Fatal Sindbis Virus Infection of Neonatal Mice in the Absence of Encephalitis

A comparative pathogenesis study was performed in neonatal mice using a molecularly cloned laboratory variant of Sindbis strain AR339, designated TRSB, and a single-site attenuated mutant of TRSB derived by site-directed mutagenesis of the E2 glycoprotein from Ser to Arg at residue 114 (TRSBr114). TRSB caused 100% mortality with an average survival time of 3.0 +/- 0.7 days, whereas mice inoculated with TRSBr114 exhibited an attenuated disease course with 46% mortality and an extended average survival time of 7.5 +/- 3.4 days for those animals that died. Reduced virulence of TRSBr114 was characterized by delayed appearance of detectable virus, relative to TRSB, and by lower peak virus titers in both sera and brains of infected mice. TRSB infection induced very high peak serum titers of interferon alpha/beta (215,000 units/ml compared to 2100 units/ml for TRSBr114). In situ hybridization analysis demonstrated replication of TRSB in brain, but only minimal histopathological changes and no evidence of encephalitis were observed. However, extensive extraneural lesions and viral replication were found in skin, connective tissue, and muscle. Moreover, dramatic involution of the thymus and loss of hematopoietic tissues were observed in the absence of virus replication at these sites, suggesting the involvement of a systemic physiological stress response in TRSB infection. TRSBr114 infection did not cause thymic lesions. Otherwise, the attenuated mutant demonstrated a similar pattern of tissue and organ involvement, but lesions and positive in situ hybridization signal were much more limited in scope and intensity compared to TRSB. TRSBr114-infected mice developed myositis and encephalomyelitis approximately 6 days postinfection. Therefore, TRSB-infected animals may succumb to an early syndrome associated with the stress response, preventing their survival for a time sufficient for the development of encephalitis. Alternatively, a systemic stress response, as evidenced by thymic involution, may result in immunosuppression, thus contributing to the absence of encephalitis. In any event, the attenuating mutation in the E2 glycoprotein significantly altered the course of Sindbis-induced disease by limiting virus replication and associated damage early in infection. Mutant-infected animals survived beyond Day 4 and progressed to a classical encephalomyelitis from which about half recovered

TNFα, Interferon, and Stress Response Induction as a Function of Age-Related Susceptibility to Fatal Sindbis Virus Infection of Mice

AbstractThe age-related acquisition of resistance to fatal Sindbis virus infection was examined using a molecularly cloned laboratory strain of the AR339 isolate designated TRSB. TRSB caused 100% mortality in mice up to 5 days of age. Resistance to fatal infection developed abruptly between 5 and 9 days of age. Lethal Sindbis virus infection of mice inoculated at 4 days of age was characterized by high levels of virus replication, induction of high levels of interferon-α/β and TNF-α and severe thymic involution indicative of a systemic stress response. These changes correlated with predominantly noninflammatory lesions. In contrast, TRSB infection of older mice was characterized by survival, more limited virus replication, reduced cytokine induction, and the development of inflammatory responses leading to encephalitis, myositis, and myocarditis. Previous studies utilized infections of neonatal mice with TRSB and an attenuated mutant of TRSB to compare fatal and nonfatal Sindbis infection (Trgovcich et al., 1996. Virology 224, 73–83). The experiments reported here utilize mouse age at the time of infection to create conditions for examination of fatal and nonfatal TRSB infections. Both experiments suggest that fatal infection is associated with a shock-like syndrome and little or no inflammatory pathology, while survival is correlated with greatly reduced cytokine levels and inflammatory lesions

Sindbis Virus Infection of Neonatal Mice Results in a Severe Stress Response

AbstractNeonatal mice were infected with virus derived from a molecular clone of a laboratory strain of Sindbis virus, TRSB. The resulting acute fatal infection was typified by few if any of the classic hallmarks of encephalitis, very high levels of interferon-alpha/beta (IFNαβ), and lesions in the thymus and hematopoietic tissues usually associated with a severe stress response. Infection with an attenuated mutant of TRSB, which harbors a single amino acid change in the E2 surface glycoprotein (TRSBr114), was characterized by encephalitis, reduced mortality, low levels of IFNαβ, and no thymic pathology (J. Trgovcich, J.F. Aronson, and R.E. Johnston, 1996,Virology224, 73–83). Here we report that infection of neonatal mice with TRSB, but not TRSBr114, resulted in induction of high levels of tumor necrosis factor-α as well as high and sustained levels of adrenalcorticotropin-releasing hormone and corticosterone. This syndrome of potentially toxic cytokine and stress hormone induction correlates with lethal Sindbis virus infection and constitutes a previously unrecognized aspect of Sindbis virus pathogenesis in mice

