Cent. Eur. J. Biol. • 3(1) • 2008 • 19-30 DOI: 10.2478/s11535-008-0002-0

Central European Journal of Biology

Comparison of pathogenic properties of the murid gammaherpesvirus (MuHV 4) strains: a role for immunomodulatory proteins encoded by the left (5’-)end of the genome

Review Article Jela Mistríková1,2, Július Rajčáni2*

1 Department of Microbiology and Virology, Faculty of Microbiology and Natural Sciences, Comenius University, 84215 Bratislava, Slovakia

2 Institute of Virology, Slovak Academy of Sciences, 84505 Bratislava, Slovakia

Received 13 September 2007; Accepted 4 January 2008

Abstract: The murid herpesvirus 4 (MuHV 4) species encompasses 7 isolates, out of which at least two (MHV-68, MHV-72) became in vitro propagated laboratory strains. Following intranasal inoculation, MuHV 4 induces an acute -like syndrome with elevated levels of peripheral blood leukocytes, shifts in the relative proportion of lymphocytes along with the appearance of atypi- cal mononuclear cells. At least two isolates exhibited spontaneous deletions at the left hand (5´-end) of their genome, resulting in the absence of M1, M2, M3 genes (strain MHV-72) and also of the M4 gene (strain MHV-76). Based on DNA sequence amplifications only, another two isolates (MHV-Šum and MHV-60) were shown to possess similar deletions of varying length. During latency (until 24 months post-infection), the mice infected with any MuHV 4 isolate (except MHV-76) developed lymphoproliferative disorders. The lack of tumor formation in MHV-76 infected mice was associated with persistent production at late post-infection intervals. In addition to careful analysis of spontaneously occurring 5´-end genome defects, our knowledge of the function of 5´-end genes relies on the behaviour of mutants with corresponding deletions and/or insertions. While M2 and M3 genes encode immune evasion proteins, M4 codes for a soluble glycopeptide acting as immunomodulator and/or immunostimulator. Keywords: Murid gammaherpesvirus 4 • Genome deletions • Immune evasion genes • Pathogenesis • Tumor formation

© Versita Warsaw and Springer-Verlag Berlin Heidelberg.

Abbreviations 1. Introduction

HSV - The history of murine herpesvirus goes back to 1980, i.n. - intranasal when several herpesvirus isolates (designated MHV-60; i.p. - intraperitoneal MHV-68; MHV-72; MHV-76; MHV-78, MHV-ŠUM) were p.i. - post-infection obtained from free-living Clethrionomys and Apodemus MHV - mouse herpesvirus (murine gammaherpesvirus) rodents captured in different regions of the former MuHV - murid herpesvirus (official nomenclature) Czechoslovakia [1,2]. All the MHV isolates grew well PCR - polymerase chain reaction in cell cultures of mouse, chick, rabbit, hamster, mink, bp - base pair swine, monkey or human origin (especially primary or kb - kilobase pair diploid embryonic cells), indicating that the new herpes nt(s) - nucleotide(s) virus was not a [3]. Later on Efstathiou w.t. - wild-type et al. [4] showed that the genome of MHV-68 had at least

* E-mail: [email protected] 19 Comparison of pathogenic properties of the murid gammaherpesvirus (MuHV 4) strains: a role for immunomodulatory proteins encoded by the left (5’-)end of the genome

