouse strains, we showed that the H-2s haplotype of SJL/J mice is not associated with susceptibility to MAV-1. Susceptibility of MAV-1-infected (BALB/cJ × SJL/J)F1 mice was intermediate between that of SJL/J mice and that of BALB/cJ mice, indicating that susceptibility is a genetically controlled quantitative trait. We mapped genetic loci involved in mouse susceptibility to MAV-1 by analysis of 192 backcross progeny in a genome scan with 65 simple sequence length polymorphic markers. A major quantitative trait locus (QTL) was detected on chromosome 15 (Chr 15) with a highly significant logarithm of odds score of 21. The locus on Chr 15 alone accounts for 40% of the total trait variance between susceptible and resistant strains. QTL modeling of the data indicated that there are a number of other QTLs with small effects that together with the major QTL on Chr 15 account for 54% of the trait variance. Identification of the major QTL is the first step in characterizing host genes involved in susceptibility to MAV-1.

Novel immunocompetent murine tumor model for evaluation of conditionally replication-competent (oncolytic) murine adenoviral vectors.

ABSTRACT Oncolytic adenoviral vectors that express immunostimulatory transgenes are currently being evaluated in clinic. Preclinical testing of these vectors has thus far been limited to immunodeficient xenograft tumor models since human adenoviruses do not replicate effectively in murine tumor cells. The effect of the immunostimulatory transgene on overall virus potency can therefore not be readily assessed in these models. Here, a model is described that allows the effective testing of mouse armed oncolytic adenovirus (MAV) vectors in immunocompetent syngeneic tumor models. These studies demonstrate that the MAV vectors have a high level of cytotoxicity in a wide range of murine tumor cells. The murine oncolytic viruses were successfully armed with murine granulocyte-macrophage colony-stimulating factor (mGM-CSF) by a novel method which resulted in vectors with a high level of tumor-specific transgene expression. The mGM-CSF-armed MAV vectors showed an improved level of antitumor potency and induced a systemic antitumor immune response that was greater than that induced by unarmed parental vectors in immunocompetent syngeneic tumor models. Thus, the oncolytic MAV-1 system described here provides a murine homolog model for the testing of murine armed oncolytic adenovirus vectors in immunocompetent animals. The model allows evaluation of the impact of virus replication and the host immune response on overall virus potency and enables the generation of translational data that will be important for guiding the clinical development of these viruses.

Requirement of Sur2 for Efficient Replication of Mouse Adenovirus Type 1

ABSTRACT Mouse adenovirus type 1 (MAV-1) early region 1A (E1A) encodes a virulence gene in viral infection of mice. To broaden our understanding of the functions of E1A in MAV-1 pathogenesis, an unbiased experimental approach, glutathione S-transferase (GST) pulldown, was used to screen for cellular proteins that interact with E1A protein. We identified mouse Sur2, a subunit of Mediator complex, as a protein that binds to MAV-1 E1A. The interaction between Sur2 and MAV-1 E1A was confirmed in virus-infected cells. Conserved region 3 (CR3) of MAV-1 E1A was mapped as the region required for Sur2-E1A interaction, as is the case for human adenovirus E1A. Although it has been proposed that human adenovirus E1A recruits the Mediator complex to transactivate transcription of viral early genes, Sur2 function in adenovirus replication has not been directly tested previously. Studies on the functions of Sur2 with mouse embryonic fibroblasts (MEFs) showed that there was a multiplicity-dependent growth defect of MAV-1 in Sur2−/− MEFs compared to Sur2+/+ MEFs. Comparison of the viral DNA and viral mRNA levels in Sur2+/+ and Sur2−/− MEFs confirmed that Sur2 was important for efficient viral replication. The viral replication defects in Sur2−/− MEFs appeared to be due at least in part to a defect in viral early gene transcription.

Mouse adenovirus type 1 infection of macrophages.

Mouse adenovirus type 1 (MAV-1) causes acute and persistent infections in mice, with high levels of virus found in the brain, spinal cord and spleen in acute infections. MAV-1 infects endothelial cells throughout the mouse, and monocytes/macrophages have also been implicated as targets of the virus. Here we determined the extent and functional importance of macrophage infection by MAV-1. Bone marrow-derived macrophages expressed MAV-1 mRNAs and proteins upon ex vivo infection. Adherent peritoneal macrophages from infected mice expressed viral mRNAs and produced infectious virus. Infected chemokine (C-C motif) receptor 2 (CCR2) knockout mice, which are defective for macrophage recruitment, did not show differences in survival or MAV-1 load compared to controls. In contrast, macrophage depletion using clodronate-loaded liposomes resulted in increased virus replication in spleens of a MAV-1-resistant mouse strain, BALB/cJ. Thus macrophages serve both as targets of infection and as effectors of the host response.