Virus-encoded microRNAs facilitate gammaherpesvirus latency and pathogenesis in vivo

Abstract

Gammaherpesviruses, including Epstein-Barr virus (EBV), Kaposi sarcoma-associated herpesvirus (KSHV, or HHV-8), and murine gammaherpesvirus 68 (MHV68, γHV68, or MuHV-4), are B cell-tropic pathogens that each encode at least 12 microRNAs (miRNAs). It is predicted that these regulatory RNAs facilitate infection by suppressing host target genes involved in a wide range of key cellular pathways. However, the precise contribution that gammaherpesvirus miRNAs make to viral life cycle and pathogenesis in vivo is unknown. MHV68 infection of mice provides a highly useful system to dissect the function of specific viral elements in the context of both asymptomatic infection and disease. Here, we report (i) analysis of in vitro and in vivo MHV68 miRNA expression, (ii) generation of an MHV68 miRNA mutant with reduced expression of all 14 pre-miRNA stem-loops, and (iii) comprehensive phenotypic characterization of the miRNA mutant virus in vivo. The profile of MHV68 miRNAs detected in infected cell lines varied with cell type and did not fully recapitulate the profile from cells latently infected in vivo. The miRNA mutant virus, MHV68.Zt6, underwent normal lytic replication in vitro and in vivo, demonstrating that the MHV68 miRNAs are dispensable for acute replication. During chronic infection, MHV68.Zt6 was attenuated for latency establishment, including a specific defect in memory B cells. Finally, MHV68.Zt6 displayed a striking attenuation in the development of lethal pneumonia in mice deficient in IFN-γ. These data indicate that the MHV68 miRNAs may facilitate virus-driven maturation of infected B cells and implicate the miRNAs as a critical determinant of gammaherpesvirus-associated disease.

Importance: Gammaherpesviruses such as EBV and KSHV are widespread pathogens that establish lifelong infections and are associated with the development of numerous types of diseases, including cancer. Gammaherpesviruses encode many small noncoding RNAs called microRNAs (miRNAs). It is predicted that gammaherpesvirus miRNAs facilitate infection and disease by suppressing host target transcripts involved in a wide range of key cellular pathways; however, the precise contribution that these regulatory RNAs make to in vivo virus infection and pathogenesis is unknown. Here, we generated a mutated form of murine gammaherpesvirus (MHV68) to dissect the function of gammaherpesvirus miRNAs in vivo. We demonstrate that the MHV68 miRNAs were dispensable for short-term virus replication but were important for establishment of lifelong infection in the key virus reservoir of memory B cells. Moreover, the MHV68 miRNAs were essential for the development of virus-associated pneumonia, implicating them as a critical component of gammaherpesvirus-associated disease.

FIG 1

Proposed MHV68-encoded miRNA and TMER nomenclature. The diagram depicts approximately 6 kb of the 5′ end of the MHV68 genome, which includes eight tRNA-miRNA-encoding RNA (TMER) genes and two unique M genes. Each TMER (gray rectangle) is composed of a tRNA-like gene (red triangle) linked to one or two pre-miRNA stem-loops (light and dark blue rectangles). Proposed naming of all putative MHV68 miRNAs, which incorporates current miRBase number designations, is indicated. (Inset) Mfold in silico prediction of TMER1 structure and corresponding miRNA locations.

FIG 2

Expression kinetics of MHV68-encoded miRNAs during lytic infection. Stem-loop qRT-PCR was used to determine the level of expression of MHV68-encoded mature miRNAs during lytic replication. NIH 3T12 fibroblasts were infected with MHV68 at MOI 5 and then harvested at the indicated time points. Stem-loop qRT-PCR was performed using TaqMan primers and probe sets (see Table S2 in the supplemental material). (A) Expression time course for all tested viral miRNAs relative (10²) to the endogenous noncoding RNA control snoRNA234. (B) Time course for all tested viral miRNAs expressed as approximate miRNA copy number per cell, based on the synthetic miR-2-5p standard curve. (C) Viral miRNAs that peak between 16 and 48 hpi, expressed as miRNA copy number per cell. (D) Viral miRNAs that peak at 48 hpi. (E) Viral miRNAs with consistent low-level expression throughout the time course, depicted on an enhanced scale. All data are means ± standard deviations (SD) from 3 experiments.

FIG 3

Expression of MHV68-encoded miRNAs during latent infection. Stem-loop qRT-PCR was used to determine the level of expression of MHV68-encoded mature miRNAs during latent infection in vitro and in vivo. (A) miRNA expression from S11 and HE2.1 B cell lines with stable latent MHV68. Following cell harvest and lysis, stem-loop qRT-PCR was performed using TaqMan primers and probe-sets (see Table S2 in the supplemental material). Displayed values are relative (10⁴) to endogenous snoRNA202 expression. Values are means ± SD from 3 experiments. (B) miRNA expression from splenocytes latently infected in vivo. For each experiment, four C57BL6/J mice were infected i.n. with 10⁴ PFU MHV68. At 16 days, splenocytes were harvested and pooled, lysed, and subjected to stem-loop qRT-PCR. Values are relative (10⁴) to endogenous snoRNA202 expression and are means ± SD from 2 experiments.