Hierarchical and Redundant Lymphocyte Subset Control Precludes Cytomegalovirus Replication during Latent Infection

Reactivation from latent cytomegalovirus (CMV) infection is often associated with conditions of immunosuppression and can result in fatal disease. Whether the maintenance of systemic CMV latency is mainly governed by factors of the infected cell or by immune control functions is unknown. Likewise, the putative immune control mechanisms which could prevent the induction and spread of recurrent CMV infection are not clearly identified. We took advantage of latently infected B cell–deficient mice and a sensitive method for virus detection to study CMV reactivation after ablation of lymphocyte subsets. A crucial role of both T lymphocytes and natural killer (NK) cells was demonstrated. Within 5 d after depletion of lymphocytes, productive infection occurred in 50% of mice, and 14 d later 100% of mice exhibited recurrent infection. A hierarchy of immune control functions of CD8+, NK, and CD4+ cells was established. Reactivation was rare if only one of the lymphocyte subsets was depleted, but was evident after removal of a further subset, indicating a functional redundancy of control mechanisms. The salivary glands were identified as the site of most rapid virus shedding, followed by the detection of recurrent virus in the lungs, and eventually in the spleen. Our findings document a previously unknown propensity of latent CMV genomes to enter productive infection immediately and with a high frequency after immune cell depletion. The data indicate that only the sustained cellular immune control prevents CMV replication and restricts the viral genome to a systemic state of latency

CD28/B7-Mediated Co-stimulation Is Critical for Early Control of Murine Cytomegalovirus Infection

Abstract Control of acute murine cytomegalovirus (MCMV) infection is dependent upon both innate and adaptive immune responses, relying primarily upon natural killer (NK) and T-cell responses for control. Although CD28/B7 plays a clear role in T-cell responses in many antigen systems including some viral infections, the importance of co-stimulation during MCMV infection is unconfirmed. In addition, recent data suggest that CD28/B7 co-stimulation might also be important to Ly49H+ NK-cell expansion. We therefore hypothesized that CD28/B7 co-stimulation is critical to viral control after MCMV infection, and further that CD28/B7 co-stimulation plays a role in MCMV-specific T- and NK-cell responses. To test these hypotheses, we utilized C57BL/6 mice lacking the co-stimulatory molecules B7-1 and B7-2 or CD28. After primary infection with MCMV, viral titers are significantly elevated in mice lacking CD28 or B7 compared with wild-type mice. Impaired viral control is associated with significant defects in peripheral T-cell responses to MCMV, which appear to be dependent upon CD28/B7 co-stimulation. Abnormal hepatic T-cell responses in CD28/ mice are preceded by impaired MCMV-specific Ly49H+ NK-cell responses. Cytokine evaluations confirm that CD28/B7 co-stimulation is not required for non-specific antiviral responses. We conclude that CD28-mediated co-stimulation is critical for early viral control during acute MCMV infection.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/78134/1/vim.2008.0080.pd

Degradation of Cellular miR-27 by a Novel, Highly Abundant Viral Transcript Is Important for Efficient Virus Replication In Vivo

Cytomegaloviruses express large amounts of viral miRNAs during lytic infection, yet, they only modestly alter the cellular miRNA profile. The most prominent alteration upon lytic murine cytomegalovirus (MCMV) infection is the rapid degradation of the cellular miR-27a and miR-27b. Here, we report that this regulation is mediated by the ∼1.7 kb spliced and highly abundant MCMV m169 transcript. Specificity to miR-27a/b is mediated by a single, apparently optimized, miRNA binding site located in its 3'-UTR. This site is easily and efficiently retargeted to other cellular and viral miRNAs by target site replacement. Expression of the 3'-UTR of m169 by an adenoviral vector was sufficient to mediate its function, indicating that no other viral factors are essential in this process. Degradation of miR-27a/b was found to be accompanied by 3'-tailing and -trimming. Despite its dramatic effect on miRNA stability, we found this interaction to be mutual, indicating potential regulation of m169 by miR-27a/b. Most interestingly, three mutant viruses no longer able to target miR-27a/b, either due to miRNA target site disruption or target site replacement, showed significant attenuation in multiple organs as early as 4 days post infection, indicating that degradation of miR-27a/b is important for efficient MCMV replication in vivo