16 ORFs, which are homologous to Epstein Barr virus epithelial cells [13]. Using the latter model, the IgM (EBV) and to Herpesvirus saimiri sequences. Based deficient CD21-positive lymphocytes became latent on these data, the murine herpesvirus strain 68 (MHV- virus carriers at a lower frequency, since macrophages 68) was classified into genus , subfamily and NK cells formed the main cell population, in which . All the above-mentioned isolates, the viral DNA survived. Obviously, in B cell-deficient which are serologically highly related [5], were classified mice, the B lymphocytes cannot account for latency [16]. as a single species of Murid herpesvirus 4 (MuHV 4) [6]. When adherent mononuclear cells (AMC) from infected Balb/c mice were T-cell depleted and enriched by means of the anti-macrophage F4/80 monoclonal antibody, this 2. Pathogenesis of the prototype strain AMC population was shown to contain MHV-68 DNA at MHV-68 in laboratory mice late p.i. intervals [17]. Thus, both lymphocytes as well as macrophages may represent the reservoir of latent Pilot studies with the MHV-68 strain showed that, in virus especially in adult animals, which did not undergo response to intranasal (i.n.) inoculation into outbred acute disease. mice, this virus spreads to the lungs and then via the The B lymphocytes producing MHV-68 or expressing bloodstream it reaches several internal organs such as MHV-68 antigen(s), may be cleared from blood, lymph the spleen, liver, and kidneys [7]. The survivors of acute nodes, spleen and lung alveoli by cytotoxic T/CD8 infection, especially adult and juvenile mice, developed cells. As a result of virus replication in the lungs, the T/ latent (and/or persistent) infection in the same organs. CD8 cells accumulate in the mediastinal lymph nodes In contrast to herpes simplex (HSV 1 and 2), [18,19]. Mice depleted of T/CD8 cells failed to resolve the absence of neural spread was a striking feature [8]. the pulmonary disease, developed a long lasting viremia Though the MuHV 4 isolates are not strictly lymphotropic, and died; this confirms that T/CD8 lymphocytes are based on their genomic structure, the new isolates were essential for virus clearance. However, the cooperation classified as members of the Gammaherpesvirinae of CD8 lymphocytes with CD4 T cells is still required subfamily [4]. Using a combination of serology and for long-term protection [20,21]. MHV-68 infection may PCR analysis, Blasdell et al. [9] found that MuHV 4 also be associated with massive proliferation of those was endemic in wood mice (Apodemus sylvaticus) but lymphocytes that are not specific for this virus. The not in two species of voles (Clethrionomys glareolus, level of “bystander-induced” cycling in a population of Microtus agrestis). More recently, a longitudinal study of influenza virus-specific T/CD8+ cells was fourfold lower MuHV 4 antibody in a mixed ensemble of bank voles (C. than was the extent of cell division driven by the specific glareolus) and wood mice (A. sylvaticus) showed that the MHV-68 antigen [22]. latter represent the more frequent reservoir, even when Following i.n. MHV-68 inoculation, viremia occurs the virus was originally isolated from the former [10]. due to virus replication in the alveolar epithelium and Studies in Balb/c mice using MHV-68 demonstrated endothelial cells of alveolar septa at acute p.i. intervals. that the lung tissue becomes primarily involved following The productive virus growth within lung epithelium is i.n. inoculation [11]. Splenomegaly occurred in the resolved at 7 to 10 days p.i. During the viremic phase, course of acute infection as well as at late post-infection the mature B cells as well as macrophages become (p.i.) intervals among healthy survivors, in which B cells infected. In the course of primary infection, the T/CD8 were predominant virus carriers [12]. Depletion of T/CD4 cells (in cooperation with T/CD4 cells) proliferate to lymphocytes prevented splenomegaly, but had no effect participate in the clearance of infected B lymphocytes on B cell-associated latency [13]. Persistent infection expressing MuHV 4 antigens. The kinetics of the T/ with MHV-68 was established in mouse myeloma B CD8 cell response may be different when tested cells (NSO cells) but not in thymoma BW5147 cells [14]. against various proteins expressed during the lytic cycle Marques et al. [15] described the frequency of MHV- or those associated with latency. Thus, the cellular 68 infection in splenic B lymphocytes, in dendritic cells immune response shows a complex pattern reflecting and macrophages at acute as well as latent stages p.i. the distinct expression of virus-coded polypeptides At early intervals, B cells within the marginal zone of in antigen presenting cells (APC) and B lymphocytes germinal centres accounted for nearly the half of the [23]. The acute infectious mononucleosis (IM) like total number of infected splenocytes. syndrome shows splenomegaly, an increased number When the µMT transgenic mice (which do not of proliferating B cells and atypical mononuclear cells. produce heavy µ-chains, and therefore, do not have Thus, it represents an animal model for EBV infection mature B cells) were used to establish latency, MHV- [24]. 68 DNA persisted in the lungs, namely in pulmonary

20 J. Mistríková, J. Rajčáni

3. Comparison of biological properties had developed in a MHV-78 infected Balb/c mouse; this cell line expressed MHV antigen in a small proportion of of other MuHV 4 strains cells at passages 43-45 [34]. Usherwood et al. (1996)

