Identification of Alternative Transcripts Encoding the Essential Murine Gammaherpesvirus Lytic Transactivator RTA

The essential immediate early transcriptional activator RTA, encoded by gene 50, is conserved among all characterized gammaherpesviruses. Analyses of a recombinant murine gammaherpesvirus 68 (MHV68) lacking both of the known gene 50 promoters (G50DblKo) revealed that this mutant retained the ability to replicate in the simian kidney epithelial cell line Vero but not in permissive murine fibroblasts following low-multiplicity infection. However, G50DblKo replication in permissive fibroblasts was partially rescued by high-multiplicity infection. In addition, replication of the G50DblKo virus was rescued by growth on mouse embryonic fibroblasts (MEFs) isolated from IFN-α/βR(−/−) mice, while growth on Vero cells was suppressed by the addition of alpha interferon (IFN-α). 5′ rapid amplification of cDNA ends (RACE) analyses of RNAs prepared from G50DblKo and wild-type MHV68-infected murine macrophages identified three novel gene 50 transcripts initiating from 2 transcription initiation sites located upstream of the currently defined proximal and distal gene 50 promoters. In transient promoter assays, neither of the newly identified gene 50 promoters exhibited sensitivity to IFN-α treatment. Furthermore, in a single-step growth analysis RTA levels were higher at early times postinfection with the G50DblKo mutant than with wild-type virus but ultimately fell below the levels of RTA expressed by wild-type virus at later times in infection. Infection of mice with the MHV68 G50DblKo virus demonstrated that this mutant virus was able to establish latency in the spleen and peritoneal exudate cells (PECs) of C57BL/6 mice with about 1/10 the efficiency of wild-type virus or marker rescue virus. However, despite the ability to establish latency, the G50DblKo virus mutant was severely impaired in its ability to reactivate from either latently infected splenocytes or PECs. Consistent with the ability to rescue replication of the G50DblKo mutant by growth on type I interferon receptor null MEFs, infection of IFN-α/βR(−/−) mice with the G50DblKo mutant virus demonstrated partial rescue of (i) acute virus replication in the lungs, (ii) establishment of latency, and (iii) reactivation from latency. The identification of additional gene 50/RTA transcripts highlights the complex mechanisms involved in controlling expression of RTA, likely reflecting time-dependent and/or cell-specific roles of different gene 50 promoters in controlling virus replication. Furthermore, the newly identified gene 50 transcripts may also act as negative regulators that modulate RTA expression. IMPORTANCE The viral transcription factor RTA, encoded by open reading frame 50 (Orf50), is well conserved among all known gammaherpesviruses and is essential for both virus replication and reactivation from latently infected cells. Previous studies have shown that regulation of gene 50 transcription is complex. The studies reported here describe the presence of additional alternatively initiated, spliced transcripts that encode RTA. Understanding how expression of this essential viral gene product is regulated may identify new strategies for interfering with infection in the setting of gammaherpesvirus-induced diseases

Murine Gamma-herpesvirus Immortalization of Fetal Liver-Derived B Cells Requires both the Viral Cyclin D Homolog and Latency-Associated Nuclear Antigen

Human gammaherpesviruses are associated with the development of lymphoproliferative diseases and B cell lymphomas, particularly in immunosuppressed hosts. Understanding the molecular mechanisms by which human gammaherpesviruses cause disease is hampered by the lack of convenient small animal models to study them. However, infection of laboratory strains of mice with the rodent virus murine gammaherpesvirus 68 (MHV68) has been useful in gaining insights into how gammaherpesviruses contribute to the genesis and progression of lymphoproliferative lesions. In this report we make the novel observation that MHV68 infection of murine day 15 fetal liver cells results in their immortalization and differentiation into B plasmablasts that can be propagated indefinitely in vitro, and can establish metastasizing lymphomas in mice lacking normal immune competence. The phenotype of the MHV68 immortalized B cell lines is similar to that observed in lymphomas caused by KSHV and resembles the favored phenotype observed during MHV68 infection in vivo. All established cell lines maintained the MHV68 genome, with limited viral gene expression and little or no detectable virus production - although virus reactivation could be induced upon crosslinking surface Ig. Notably, transcription of the genes encoding the MHV68 viral cyclin D homolog (v-cyclin) and the homolog of the KSHV latency-associated nuclear antigen (LANA), both of which are conserved among characterized γ2-herpesviruses, could consistently be detected in the established B cell lines. Furthermore, we show that the v-cyclin and LANA homologs are required for MHV68 immortalization of murine B cells. In contrast the M2 gene, which is unique to MHV68 and plays a role in latency and virus reactivation in vivo, was dispensable for B cell immortalization. This new model of gammaherpesvirus-driven B cell immortalization and differentiation in a small animal model establishes an experimental system for detailed investigation of the role of gammaherpesvirus gene products and host responses in the genesis and progression of gammaherpesvirus-associated lymphomas, and presents a convenient system to evaluate therapeutic modalities

