The Murine Gammaherpesvirus 68 v-Cyclin Is a Critical Regulator of Reactivation from Latency

Abstract

Murine gammaherpesvirus 68 (␥HV68; also referred to as MHV68) infection of mice is a developing small-animal model for analysis of gammaherpesvirus pathogenesis (reviewed in references 18, 19, 45, 46, 64, and 69). ␥HV68 is a gamma-2 herpesvirus which is closely related to the human and primate gammaherpesviruses Epstein-Barr virus (EBV), Kaposi's sarcoma-associated herpesvirus (KSHV), and herpesvirus saimiri (HVS) (22,67). Acute ␥HV68 infection of laboratory mice is cleared by 2 to 3 weeks postinfection, with the concomitant establishment of a presumably life-long latent infection. Notably, several ␥HV68 genes that are also present in HVS, KSHV, and/or EBV have been identified as candidate latent genes by transcriptional analysis of latently infected mice (68). These genes include those encoding the viral bcl-2 (v-bcl2) homolog and the viral G-protein-coupled receptor (v-GPCR) homolog as well as gene 73, which in KSHV encodes the latency-associated nuclear antigen (LANA) (11,35,50). In addition, KSHV, HVS, and ␥HV68 have in common a complement-regulatory protein homolog (1, 2, 26, 33, 67), and in both HVS and ␥HV68 this protein occurs in membrane-bound and soluble isoforms (26,33). These studies argue strongly that ␥HV68 shares mechanisms of pathogenesis with other gammaherpesviruses.Manipulation of the host cell cycle is a shared feature of gammaherpesviruses, and one focus for this regulation is the D-type cyclins. EBV infection (or expression of the EBV latency-associated membrane protein 1) upregulates expression of host cyclin D2 (3, 9, 63). In contrast, the gamma-2 herpesviruses HVS (32, 47), KSHV (2, 47, 52), and ␥HV68 (67) all contain open reading frames (ORFs) predicted to encode homologs of mammalian D-type cyclins. The viral cyclins (v-cyclins) exhibit 25 to 31% identity to mammalian D-type cyclins and 26 to 32% identity to each other, and they are positionally conserved in the gamma-2 herpesvirus genomes (67). Notably, the highest level of sequence conservation among these homologs is within the cyclin box, a domain demonstrated to be essential for cyclin-dependent kinase (cdk) binding (29,31,37,38,43,51).The biochemistry of the KSHV and HVS proteins has been intensively studied. The KSHV and HVS v-cyclins, in conjunction with cdk's, have been demonstrated to phosphorylate the retinoblastoma protein (pRb) and promote cell cycle progression, like their mammalian homologs (2, 8). The KSHV and HVS v-cyclins predominantly bind cdk6. Overexpression of the KSHV v-cyclin in cells expressing high levels of cdk6 results in apoptosis (48), suggesting that there may be other viral genes responsible for preventing apoptosis (e.g., v-bcl2). Several unusual properties of the v-cyclins have also been noted. The v-cyclin ORFs lack an LXCXE motif present in mammalian cyclin D proteins and thought to be important in direct binding to pRb (20,25). The HVS and KSHV v-cyclins are able to bind multiple cdk's, rather than being restricted to binding cdk4 and cdk6 (27,32,39), and have a broadened substrate range, being capa...

Latent Herpesvirus Infection Augments Experimental Pulmonary Fibrosis

“…In our model, we found that FITC reactivated transcription of lytic viral RNA in the lungs (although it was to a level two orders of magnitude below peak viral gene expression 5 days after the primary infection [ Figure 5 and data not shown]). To further investigate this question, we infected mice with a mutant strain of gHV-68 (DORF72) with a 100-fold reduced ability to reactivate (15). There is no evidence that infection with DORF72 results in lytic or persistent infection during long-term latency (33).…”

“…As a result of this mutation, DORF72 has a greatly diminished ability to reactivate from latency (approximately 100-fold reduction) (15). Mice were inoculated intranasally with 5 3 10 4 PFU of DORF72, 5 3 10 4 PFU of a v-cyclin marker-rescue virus (''MR,'' essentially a wild-type control), or were mock infected with saline on Day 0.…”

“…Mice were anesthetized and 5 3 10 4 plaque forming units (PFU) of gHV-68 clone WUMS (American Type Culture Collection, Manassas, VA), DORF72 (a v-cyclin mutant virus described previously (15)), or v-cyclin marker rescue virus (''MR'') generated by recombination of wild-type gHV-68 sequence, including the v-cyclin DNA, with a LacZ cassette-containing virus, thus reconstituting an essentially wild-type virus control (15), were suspended in 20 ml saline and delivered intranasally to each mouse. Mock infection was 20 ml of saline.…”

Infection of neonates with murine gammaherpesvirus 68 results in enhanced viral persistence in lungs and absence of infectious mononucleosis syndrome

“…MEFs were seeded in 200 ml volumes in 96-well plates at a concentration of 5610 4 cells ml 21 in DMEM containing 2.5 % FBS (Invitrogen), L-glutamine, penicillin and streptomycin. Lung cells were resuspended at a concentration of 10 6 cells ml 21 , and LDA was performed as described elsewhere (van Dyk et al, 2000;Weck et al, 1996) with the exception that threefold serial dilutions were made. To determine the levels of preformed lytic virus, a duplicate aliquot of cells was subjected to three rounds of freeze-thawing and lysates were plated in 100 ml volumes onto MEF monolayers.…”

Specific Mutation of a Gammaherpesvirus-Expressed Antigen in Response to CD8 T Cell SelectionIn Vivo

“…This insertion removes most of ORF 72, leaving behind only 281 nucleotides at its 3= end, but does not alter ORFs upstream of ORF 72. ␥HV68.OVA replicates with kinetics similar to those of wild-type ␥HV68 in vitro (4), and disruption of ORF 72 via gene insertion does not impair either viral acute replication or establishment of latent infection in vivo (24). (A) OTI-RAG and RAG mice were infected intraperitoneally (i.p.)…”