reported that the S11 B-lymphoma derived cell line The role for macrophages in the spread of MuHV 4 was contains MHV-68 genomes, either linear or episomal [35]. first postulated by Mistrikováet al. [25], who claimed that Tarakanova et al. (2005) found abundant positive cells adherent lung and/or peripheral blood mononuclear cells by in situ hybridization with probes specific for vtRNA in (mainly macrophages) participate in virus dissemination the samples from lymphoproliferative lesions of MHV-68 during acute infection with the MHV-72 strain. Plaque infected Balb/c mice deficient for the b2- microglobulin assay combined with nested PCR confirmed the subunit of MHC [36]. On the other hand, the frequency of hematogenic spread of MHV-72 to the mammary glands vtRNA positive cells was low in lymphomas of the same of female nu/nu Balb/c mice and its transmission to the origin and scarce in the spleen of infected animals. offspring via breast milk [26,27]. In Balb/c mice infected When athymic nude mice (BALB/c) were injected with MHV-72, the number of T/CD8 cells reached subcutaneously with MHV-72, the animals died 1 maximum by day 11 p.i., and remained elevated till month p.i. showing a high frequency (48%) of atypical day 30 p.i.. This correlated with increased levels of T/ lymphoblast-like B cells in the blood, but showing no CD4 cells and elevated numbers of activated B/CD19 increase in the number of natural killer (NK) cells. The lymphocytes and NK cells [28]. The atypical leukocytes, use of UV-irradiated virus increased mouse survival which could be found in the mouse IM-like syndrome time (by up to 3 months p.i.). The atypical lymphocytes following acute MHV-72 infection, were classified as in the peripheral blood were similar in shape to those lymphoblasts. The population of peritoneal, alveolar seen following infection with the non-irradiated virus. and bone marrow cells occasionally yielded virus for However, nude mice did not develop splenomegaly, up to eight months p.i., indicating that mononuclear probably due to the lack of the T cell subset. These phagocytes could harbour the latent MHV-72 for a findings suggest that NK cells do not play an important considerable period of time and that they occasionally role in immune defences against MuHV 4 infection in produce infectious virus. Some mice with latent MHV- nude mice [37]. 72 infection showed signs resembling a leukemia-like BALB/c mice inoculated by i.n. route with the isolate syndrome with splenomegaly and lymphadenopathy, Šumava (MHV-ŠUM) showed increased number of with an extremely high number of peripheral blood leukocytes and appearance of atypical peripheral blood leukocytes and an increased proportion of immature cells during acute infection. The infiltration of spleen with blastic leukocytes [29]. atypical cells resulted in splenomegaly. In the course of Lymphoproliferative disease and lymphomas latent infection predominantly non-infectious virus (viral were first described in MHV-68 infected mice 30[ ]. We DNA) persisted in lungs, spleen, thymus, bone marrow, reported tumors histologically verified as lymphomas, mammary glands, peritoneal macrophages and liver. A non-differentiated lymphoblastomas and/or sarcomas in leukemia-like syndrome associated with strain MHV- 10 out of 100 Balb/c mice which had been inoculated ŠUM developed at remote intervals (day 350 to 810 i.n. with MHV-72 and then kept for over 2 years p.i.) in Balb/c mice, showing leukocytosis ranging from [31]. The lymphoid cells present in lymphomas or 8x104 to 5x105 cells/ml and as many as 60% atypical lymphoblastomas expressed the pan-T/CD45 marker lymphocytes in the total peripheral blood cell count [38]. [32]. In addition to lymphomas, neoplasms such as In Balb/c mice infected i.n. with MHV-78 strain skin tumors (squamous cell carcinoma, hemangioma), the distribution of infectious virus was followed for up were also seen in long term observed animals, including to 180 days. High virus levels (over 100 TCID /ml) controls. Statistical analysis confirming the relevance of 50 were reached in lungs on days 9-28 p.i. and in blood the association between MHV-72 and various tumors leukocytes on day 7. Similar virus levels were detected in CB17scid/scid mice was reported by Oda et al. [33]. in the spleen at remote intervals (days 60-180 p.i.) by The latter authors, however, did not find viral DNA in the means of a co-cultivation technique. The persisting tumors of MHV-72 infected mice. In our hands, infectious virus was detected in bone marrow, thymus, peritoneal virus was recovered by explantation (or co-cultivation) macrophages and mammary glands [39]. Elevated from nearly the half of lymphoma and sarcoma tissues. numbers of blood leukocytes and atypical mononuclear One sarcoma was used to establish a cell fibroblast cell cells in blood smears were seen on day 15 p.i. (acute line, which yielded scattered IF-positive MHV plaques at IM-like syndrome) and on day 122 p.i. (late leukemia- the 42nd passage (not published). Later on, we referred like disease). Approximately 7% of animals inoculated to the cell line NB-78, derived from a lymphoma, which with the MHV-78 strain developed solid tumors from

