CD4⁺ T cells specific for a model latency‐associated antigen fail to control a gammaherpesvirus in vivo

Abstract

CD4+ T cells play a major role in containing herpesvirus infections. However, their cellular targets remain poorly defined. In vitro CD4+ T cells have been reported to kill B cells that harbor a latent gammaherpesvirus. We used the B cell‐tropic murine gammaherpesvirus‐68 (MHV‐68) to test whether this also occurred in vivo. MHV‐68 that expressed cytoplasmic ovalbumin (OVA) in tandem with its episome maintenance protein, ORF73, stimulated CD8+ T cells specific for the H2‐Kb‐restricted OVA epitope SIINFEKL and was rapidly eliminated from C57BL/6 (H2b) mice. However, the same virus failed to stimulate CD4+ T cells specific for the I‐Ad/I‐Ab‐restricted OVA323–339 epitope. We overcame any barrier to the MHC class II‐restricted presentation of an endogenous epitope by substituting OVA323–339 for the CLIP peptide of the invariant chain (ORF73‐IRES‐Ii‐OVA), again expressed in tandem with ORF73. This virus presented OVA323–339 but showed little or no latency deficit in either BALB/c (H2d) or C57BL/6 mice. Latent antigen‐specific CD4+ T cells therefore either failed to recognize key virus‐infected cell populations in vivo or lacked the effector functions required to control them.

Use of a Virus-Encoded Enzymatic Marker Reveals that a Stable Fraction of Memory B Cells Expresses Latency-Associated Nuclear Antigen throughout Chronic Gammaherpesvirus Infection

“…1A). Although such a fusion could interfere with the functions of mLANA during viral infection, previous work has demonstrated that the fusion of a heterologous protein epitope to the C terminus had no significant effect on latency establishment (7,52). Although the MHV68 mLANA protein has not been carefully mapped, the orf73 coding region is conserved among the gamma-2-herpesviruses KSHV, herpesvirus saimiri (HVS), and MHV68 (25), and the products of these genes share C-terminal homology with EBNA-1 (20,25).…”

“…The episomal tethering function of KSHV LANA is mediated by portions of both the N and C termini, which bind nucleosomes and viral terminal repeats, respectively (24,36,43). The central repeat regions of LANA and EBNA-1 are believed to be responsible for cis-acting cytotoxic T-cell evasion by inhibiting translation and proteosomal degradation (7,34,48,68), and fusion to the mLANA C terminus appears to prevent the proteosomal degradation of heterologous fusion proteins (7,52).…”

Activated Mouse B Cells Lack Expression of Granzyme B

“…injection 45 d later of 10 mg NP-KLH suspended in PBS. For viral infections, mice were infected intranasally with 10 4.5 PFU HKx31 (H3N2) influenza virus (21) or with 3 3 10 4 PFU murine g-herpesvirus-68-OVA (MHV-OVA) (22).…”

Interference with Dendritic Cell Populations Limits Early Antigen Presentation in Chronic γ-Herpesvirus-68 Infection

“…Virus stocks were grown and titered on BHK-21 cells. All of the introduced mutations were confirmed as correct by viral DNA sequencing (11).…”

“…The generation of MHV-68 engineered to express OVA from an intergenic expression cassette under a lytic cycle promoter has been described previously (11). This approach incorporates an ectopic copy of the 500 bp upstream of the MHV-68 M3 open reading frame (ORF) as a strong, ORF50-dependent lytic cycle promoter (12).…”