FIG 4

miRNA mutations incorporated in MHV68.Zt6 and corresponding expression of miRNAs and surrounding genes. (A) Depiction of MHV68.Zt6 mutations, including deletion of TMER1 to TMER5, TMER7, and TMER8 miRNA stem-loops (red X) and insertion of the PolIII stop site (red line) following vtRNA6 in TMER6. (B) Expression of viral miRNAs from wild-type MHV68 marker virus or MHV68.Zt6, relative (10²) to the endogenous noncoding RNA control snoRNA202. NIH 3T12 fibroblasts were infected with MHV68.ORF73βla or MHV68.Zt6 at an MOI of 5 and then harvested at 48 hpi. Stem-loop qRT-PCR was performed as described for Fig. 1. Values are means ± SD from 2 experiments. (C) Expression of viral coding genes surrounding miRNA mutations. NIH 3T12 fibroblasts were infected with MHV68.ORF73βla or MHV68.Zt6 at an MOI of 5 for 24 h. M1, M2, and M3 expression was quantified using qRT-PCR. Values are relative (10³) to endogenous GAPDH expression and are means ± SD from 3 experiments.

FIG 5

MHV68 miRNAs are dispensable for lytic replication. Plaque assays were used to determine the titer of wild-type or miRNA deletion mutant viruses during acute replication in vitro and in vivo. (A) Lytic replication in fibroblasts in vitro. Single-step and multistep growth curves were generated following infection of NIH 3T12 fibroblasts with MHV68.ORF73βla or MHV68.Zt6 at an MOI of 5 or 0.05, respectively. At the specified time points, cells and supernatant fluid were collected, and titers were determined by plaque assay. Data are means ± SD of 3 experiments. (B) Lytic replication in lungs in vivo. C57BL6/J mice were infected i.n. with 10⁴ PFU MHV68.ORF73βla or MHV68.Zt6. At 5 or 8 dpi, lungs were harvested and viral titers were determined by plaque assay. Lines represent the mean titers for eight individual mice. For all experiments, statistical significance was determined by Student’s t test. *, P < 0.05; **, P < 0.01.

FIG 6

MHV68 miRNAs modulate latency and reactivation in vivo. Parameters of wild-type and miRNA mutant virus latency and reactivation were assessed 16 to 19 dpi. For each experiment, four C57BL6/J mice were infected i.n. with MHV68.ORF73βla or MHV68.Zt6 at (A) 10⁴ PFU (n = 7 experiments) or (B) 100 PFU (n = 4 experiments). Sixteen (10⁴ PFU) or nineteen (100 PFU) days later, splenocytes from each mouse were harvested, pooled within groups, and then subjected to limiting dilution assays to detect the presence of viral genome, ex vivo reactivation from latency, or the presence of preformed infectious virus. For viral genome assays, splenocytes were serially diluted 3-fold in a background of uninfected cells, plated at 12 reactions per cell dilution, lysed, and then subjected to nested PCR specific for MHV68 ORF72. This assay is specific for a single copy of viral genome in a background of 10,000 uninfected cells. The frequency of cells harboring viral DNA was determined using a Poisson distribution, indicated by the line at 63.2%. For reactivation assays, splenocytes were serially diluted 2-fold and then plated at 24 wells per cell dilution over a monolayer of mouse embryonic fibroblasts (MEFs). After 3 weeks, monolayers were assessed for cytopathic effect (CPE) following spontaneous reactivation from latency. To detect preformed infectious virus, parallel cell samples were mechanically disrupted prior to plating on MEF monolayers. The frequencies of splenocytes reactivating from latency or carrying preformed virus was determined using a Poisson distribution, as indicated by the line at 63.2% (n = 3 experiments).

FIG 7

MHV68 miRNAs contribute to efficient infection of memory B cells in vivo. Flow-cytometric detection of β-lactamase activity and cell surface marker costaining were used to determine the phenotype of B cells infected in vivo. Three C57BL6/J mice per sample group per experiment were infected i.n. with parental wild-type MHV68.ORF73βla, or miRNA mutant MHV68.Zt6. After 16 days, splenocytes were harvested, pooled, and then stained with antibodies for CD19, IgM, and CD38, in addition to the β-lactamase substrate CCF4/AM. Single-cell suspensions were then subjected to flow-cytometric analyses. (A) Schematic of representative flow cytometry gating scheme to quantify the percentage of infected naive (CD19⁺ IgM⁺), germinal-center (CD19⁺ IgM⁻ CD38^lo) and memory (CD19⁺ IgM⁻ CD38^hi) B cells. (B and C) Mice were infected i.n. with 10⁴ (4 experiments) or 100 (5 experiments) PFU of parental wild-type MHV68.ORF73βla or the miRNA mutant MHV68.Zt6. Values are percentages of infected CD19⁺ cells from each sample group displaying a naive, germinal-center, or memory B cell surface marker phenotype. Statistical significance was determined using Student’s t test. **, P < 0.01; ***, P < 0.005.

FIG 8

MHV68 miRNAs are essential for lethal pneumonia. Survival of IFN-γ-deficient mice from MHV68-associated lethal pneumonia during a 14-day infection time course is shown. BALB/c.IFNγ^−/− mice were infected i.n. with 4 × 10⁵ PFU of MHV68, MHV68.ORF73βla, or MHV68.Zt6 and monitored daily for signs of disease. Data are the percentage of mice surviving infection at each time. Parenthetical values are the numbers of mice surviving 14 dpi/number of mice tested.

FIG 9

MHV68.Zt6 displays reduced lung inflammation in IFN-γ^−/− mice. Hematoxylin and eosin (H&E) stains of lung sections from infected mice are shown. Following harvest, lungs from infected mice were fixed, embedded in paraffin, sectioned, and stained with H&E. (Top) Magnification, ×10. (Bottom) Higher (×40) magnifications of boxed portions in top row.