21 Comparison of pathogenic properties of the murid gammaherpesvirus (MuHV 4) strains: a role for immunomodulatory proteins encoded by the left (5’-)end of the genome

day 240 p.i. A tumor cell line, NB-78 derived from a were accompanied by atypical leukocytes (ranging from MHV-78 induced lymphoma, has undergone more than 3 to 13%) at early as well as late (by 5 and 15 months) 100 passages in culture. The NB-78 cells displayed intervals p.i. Despite this, the MHV-76 infected Balb/c an epitheloid cell morphology and a diploid set of 40 mice developed neither splenomegaly nor showed solid chromosomes. MuHV 4-specific antigen was detected tumors. by immunofluorescence in about 2% of monolayer cells. Reactivation of latent MHV-78 was proven by detecting the infectious virus in the culture medium at passages 4. The MuHV 4 genome and spontanous 43–45 only. Summing up, NB-78 is a continuous cell deletions at its 5´-end line derived from a tumor which developed in the MHV- 78 infected host; it harbors the latent viral genome and The MHV-68 DNA has sequences homologous to EBV rarely produces infectious virus upon spontaneous [4], KHSV and HVS viruses [43]. The MHV-68 genome reactivation [34]. (GenBank Accession number: U 97553) is a linear Following infection with the MHV-60 strain, the lungs double stranded DNA molecule, which contains 118,823 again represented the main portal of entry [40]. The bp flanked by multiple copies of terminal repeats with pathological features of the acute phase were similar a total length of 1,213 bp [44]. In addition, the genome to those elicited by other MuHV 4 isolates, including has two internal repeats. At the left hand side, there the prototype MHV-68 virus. Nevertheless, the MHV- is a series of 40 bp repeats (each contains a BamHI 60 strain differed from others as follows: (A) The acute restriction site called a Bam repeat), located between phase showed fast dynamics peaking on days 3–5 nts 26,778–28,191. The second series of several 100 bp p.i., never exceeding a total of 10 days (in contrast repeats is located between nts 98,981–101,170. The left- to the 14–28 day duration of acute viremic phase in end sequence of MHV-68 starts with four virus specific the above mentioned isolates). At given intervals, an ORFs (M1, M2, M3 and M4). The given region also increased number of leukocytes in peripheral blood had encodes a family of eight tRNA-like molecules, sharing been observed along with the appearance of atypical secondary loop structures, and promoter elements leukocytes. In addition to lungs, infectious virus could for RNA polymerase III. The tRNA-like sequences are be recovered from spleen, thymus and kidneys, but not transcribed at high levels during lytic infection as well as from other organs. Acute splenomegaly was prominent during latency and are processed into mature vtRNAs in contrast to infection with the MHV-78 strain. (B) [45,46]. The expression of murine herpesvirus tRNAs Massive splenomegaly was characteristic of the chronic is driven by the cellular RNA polymerase III, which is leukemia-like disease. Despite the quick clearance of active in any mammalian cell [47]. Though their exact infectious virus, MHV-60 latency was associated with a function is still unknown, some workers claim that they high probability of tumor formation (as high as 22%) even might represent analogues to EB early RNA (EBER) when considering the 5% frequency of spontaneous molecules encoded by EBV [48]. Recently, it was shown tumors that appeared in non-infected controls kept for that the small non-coding tRNAs transcribed from the the same observation period. viral genome reveal a segment with silencer function The frequency of tumors and other important data identified as microRNA (miRNA) 49[ ]. This may point at concerning the Balb/c mice infected with various MuHV the possible role of MHV-68 tRNA, when expressed in 4 strains (or isolates) are shown on Table 1. From spleen germinal centres during latent infection. In any this Table it is that clear that the highest frequency of case, the presence of tRNA is regarded as a hallmark of tumor formation was observed with MHV-60, while no latent infection [45,47]. tumors developed in MHV-76 infected animals. After i.n. Although the complete sequence of the MHV-68 inoculation of MHV-76, the infectious virus spread to genome has been determined, our knowledge of the lungs, where it replicated to relatively high titers until day function of individual genes is still limited. Out of a 7 p.i. [41]. This agrees with the results of Macrae et al. total of 82 genes, only 48 encode proteins with clearly [42], who describe the rapid clearance of MHV-76 within identified function. The MHV ORFs fall into 3 categories: lungs. Via blood macrophages small amounts of MHV- (A) genes common to the gammaherpesvirus subfamily, 76 virus could be disseminated to many organs, since (B) genes common to the herpesvirus family and (C) the virus reappeared at remote intervals (from 1 to 27 unique murine herpesvirus genes (designated M1-M9, months p.i.), and unexpectedly persisted in the spleen, M10a,b,c, M11-M14). From the latter, we attempted to liver, bone marrow, lymph nodes, kidneys, thymus as well define the function of M1- to M4-encoded polypeptides as in the small intestine, spinal cord and brain. Slightly based on a survey of the pathogenic behaviour of elevated numbers of leukocytes in peripheral blood spontaneous and artificial mutants lacking various