A Role for Cytoplasmic PML in Cellular Resistance to Viral Infection

PML gene was discovered as a fusion partner with retinoic acid receptor (RAR) α in the t(15:17) chromosomal translocation associated with acute promyelocytic leukemia (APL). Nuclear PML protein has been implicated in cell growth, tumor suppression, apoptosis, transcriptional regulation, chromatin remodeling, DNA repair, and anti-viral defense. The localization pattern of promyelocytic leukemia (PML) protein is drastically altered during viral infection. This alteration is traditionally viewed as a viral strategy to promote viral replication. Although multiple PML splice variants exist, we demonstrate that the ratio of a subset of cytoplasmic PML isoforms lacking exons 5 & 6 is enriched in cells exposed to herpes simplex virus-1 (HSV-1). In particular, we demonstrate that a PML isoform lacking exons 5 & 6, called PML Ib, mediates the intrinsic cellular defense against HSV-1 via the cytoplasmic sequestration of the infected cell protein (ICP) 0 of HSV-1. The results herein highlight the importance of cytoplasmic PML and call for an alternative, although not necessarily exclusive, interpretation regarding the redistribution of PML that is seen in virally infected cells

Cell Surface Major Histocompatibility Complex Class II Proteins Are Regulated by the Products of the γ(1)34.5 and U(L)41 Genes of Herpes Simplex Virus 1

Modulation of host immune responses has emerged as a common strategy employed by herpesviruses both to establish life-long infections and to affect recovery from infection. Herpes simplex virus 1 (HSV-1) blocks the major histocompatibility complex (MHC) class I antigen presentation pathway by inhibiting peptide transport into the endoplasmic reticulum. The interaction of viral gene products with the MHC class II pathway, however, has not been thoroughly investigated, although CD4(+) T cells play an important role in human recovery from infection. We have investigated the stability, distribution, and state of MHC class II proteins in glioblastoma cells infected with wild-type HSV-1 or mutants lacking specific genes. We report the following findings. (i) Wild-type virus infection caused a decrease in the accumulation of class II protein on the surface of cells and a decrease in the endocytosis of lucifer yellow or dextran conjugated to fluorescein isothiocyanate but no decrease in the total amount of MHC class II proteins relative to the levels seen in mock-infected cells. (ii) Although the total amount of MHC class II protein remained unchanged, the amounts of cell surface MHC class II proteins were higher in cells infected with the U(L)41-negative mutant, which lacks the virion host shutoff protein, and especially high in cells infected with the γ(1)34.5-negative mutant. We conclude that infected cells attempt to respond to infection by increased acquisition of antigens and transport of MHC class II proteins to the cell surface and that these responses are blocked in part by the virion host shutoff protein encoded by the U(L)41 gene and in large measure by the direct or indirect action of the infected cell protein 34.5, the product of the γ(1)34.5 gene

The Carboxy Terminal Region of the Human Cytomegalovirus Immediate Early 1 (IE1) Protein Disrupts Type II Inteferon Signaling

Interferons (IFNs) activate the first lines of defense against viruses, and promote innate and adaptive immune responses to viruses. We report that the immediate early 1 (IE1) protein of human cytomegalovirus (HCMV) disrupts signaling by IFNγ. The carboxyl-terminal region of IE1 is required for this function. We found no defect in the initial events in IFNγ signaling or in nuclear accumulation of signal transducer and activator of transcription 1 (STAT1) in IE1-expressing cells. Moreover, we did not observe an association between disruption of IFNγ signaling and nuclear domain 10 (ND10) disruption. However, there is reduced binding of STAT1 homodimers to target gamma activated sequence (GAS) elements in the presence of IE1. Co-immunoprecipitation studies failed to support a direct interaction between IE1 and STAT1, although these studies revealed that the C-terminal region of IE1 was required for interaction with STAT2. Together, these results indicate that IE1 disrupts IFNγ signaling by interfering with signaling events in the nucleus through a novel mechanism