Association of acute toxic encephalopathy with litchi consumption in an outbreak in Muzaffarpur, India, 2014: a case-control study

Background Outbreaks of unexplained illness frequently remain under-investigated. In India, outbreaks of an acute neurological illness with high mortality among children occur annually in Muzaffarpur, the country’s largest litchi cultivation region. In 2014, we aimed to investigate the cause and risk factors for this illness. Methods In this hospital-based surveillance and nested age-matched case-control study, we did laboratory investigations to assess potential infectious and non-infectious causes of this acute neurological illness. Cases were children aged 15 years or younger who were admitted to two hospitals in Muzaffarpur with new-onset seizures or altered sensorium. Age-matched controls were residents of Muzaffarpur who were admitted to the same two hospitals for a non-neurologic illness within seven days of the date of admission of the case. Clinical specimens (blood, cerebrospinal fluid, and urine) and environmental specimens (litchis) were tested for evidence of infectious pathogens, pesticides, toxic metals, and other non-infectious causes, including presence of hypoglycin A or methylenecyclopropylglycine (MCPG), naturally-occurring fruit-based toxins that cause hypoglycaemia and metabolic derangement. Matched and unmatched (controlling for age) bivariate analyses were done and risk factors for illness were expressed as matched odds ratios and odds ratios (unmatched analyses). Findings Between May 26, and July 17, 2014, 390 patients meeting the case definition were admitted to the two referral hospitals in Muzaffarpur, of whom 122 (31%) died. On admission, 204 (62%) of 327 had blood glucose concentration of 70 mg/dL or less. 104 cases were compared with 104 age-matched hospital controls. Litchi consumption (matched odds ratio [mOR] 9·6 [95% CI 3·6 – 24]) and absence of an evening meal (2·2 [1·2–4·3]) in the 24 h preceding illness onset were associated with illness. The absence of an evening meal significantly modified the effect of eating litchis on illness (odds ratio [OR] 7·8 [95% CI 3·3–18·8], without evening meal; OR 3·6 [1·1–11·1] with an evening meal). Tests for infectious agents and pesticides were negative. Metabolites of hypoglycin A, MCPG, or both were detected in 48 [66%] of 73 urine specimens from case-patients and none from 15 controls; 72 (90%) of 80 case-patient specimens had abnormal plasma acylcarnitine profiles, consistent with severe disruption of fatty acid metabolism. In 36 litchi arils tested from Muzaffarpur, hypoglycin A concentrations ranged from 12·4 μg/g to 152·0 μg/g and MCPG ranged from 44·9 μg/g to 220·0 μg/g. Interpretation Our investigation suggests an outbreak of acute encephalopathy in Muzaffarpur associated with both hypoglycin A and MCPG toxicity. To prevent illness and reduce mortality in the region, we recommended minimising litchi consumption, ensuring receipt of an evening meal and implementing rapid glucose correction for suspected illness. A comprehensive investigative approach in Muzaffarpur led to timely public health recommendations, underscoring the importance of using systematic methods in other unexplained illness outbreaks

Tidal and atmospheric forcing of the upper ocean in the Gulf of California, Part 2: Surface heat flux