22 J. Mistríková, J. Rajčáni

Mouse species Clethrionomys Clethrionomys Apodemus flavicollis Apodemus flavicollis Apodemus flavicollis glareolus glareolus Isolate (strain) MHV-60 MHV-72 MHV-76 MHV-78 MHV-Šumava Tumor formation 22% 9% none 7% 14.6% Acute IM-like syn- 30 000/ml26% atypi- 6 300/ ml10% atypical 9 000/ ml9% atypical 15 000/ ml13% 15 000/ ml15% drome cal atypical atypical Leukemia-like syn- 20 000/ ml12% atypi- 118 000/ ml48% atypi- 16 800/ ml13% 15 000/ ml9% atypical 500 000/ ml80% atypi- drome (late) cal PBC cal PBC atypical PBC(no PBC cal PBC splenomagaly) Splenomegaly* 60x106 (yes) 52 x106 (yes) 20 x106 (no) 60 x106 (yes) 50 x106 (yes) Virus growth at 2–24 d.p.i. 7–240 d.p.i. 1–870 d.p.i. 2–180 d.p.i. 2–180 d.p.i. intervals Brain Negat. Negat. 210, 450, 810 d.p.i. Negat. Negat. Lungs 2-7 d.p.i. 2-28 d.p.i. 1-7 d.p.i, then at 150, 2-60 d.p.i. 2-14 d.p.i. 270, 330 d.p.i. Thymus 5 d.p.i. 2-90 d.p.i. 210 and 720 d.p.i. 3-180 d.p.i. 3-90 d.p.i. Spleen 3-10 d.p.i. 7-28 d.p.i. 210–720 d.p.i. 15-60 d.p.i. 10-180 d.p.i. Peritoneal mac- Negat. 7-240 d.p.i. 210 d.p.i. 28-60 d.p.i. 7-180 d.p.i. rophages Bone marrow Negat. 7-90 d.p.i. 150, 270, 330 and 660 15-120 d.p.i. 7-180 d.p.i. d.p.i. Lymph nodes Negat. 10-28 d.p.i. 150, 390 and 450 15-60 d.p.i. ND d.p.i. Kidneys 18-24 d.p.i. Negat. 150, 270, 330, 390, 7-15 d.p.i. Negat. 450, 720 and 810 d.p.i. Liver Negat. Negat. 150–810 d.p.i. Negat. 180 d.p.i. VNT antibodies 1:512 1:1024 1:64 1:12-256 1:256 – 1:512

Table 1. Comparison of the basic biological properties of MuHV 4 strains. * expressed as the number of splenocytes per organ (during late leukemia-like syndrome); ND – not done; PBC = peripheral blood cells proportions of the 5´-end of the MuHV 4 genome. Microbiology and Virology, Faculty of Natural Science of Analysis of MHV-76 DNA showed a spontaneous CU in Bratislava, was studied in several laboratories. As 9,538 bp deletion at the 5´-end resulting in the loss of M1, described by Oda et al. [33] an approximately 7000 bp M2, M3 and M4 genes as well as the ORFs from which deletion was found at the 5´-end of the MHV-72 genome the tRNAs are ordinarily transcribed [42]. The deletion as confirmed by extensive PCR amplification studies in question was confirmed by restriction endonuclease (including 15 ORFs), Southern blot analysis and direct cleavage, by PCR amplification of the genes in question sequencing. The deletion in question encompassed and, finally, by Southern blot hybridization using the vtRNA sequences and the sequences encoding radiolabeled probes derived from MHV-68 and MHV- the M1, M2 and M3 genes. Biological studies in an 76 DNAs. Repeated and independent studies on the independent laboratory confirmed the ability of MHV- pathogenesis of MHV-76 showed that despite quick 72 to form tumors of histological appearance similar clearance from lungs it established latency, though at to that previously described [31]. Studies using SCID a lower rate. Quite surprisingly, MHV-76 reactivated mice versus immunocompetent CB17 w.t. mice showed and replicated to high titres at remote intervals up to 27 that the rate of tumor formation under conditions of months p.i., fulfilling the criterion of persistent infection, immunosuppression increased three-fold. As already characterized by continuous or interrupted production described, FK-506 treatment increased the rate of of infectious virus [7]. In contrast, “true” non-productive tumor formation two-fold [32]. In contrast to the highly latency means long term presence of MHV DNA with passaged MHV-72 virus, its low passaged clone h.3.7 very limited transcription of latency associated genes, from the same isolate was shown to possess the M1- not resulting in virus replication. M4 gene block as well as the vtRNA ORFs. The latter Another 5´-end spontaneous deletion, though less finding was demonstrated by restriction endonuclease extensive, was later described with MHV-72. This analysis, PCR amplification and by sequencing of virus, originally isolated in the laboratory of the Chair of the MHV-72 M3 gene, present in the h.3.7 clone

