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. 2020 Aug 26:10:454.
doi: 10.3389/fcimb.2020.00454. eCollection 2020.

Positive Role of the MHC Class-I Antigen Presentation Regulator m04/gp34 of Murine Cytomegalovirus in Antiviral Protection by CD8 T Cells

Sara Becker  1 , Annette Fink  1 , Jürgen Podlech  1 , Irina Giese  2 , Julia K Schmiedeke  1 , Thomas Bukur  2 , Matthias J Reddehase  1 , Niels A Lemmermann  1
Affiliations

Positive Role of the MHC Class-I Antigen Presentation Regulator m04/gp34 of Murine Cytomegalovirus in Antiviral Protection by CD8 T Cells

Sara Becker et al. Front Cell Infect Microbiol. .

Abstract

Murine cytomegalovirus (mCMV) codes for MHC class-I trafficking modulators m04/gp34, m06/gp48, and m152/gp40. By interacting with the MHC class-Iα chain, these proteins disconnect peptide-loaded MHC class-I (pMHC-I) complexes from the constitutive vesicular flow to the cell surface. Based on the assumption that all three inhibit antigen presentation, and thus the recognition of infected cells by CD8 T cells, they were referred to as "immunoevasins." Improved antigen presentation mediated by m04 in the presence of m152 after infection with deletion mutant mCMV-Δm06W, compared to mCMV-Δm04m06 expressing only m152, led us to propose renaming these molecules "viral regulators of antigen presentation" (vRAP) to account for both negative and positive functions. In accordance with a positive function, m04-pMHC-I complexes were found to be displayed on the cell surface, where they are primarily known as ligands for Ly49 family natural killer (NK) cell receptors. Besides the established role of m04 in NK cell silencing or activation, an anti-immunoevasive function by activation of CD8 T cells is conceivable, because the binding site of m04 to MHC class-Iα appears not to mask the peptide binding site for T-cell receptor recognition. However, functional evidence was based on mCMV-Δm06W, a virus of recently doubted authenticity. Here we show that mCMV-Δm06W actually represents a mixture of an authentic m06 deletion mutant and a mutant with an accidental additional deletion of a genome region encompassing also gene m152. Reanalysis of previously published experiments for the authentic mutant in the mixture confirms the previously concluded positive vRAP function of m04.

Keywords: BAC mutagenesis; CD8 T cells; adoptive cell transfer; antigen presentation; immune evasion; immunoevasin; next-generation sequencing (NGS); recombinant virus.

