A Recombinant Rhesus Monkey Rhadinovirus Deleted of Glycoprotein L Establishes Persistent Infection of Rhesus Macaques and Elicits Conventional T Cell Responses

Abstract

A replication-competent, recombinant strain of rhesus monkey rhadinovirus (RRV) expressing the Gag protein of SIVmac239 was constructed in the context of a glycoprotein L (gL) deletion mutation. Deletion of gL detargets the virus from Eph family receptors. The ability of this gL-minus Gag recombinant RRV to infect, persist, and elicit immune responses was evaluated after intravenous inoculation of two Mamu-A*01+ RRV-naive rhesus monkeys. Both monkeys responded with an anti-RRV antibody response, and quantitation of RRV DNA in peripheral blood mononuclear cells (PBMC) by real-time PCR revealed levels similar to those in monkeys infected with recombinant gL+ RRV. Comparison of RRV DNA levels in sorted CD3+ versus CD20+ versus CD14+ PBMC subpopulations indicated infection of the CD20+ subpopulation by the gL-minus RRV. This contrasts with results obtained with transformed B cell lines in vitro, in which deletion of gL resulted in markedly reduced infectivity. Over a period of 20 weeks, Gag-specific CD8+ T cell responses were documented by major histocompatibility complex class I (MHC-I) tetramer staining. Vaccine-induced CD8+ T cell responses, which were predominantly directed against the Mamu-A*01-restricted Gag181-189CM9 epitope, could be inhibited by blockade of MHC-I presentation. Our results indicate that gL and the interaction with Eph family receptors are dispensable for the colonization of the B cell compartment following high-dose infection by the intravenous route, which suggests the existence of alternative receptors. Further, gL-minus RRV elicits cellular immune responses that are predominantly canonical in nature. IMPORTANCE Kaposi’s sarcoma-associated herpesvirus (KSHV) is associated with a substantial disease burden in sub-Saharan Africa, often in the context of human immunodeficiency virus (HIV) infection. The related rhesus monkey rhadinovirus (RRV) has shown potential as a vector to immunize monkeys with antigens from simian immunodeficiency virus (SIV), the macaque model for HIV. KSHV and RRV engage cellular receptors from the Eph family via the viral gH/gL glycoprotein complex. We have now generated a recombinant RRV that expresses the SIV Gag antigen and does not express gL. This recombinant RRV was infectious by the intravenous route, established persistent infection in the B cell compartment, and elicited strong immune responses to the SIV Gag antigen. These results argue against a role for gL and Eph family receptors in B cell infection by RRV in vivo and have implications for the development of a live-attenuated KSHV vaccine or vaccine vector.

Plxdc family members are novel receptors for the rhesus monkey rhadinovirus (RRV)

“…To ultimately address the correlation between receptor- and tissue-tropism, in vivo studies using receptor-de-targeted mutants to analyze cell and tissue tropisms will be required. The importance of in vivo studies is also supported by a recent report that showed that a gL-null RRV mutant still established persistent infection in the B cell compartment upon intravenous inoculation, while infection of B cells in vitro was drastically reduced [ 24 ], a finding that could be explained by the gL-independent usage of Plxdc1/2 for B cell infection in vivo . At a minimum, our data on RRV-YFP ΔgL infection of Plxdc1 and Plxdc2 overexpressing Raji cells ( Fig 6B ) confirms that RRV can efficiently use Plxdc1 and Plxdc2 as entry receptors in the absence of gL.…”

“…While for KSHV, in addition to Eph family receptors, several membrane proteins have been proposed as cellular receptors for different viral glycoproteins mediating either attachment or entry on a range of target cells (reviewed in [ 21 ]) the receptor usage of RRV is comparatively less well characterized. Nevertheless, studies using receptor knock-down or knockout [ 14 , 22 ], receptor- and ligand-mediated blocking [ 15 , 23 ], and Eph de-targeted virus mutants [ 23 , 24 ] showed that both viruses and in particular RRV can infect various cells partially or completely independently of the Eph-interaction, which suggests that RRV engages at least one additional entry receptor that can functionally substitute for the Eph-interaction. This notion is also supported by a recent in vivo study that demonstrated that an RRV mutant deleted of gL and therefore unable to interact with Eph receptors still establishes persistent infection in RRV-naïve rhesus macaques upon intravenous inoculation [ 24 ].…”

Plxdc family members are novel receptors for the rhesus monkey rhadinovirus (RRV)

“…To ultimately address the correlation between receptor-and tissuetropism, in vivo studies using receptor-de-targeted mutants to analyze cell and tissue tropisms will be required. The importance of in vivo studies is also supported by a recent report that showed that a gLnull RRV mutant still established persistent infection in the B cell compartment upon intravenous inoculation, while infection of B cells in vitro was drastically reduced (24), a finding that could be explained by the gL-independent usage of Plxdc1/2 for B cell infection in vivo. At a minimum, our data on RRV-YFP ΔgL infection of Plxdc1 and Plxdc2 overexpressing Raji cells (Fig 6) confirms that RRV can efficiently use Plxdc1 and Plxdc2 as entry receptors in the absence of gL.…”

“…While for KSHV, in addition to Eph family receptors, several membrane proteins have been proposed as cellular receptors for different viral glycoproteins mediating either attachment or entry on a range of target cells (reviewed in (21)) the receptor usage of RRV is comparatively less well characterized. Nevertheless, studies using receptor knock-down or knockout (14,22), receptor-and ligand-mediated blocking (15,23), and Eph de-targeted virus mutants (23,24) showed that both viruses and in particular RRV can infect various cells partially or completely independently of the Eph-4 interaction, which suggests that RRV engages at least one additional entry receptor that can functionally substitute for the Eph-interaction. This notion is also supported by a recent in vivo study that demonstrated that an RRV mutant deleted of gL and therefore unable to interact with Eph receptors still establishes persistent infection in RRV-naïve rhesus macaques upon intravenous inoculation (24).…”

“…Nevertheless, studies using receptor knock-down or knockout (14,22), receptor-and ligand-mediated blocking (15,23), and Eph de-targeted virus mutants (23,24) showed that both viruses and in particular RRV can infect various cells partially or completely independently of the Eph-4 interaction, which suggests that RRV engages at least one additional entry receptor that can functionally substitute for the Eph-interaction. This notion is also supported by a recent in vivo study that demonstrated that an RRV mutant deleted of gL and therefore unable to interact with Eph receptors still establishes persistent infection in RRV-naïve rhesus macaques upon intravenous inoculation (24). We therefore aimed to identify additional rhadinovirus receptors that bind the gH/gL complex or gH and identified Plexin domain containing protein 2 (Plxdc2) as novel interaction partner of the gH/gL complex of RRV, but not KSHV.…”