23 Comparison of pathogenic properties of the murid gammaherpesvirus (MuHV 4) strains: a role for immunomodulatory proteins encoded by the left (5’-)end of the genome

Virus strain The gene sequence present Frequency References M1 tRNA M2 M3 M4 of tumors MHV-68 yes yes yes yes yes 11% [44], [75] MHV-72 no no no no yes 10% [33], [31] MHV-76 no no no no no None [42], [41] MHV-78* yes yes yes yes yes 7% [39]*, [39] MHV-60* no ND No no yes 22% [40]* MHV-Šum* no no No no ? 14.6% [52]*, [76] Table 2. Survey of MuHV 4 strains showing deletions at the 5´-end of their genome. * = deletion detected by PCR only (the presence of the gene in question is convincing when detected by PCR; its absence as found by PCR may be questionable, if not analyzed by alternative techniques). ND = not done Notice: the M1-M3 genes were absent in the MHV-Sum and MHV-60 strains; preliminary amplifications of the 5‘-end of the MHV-60 genome showed the presence of the M4 gene. ? = pilot results showed a partial deletion of the M4 gene. derived from the original MHV-72 isolate [50]. It is unclear 5. Characterisation of unique (MuHV whether similar 5´-end deletions may occur in any other frequently passaged MuHV 4 strain. Clambey et al. 4-specific) genes located within (2002) described a spontaneous 9.5 kb deletion within the 5´-end of the genome the MHV-68 genome accompanied by the loss of M1, The M1/ORF1 is claimed to display homology to the M2, M3 and M4 genes and eight tRNA-like genes [51]. poxvirus serin protease inhibitor serpin, SPI-2 [46]. Studies following i.n. inoculation of this spontaneous Nevertheless, the presence of M1 protein was not MHV-68 mutant revealed a defect in splenic latency at shown to hamper the development of cytotoxic and late stages p.i. No such defect was observed following NK cell responses nor to interfere with the induction of intraperitoneal inoculation. The authors concluded that apoptosis, functions attributed to the family of serpin the establishment of latency may depend upon the route poteins [53]. The possible function of MuHV 4 M1 of infection. In the case of both aforementioned strains was investigated by inserting a LacZ casette into M1 there is strong evidence that spontaneous deletions had ORF in order to create a recombinant M1.LacZ virus. occurred during repeated passages in culture especially The M1.LacZ recombinant exhibited decreased virus under conditions of high multiplicity of infection titres in the spleen of immunocompetent as well as (Medveczky, P., personal communication). immunodeficient mice during acute stages p.i. Despite To summarize: in the case of MHV-68 and MHV-72 this, it established latent infection comparable to that of strains there is strong evidence that the original isolates w.t. virus, indicating that the establishment of latency was had possessed a complete genome and that the deletion not determined by the extent of acute virus replication. appeared during in vitro passaging. No such evidence Finally, the M1 deficient virus showed an increased rate exists in the case of MHV-76 and MHV-Šum viruses. of reactivation [54]. Recently, amplification of the MHV-Šum DNA using Initially, based on its genomic position, the MHV- primers for the M1, M2, M3 and M4 genes showed the 68 M2 was identified as a latency candidate gene that it lacks approximately a 9.3 kb genomic region from [55]. Expression of M2 protein has been detected in the left end of the viral genome [52]. The PCR analyses splenocytes during latency and in peritoneal exudate performed with the MHV-Šum isolate revealed the cells (PECs) without detectable lytic-gene expression absence of M1, M2 and M3 sequences, while the ORF4 [56]. The M2 transcripts have been found in the sequences were in part amplified. When mapping more spleen and lungs during the first month p.i. The latter precisely the 3´-end of the deletion within the M4 ORF of study raised the question of whether M2 may be the MHV-Šum genome, PCR amplification of ORF4-M4 expressed during productive virus replication as well region suggested that the deletion extended beyond the as during latency. During latency, M2 expression was M3 ORF, thus encompassing a portion of the M4 gene. shown to be restricted to spleen [57]. In addition, M2 Since the latter result was not confirmed by Southern blot was expressed in latently infected murine B cells of or direct sequencing, the precise length of the MHV-Šum lymphoma line S11, which was derived from a tumor 5´-end deletion must still be determined. Most unclear found in a persistently infected mouse. Liang et al. [58] is the deletion within the MHV-60 genome (Table 2). described the cell type dependent localization pattern for MHV-68 M2 expression. In lymphocytes, this protein was seen in the cytoplasm and cell membranes, while in