Satellite infrared imagery and coastal meteorological data for March 1984 through February 1985 are used to estimate the net annual surface heat flux for the northern Gulf of California. The average annual surface heat flux for the area north of Guaymas and Santa Rosalia is estimated to be +74 W m-2 for the 1984–1985 time period. This is comparable to the +20–50 W m-2 previously obtained from heat and freshwater transport estimates made with hydrographic surveys from different years and months. The spatial distribution of the net surface heat flux shows a net gain of heat over the whole northern gulf. Except for a local maximum near San Esteban Island, the largest heat gain (+110–120 W m-2) occurs in the Ballenas and Salsipuedes channels, where strong tidal mixing produces anomalously cold sea surface temperatures (SSTs) over much of the year. The lowest heat gain occurs in the Guaymas Basin (+40–50 W m-2), where SSTs are consistently warmer. In the relatively shallow northern basin the net surface heat flux is fairly uniform, with a net annual gain of approximately +70 W m-2. A local minimum in heat gain (approximately +60 W m-2) is observed over the shelf in the northwest, where spring and summer surface temperatures are particularly high. A similar minimum in heat gain over the shelf was observed in a separate study in which historical SSTs and 7 years (1979–1986) of meteorological data from Puerto Penasco were used to estimate the net surface heat flux for the northern basin. In that study, however, the heat fluxes were higher, with a gain of +100 W m-2 over the shelf and +114 W m-2 in the northern basin. These larger values are directly attributable to the higher humidities in the 1979–1986 study compared to the 1984-1985 satellite study. Significant interannual variations in humidity appear to occur in the northern gulf, with relatively high humidities during El Niño years and low humidities during anti-El Niño years. High humidities reduce evaporation and the associated latent heat loss, promoting a net annual heat gain. In the northern Gulf of California, however, tidal mixing appears to play a key role in the observed gain of heat. Deep mixing in the island region produces a persistent pool of cold water which is mixed horizontally by the large-scale circulation, lowering surface temperatures over most of the northern gulf. These cold SSTs decrease evaporation by reducing the saturation vapor pressure of the overlying air. As a result, heat loss is substantially reduced, even when humidities are low. By removing heat from the surface, tidal mixing alters the time scale of air-sea interaction and reduces or possibly even inhibits the formation of deep water masses via convection. Over climatological timescales, it may be tidal mixing that ultimately maintains the estuarinelike circulation in the northern Gulf of California, differentiating it from the Mediterranean and Red seas, which lose heat to the atmosphere

Unbiased mutagenesis of MHV68 LANA reveals a DNA-binding domain required for LANA function in vitro and in vivo.

The Latency-Associated Nuclear Antigen (LANA), encoded by ORF73, is a conserved gene among the γ2-herpesviruses (rhadinoviruses). The Kaposi's Sarcoma-Associated Herpesvirus (KSHV) LANA is consistently expressed in KSHV-associated malignancies. In the case of the rodent γ2-herpesvirus, murine gammaherpesvirus 68 (MHV68), the LANA homolog (mLANA) is required for efficient virus replication, reactivation from latency and immortalization of murine fetal liver-derived B cells. To gain insights into mLANA function(s), knowing that KSHV LANA binds DNA and can modulate transcription of a variety of promoters, we sought out and identified a mLANA-responsive promoter which maps to the terminal repeat (TR) of MHV68. Notably, mLANA strongly repressed activity from this promoter. We extended these analyses to demonstrate direct, sequence-specific binding of recombinant mLANA to TR DNA by DNase I footprinting. To assess whether the DNA-binding and/or transcription modulating function is important in the known mLANA phenotypes, we generated an unbiased library of mLANA point mutants using error-prone PCR, and screened a large panel of mutants for repression of the mLANA-responsive promoter to identify loss of function mutants. Notably, among the mutant mLANA proteins recovered, many of the mutations are in a predicted EBNA-1-like DNA-binding domain. Consistent with this prediction, those tested displayed loss of DNA binding activity. We engineered six of these mLANA mutants into the MHV68 genome and tested the resulting mutant viruses for: (i) replication fitness; (ii) efficiency of latency establishment; and (iii) reactivation from latency. Interestingly, each of these mLANA-mutant viruses exhibited phenotypes similar to the mLANA-null mutant virus, indicating that DNA-binding is critical for mLANA function

ORF73-Null Murine Gammaherpesvirus 68 Reveals Roles for mLANA and p53 in Virus Replication▿

Gammaherpesviruses establish lifelong, latent infections in host lymphocytes, during which a limited subset of viral gene products facilitates maintenance of the viral episome. Among the gamma-2-herpesvirus (rhadinovirus) subfamily, this includes expression of the conserved ORF73-encoded LANA proteins. We previously demonstrated by loss-of-function mutagenesis that the murine gammaherpesvirus 68 (MHV68) ORF73 gene product, mLANA, is required for the establishment of latency following intranasal inoculation of mice (N. J. Moorman, D. O. Willer, and S. H. Speck, J. Virol. 77:10295-10303, 2003). mLANA-deficient viruses also exhibited a defect in acute virus replication in the lungs of infected mice. The latter observation led us to examine the role of mLANA in productive viral replication. We assessed the capacity of mLANA-deficient virus (73.Stop) to replicate in cell culture at low multiplicities of infection (MOIs) and found that 73.Stop growth was impaired in murine fibroblasts but not in Vero cells. A recombinant virus expressing an mLANA-green fluorescent protein (GFP) fusion revealed that mLANA is expressed throughout the virus replication cycle. In addition, 73.Stop infection of murine fibroblasts at high MOIs was substantially more cytotoxic than infection with a genetically repaired marker rescue virus (73.MR), a phenotype that correlated with enhanced kinetics of viral gene expression and increased activation of p53. Notably, augmented cell death, viral gene expression, and p53 induction were independent of viral DNA replication. Expression of a mLANA-GFP fusion protein in fibroblasts correlated with both reduced p53 stabilization and reduced cell death following treatment with p53-inducing agonists. In agreement, accentuated cell death associated with 73.Stop infection was reduced in p53-deficient murine embryonic fibroblasts. Additionally, replication of 73.Stop in p53-deficient cells was restored to levels comparable to those of 73.MR. More remarkably, the absence of p53 led to an overall delay in replication for both 73.Stop and 73.MR viruses, which correlated with delayed viral gene expression, indicating a role for p53 in MHV68 replication. Consistent with these findings, the expression of replication-promoting viral genes was positively influenced by p53 overexpression or treatment with the p53 agonist etoposide. Overall, these data demonstrate the importance of mLANA in MHV68 replication and suggest that LANA proteins limit the induction of cellular stress responses to regulate the viral gene expression cascade and limit host cell injury