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Figures

Figure 1
Figure 1
Comparison of Illumina-sequenced viral genomes. Viral DNA was purified from stocks of viruses mCMV-WT.BAC, mCMV-Δm06W, and mCMV-Δm06L, sequenced on Illumina MiSeq, and aligned to the mCMV WT reference sequence (RefSeq: NC_004065.1, INSDC: U68299.1). The Integrative Genomics Viewer was used for visualization. Gray areas indicate matches to the reference sequence, whereas colored spots indicate SNVs or INDELs. Regions with no successful alignment to the reference sequence are left white. Arrows represent the positions of the indicated ORFs. (A) Comparison of the viral genomes at 4.8–6.8 kbp. (B) Comparison of the viral genomes at 203–219 kbp.
Figure 2
Figure 2
Mapping of the deletion in mCMV-Δm06W. (A) Map of the primer design. Gray arrowheads demarcate the suspected deletion in the m145 gene family, numbers indicate nucleotide positions in the mCMV genome. Light gray arrows represent primers flanking the site of deletion, dark gray arrows represent primers flanking the m145 gene. Expected PCR product sizes are shown on the right side. (B) PCR products of the m145-flank PCR on an agarose gel. (C) PCR products of the deletion-flank PCR on an agarose gel. Purified DNA from mCMV-WT.BAC, mCMV-Δm06W, and mCMV-Δm06L virus stocks was used as template in both (B,C). (D) Sanger sequencing of the 4.3 kbp-sized deletion-flank PCR product in mCMV-Δm06W. The PCR product was gel-purified and used for Sanger sequencing. In total, four primers were used. Black arrows indicate their map positions. Sequences (Seq 1–4, blue lines) were aligned with the mCMV genome and the matching regions are displayed by the boxes. The gray linker between the second and third box represents a short (11 bp) unmatched region. The lower black bar represents the ISH probe used to identify the “large deletion” mutant mCMV-Δm06m145-158 (probe BAC-P). Numbers indicate nucleotide positions in the mCMV genome.
Figure 3
Figure 3
2C-ISH analysis of liver infection. Reanalysis of stored liver specimens from a previously performed experiment [lung virus titers shown in Holtappels et al. (2006), Figure 8B]. BALB/c mice were immunocompromised by γ-irradiation (6.5 Gy) and infected at one footpad. Liver tissue sections were taken on day 12 after infection (A,B) Virus spread in liver tissue. Infection was performed with 105 PFU of mCMV-Δm06W, now identified to represent a mixture of correct virus mCMV-Δm06 and a “large deletion” mutant mCMV-Δm06m145-158 that includes deletion of the m152 gene. (A) 2C-ISH performed with probe m152-P (red staining) specific for mCMV-Δm06 and probe BAC-P (black staining) specific for mCMV-Δm06m145-158. (Center panel) overview image showing foci of infection for both viruses in the mixture representing mCMV-Δm06W. (Left panel) red-stained mCMV-Δm06-infected cell resolved to greater detail. (Right panel) black-stained mCMV-Δm06m145-158-infected cell resolved to greater detail. (B) 2C-ISH performed with probe M55/gB (red staining) specific for both viruses and probe BAC-P (black staining) specific for mCMV-Δm06m145-158. (Center panel) overview image showing foci of infection for both viruses in the mixture representing mCMV-Δm06W. (Left panel) red-stained mCMV-Δm06-infected cell resolved to greater detail. (Right panel) red-black speckled cells infected with mCMV-Δm06m145-158, resolved to greater detail. Frames in the overview images indicate tissue section areas resolved to greater detail in the left and right images. Note the stained CMV-typical inclusion bodies in the nuclei of infected hepatocytes. Counterstaining was performed with hematoxylin. Bar markers: 50 μm. (C) vRAP expression-dependent control of liver tissue infection with the indicated viruses on day 12 after adoptive transfer of 104 antiviral CD8 T cells specific for the viral epitope M45-Dd. 2C-ISH was performed with probes M55/gB-P (red symbols) and BAC-P (black symbols). (no AT) no adoptive transfer. (AT) adoptive transfer. vRAPs actually expressed by the viruses as well as their proposed impact on antigen presentation (AP, arrows up or down) are indicated. Data represent counts of infected liver cells in representative 10 mm2 tissue section areas. The dashed line indicates the detection limit of the assay, which is one infected cell per selected counting area. Symbols represent mice tested individually. Median values are marked and connected for the groups “no AT” and “AT” to highlight the strength of antiviral control. For statistical analysis, data were log-transformed and P-values were calculated by using the two-sided unpaired t-test with Welch's correction of unequal variances. P < 0.05 indicates statistical significance of the difference between “no AT” and “AT” groups. Linked data are connected by dotted lines, which reveals a correlation between the numbers of cells infected with the correct mutant mCMV-Δm06 and the “large deletion” mutant mCMV-Δm06m145-158 after infection with mCMV-Δm06W.
Figure 4
Figure 4
2C-IHC analysis of liver infection. Reanalysis of stored liver specimens from a previously performed experiment [lung virus titers shown in Holtappels et al. (2006), Figure 8A]. C57BL/6 mice were immunocompromised by γ-irradiation (7.5 Gy) and infected at one footpad. Liver tissue sections were taken on day 12 after infection. (A) Virus spread in liver tissue. Infection was performed with 105 PFU of mCMV-Δm06W, now identified to represent a mixture of correct virus mCMV-Δm06 and a “large deletion” mutant mCMV-Δm06m145-158 that includes deletion of the m152 gene. 2C-IHC was performed to detect cytoplasmic m152 protein (red staining) specific for mCMV-Δm06 and intranuclear IE1 protein (black staining) expressed by both viruses. (Center panel) overview image showing foci of infection for both viruses in the mixture representing mCMV-Δm06W. (Left panel) detail image of cells infected with mCMV-Δm06 identified by red cytoplasmic staining of m152. (Right panel) Detail image of cells infected with mCMV-Δm06m145-158 characterized by absence of red cytoplasmic staining. Counterstaining was performed with hematoxylin. Bar markers: 50 μm. (B) vRAP expression-dependent control of liver tissue infection with the indicated viruses on day 12 after adoptive transfer of 105 antiviral CD8 T cells specific for the viral epitope M45-Db. 2C-IHC was performed to identify infected cells expressing m152 (red symbols) or lacking m152 (black symbols). (no AT) no adoptive transfer. (AT) adoptive transfer. Symbols represent mice tested individually. Data represent counts of infected liver cells in representative 50 mm2 tissue section areas. For further explanation, see the legend to Figure 3.
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