24 J. Mistríková, J. Rajčáni

Protein EBV ORF MuHV 4 ORF Function of corresponding protein / behaviour of the deletion mutant Serpin BARF1 M1 caspase 1 inhibitor?/increased rate of reactivation during latency None M2 Latency associated protein, IFN type I antagonist / important for establishing B-cell latency and reactivation (following i.n. administration) None M3 Soluble chemokine antagonist, binds to several chemokines / the M3del virus cannot grow at the portal of entry None M4 Soluble immunomodulatory glycoprotein, which binds to receptors recognizing MuHV 4-specific antigen(s) / extremely low level of latency at day 100 p.i. in the case of the M4stopMHV-68 mutant Table 3. Possible function of proteins encoded by the 5´-end of the MuHV 4 genome. ? = no anti-apoptotic function has been attributed to M1 protein epithelial cells and fibroblasts it was detected solely as compared to wild-type and marker rescue viruses. within the nucleus. Mutational analysis indicated that M2 In contrast, after intraperitoneal inoculation of the has positively charged internal amino acids. Purification M2 mutant virus, no significant defect was observed of M2 showed that it interacts with the cellular p32 in latency establishment or reactivation. From these protein, which recruits the complex into the nucleus. considerations it was concluded that M2 plays some M2 inhibits the IFN action triggered by its binding to IFN role in the establishment of and reactivation from latency receptor and resulting in the activation of corresponding in the spleen soon after i.n. inoculation. However, M2 intracellular pathways. Nuclear M2 downregulates the may be not be crucial for establishing splenic latency expression of the signal transduction and transactivation following intraperitoneal application. The significance (STAT) proteins 1 and 2, which mediate the transcription of the latter finding is unclear, at least regarding to the of IFN-reactive cellular proteins. In MHV-68 infected ability of M2 to block IFN signaling. fibroblasts the M2 protein acts as an IFN antagonist, The MHV M3 gene encodes a soluble chemokine representing an immune evasion mechanism for the binding protein, which potentially interferes with natural maintenance of latent infection (Table 3). chemokines and their interactions with corresponding The expression of the M2 gene was examined in receptors [61,62]. The 46K M3 polypeptide is secreted B lymphocytes during acute infection as well as during from MHV-infected cells. This protein is encoded by latency in mice inoculated by the i.n. route [15]. While an early-late 1.4 kb mRNA that initiates at nt 7294 and the proportion of M2-positive splenocytes was 311:1 terminates downstream at nt 6007 followed by a canonic (day 14 p.i), their proportion decreased sharply (by 47 polyA signal 9 bp apart. Recently, the M3 protein has fold) by day 21 p.i., to decrease even further (nearly been shown to be capable of binding a broad variety 2400 fold) by day 153 p.i. It was shown that vaccination of chemokines and neutralizing the cellular responses with M2 reduces the M2 protein load of latently infected to them in vitro [63]. In vivo it probably attenuates the B cells in the spleen at early intervals and abolished immune response and assists in regulation of leukocyte latency at late intervals p.i. [59]. The specific T/CD8 cell recruitment that could lead to enhanced virus replication response appearing from day 14 p.i. was undetectable [64]. As recently shown, M3 is a secreted protein; its during long-term latency. Adoptive-transfer studies soluble form interacts with many residues of CCL2 also demonstrated that the T/CD8 cells specific for chemokines, including monocyte chemotactic proteins M2 initially reduced, but did not limit the long-term (MCP) and macrophage inflammatory proteins (MIP), all load of that protein. The splenic lymphocytosis, which of which are recognized by the cellular receptor CCR2. was found to be independent of M2 expression, could Interestingly, the interaction of M3 with CCL2 chemokines be separated from the establishment of latency [57]. mimics their binding with the corresponding CCR2 Mutation of the M2 gene did not affect the ability of the receptor. M3 was also shown to interact with some CXC virus to replicate in tissue culture, nor did it affect MHV- chemokines with relatively high affinities 65 [ ]. It also 68 virulence. However, M2 was differentially required binds to many inflammatory and chemotactic proteins, for acute replication in vivo, being indispensable for like eotaxin, fractalkine, lymphotactine, RANTES, etc. virus production in the spleen but not in the lungs M3 is an example of a soluble chemokine modulator [60]. The M2 mutant virus was defective in its ability to acting as immune evasion tool. DNA vaccination of cause the transient sharp rise in latently infected cells mice with an M3 expressing plasmid yielded partial normally seen in the spleen after w.t. MHV-68 infection, protection lowering the infectious virus titre in the lungs but showed no defect in productive replication within of challenged animals [66]. The latter authors showed lungs. Following i.n. inoculation, the cells positive for that M3 elicited a cytotoxic T cell response during acute the M2 mutant inefficiently reactivated from latency infection, which persisted at later stages of latency. It