Deletion analysis of TR reveals DNA sequence required for mLANA-mediated repression.

<p>(A) Schematic of pGL-TR, noting the numbering scheme with regard to the NotI site. Coordinate 1 is corresponds to MHV68 WUMS <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002906#ppat.1002906-Virgin1" target="_blank">[31]</a> bp 119,105, proceeding in order towards the right end of the unique sequence. Coordinate 894 is the end of one TR unit fused to the beginning of the next. Indicated on the diagram are two known sites of transcription initiation: (i) (73p1) starts transcription between coordinates 423–466 (118,683–118,640) depending on cell type <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002906#ppat.1002906-Allen1" target="_blank">[29]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002906#ppat.1002906-Coleman1" target="_blank">[32]</a> and the partial exon splices to the full 73E1 exon (118,695–118,605) in the next repeat unit; (ii) initiates transcription of one species of ORF75a beginning between coordinates 885–891 (118221–118216) <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002906#ppat.1002906-Coleman1" target="_blank">[32]</a> (promoter elements are likely shared between ORF75a and ORF73 transcripts that initiate at 73E2 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002906#ppat.1002906-Allen1" target="_blank">[29]</a>, and in distal copies may splice into 73E1). (B) Serial deletions of the TR were made in the pGL-TR vector by PCR and are named accordingly. Each TR deletion, including the full-length TR (FL) was co-transfected into 293T cells, along with either an mLANA-GFP or empty GFP expression vector, as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002906#ppat-1002906-g001" target="_blank">Fig. 1</a>, in triplicate. Here, data are normalized by taking the ratio of each deletion construct over its respective pGL4.10 empty reporter control, setting pGL4.10 in each case to 1. Deleting the 3′ end gives a large uptick in activity, which is still repressed by mLANA, until the second deletion removes mLANA sensitivity. 5′ deletions had no effect on mLANA sensitivity.</p

Repression activity of mLANA mutants in the MHV68 TR-driven luciferase reporter assay.

*<p>−, 0–20% repression; +, 20–50% repression; ++, 50–70% repression; +++, 80–100% repression.</p

Random mutagenesis of mLANA reveals residues important for transcriptional repression.

<p>Loss of function mutants generated at random by error-prone PCR and defined by their failure to repress TR-luciferase reporter activity are shown here. (A) The mLANA primary amino acid sequence is shown, and underneath, each amino acid difference shown represents an amino acid changed in a mutant that had lost its capacity to repress transcription. Highlighted in grey is the region that has homology to other LANA proteins and is the domain that is predicted to fold like the EBV EBNA1 DNA binding domain, as shown in panel C. Underlined are mutations that were introduced into the MHV68-YFP BAC in subsequent experiments. (B) Sequence alignment of the conserved C-terminal domains of mLANA and KSHV LANA (kLANA). Shown in red are mutations in mLANA and kLANA that diminish LANA-mediated repression of transcription (those mutations introduced into kLANA that altered more than 1 residue are underlined). (C) The PHYRE protein structure prediction algorithm modeling of the conserved C-terminal domain (shaded in grey in panel A) of mLANA based on the solved structure of the EBNA-1 DNA-binding domain <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002906#ppat.1002906-Kelley1" target="_blank">[33]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002906#ppat.1002906-Bochkarev1" target="_blank">[34]</a>. This depiction of the predicted three-dimensional structure of the mLANA conserved C-terminal domain has highlighted in green mutations that ablate the transcription repression function of mLANA. Also shown in red is a mutation, A171V, that does not impact mLANA transcriptional repression (has wild type phenotype) and was used in subsequent experiments as a negative control mLANA mutant.</p