25 Comparison of pathogenic properties of the murid gammaherpesvirus (MuHV 4) strains: a role for immunomodulatory proteins encoded by the left (5’-)end of the genome

may be suggested that M3 itself can act as an immune virus cannot grow to high titres at the portal of entry target for virus specific TCRs. Summing up, the 5’-end of [69]. The level of spleen virus load was similar with the the MHV genome encodes at least two immune evasion M4 negative mutants and the w.t. virus at day 10 p.i., proteins, namely M2 (a blocker of IFN type I responsive but later on a nearly 100 fold difference was found. At genes), and M3 (a soluble chemokine inhibitor active day 30 p.i., the M4stop virus was completely cleared. during latency). Considerably decreased levels of the M4stop virus DNA The function of the next MHV specific gene, were found at the late p.i. intervals as detected by PCR designated M4, was long obscure. Recently, it has (using M4-specific primers). been followed by means of MHV-76inM4, a mutant with As shown by Evans et al. [70], M4 is a secreted the M4 gene inserted. To understand why this MHV- glycoprotein which might bind to receptors of cells 76 construct was prepared, it should be mentioned involved in the immune response. Alternatively, the that MHV-76 is a spontaneous deletion mutant lacking M4 glycoprotein could interfere with the action of a 9.53 kb segment from the 5’-end of the genome chemokines and/or cytokines. While M3 as well as M2 (Table 2). The missing DNA region encompasses proteins are immune evasion tools used to increase the the M1, M2, M3 and M4 ORFs as well as sequences rate of virus replication, M4 might delay virus clearance coding for the vtRNA transcripts [42]. As described and/or stimulate proliferation of cells involved in immune above, MHV-76 is more rapidly cleared from lungs as response. Therefore, the latent load of the M4 negative compared with the protoptype MHV-68 strain. Acute strains is consistently lower than of the w.t. virus. This splenomegaly was found to be reduced following i.n. was evident in the case of the M4 disruption mutant, MHV-76 inoculation and, furthermore, long-term latency which was most severely limited in establishment of could be established at considerably lower frequency latency. Nevertheless, a minimum copy number of compared with the prototype strain. The MHV-76inM4 the MuHV 4 genome is required for initiation of false construct (with M4 reinserted) grew similarly in vitro as positive signaling involved in cellular proliferation [71]. did the w.t. MHV-76. However, in MHV-76inM4 infected IFN-g deficient mice developed very severe splenic, Balb/c mice, acute virus titres, especially in lungs, were mediastinal lymph node and lung pathologies when higher than in animals inoculated with the w.t. virus infected with the MHV-68 strain, but not when infected (in which the virus was quickly cleared). At days 17 with mutants showing artificial left end deletions of and 21 p.i., the persisting virus load of MHV-76inM4 varying lengths. It became clear from these studies, – as detected by co-cultivation – was higher also in that the severe pathology seen in IFN-g deficient mice splenocytes. During acute infection, the M4 polypeptide infected with MHV-68 containing the complete 5-end of the prototype MHV-68 strain was expressed in lungs sequence could not be explained by the presence of any and within spleen. The M4 protein encoded by the MHV- presently defined genes in the given region. 76inM4 construct was also expressed in the lungs during The survey of known virus-specific proteins encoded acute infection and within the spleen at day 21 p.i., but by the 5’-end of the MuHV 4 genome is given in Table 3. became undetectable at late latency intervals, i.e. from It should be noted in this context that the left hand (5’- day 100 p.i. [67]. These data would indicate that M4 may end) region of the MuHV 4 genome might encode at least have some role in the modulation of immune response. two additional genes that remain uncharacterized [72]. Nevertheless, the interpretation of the experiments with MHV-76inM4 is difficult due to the absence of the M2 and M3 gene products. To overcome this problem, two 6. Conclusions and perspectives deletion mutants of MHV-68 were constructed, namely M4stop (a stop codon inserted at nt 8672 allowing The growth of MuHV-4 strains (or isolates), which bear expression of a 22 aa oligopeptide instead of the 393 aa deletions in the 5´-end region of their genome, was not full protein) and an independent deletion mutant lacking hampered in cell cultures. All MHV viruses were able to nt 8385–9155 (M4del). No differences in the acute infect Balb/c mice. The deletions in question had varying phase virus titres within lungs were seen with all M4 impact on the virus load at early stages p.i. and at least negative MHV-68 mutants in comparison with the w.t. minimal latency could be established even when the 5’- virus [68]. It should be noted in this context that at early end of the genome was missing. Paradoxically, the rate intervals following i.n. inoculation (days 1–3) the MHV- and extent of reactivation (despite of the low number 76inM4 construct grew to higher titres in lungs than the of cells carrying the latent virus), was the highest MHV-76 strain. It is possible that the MHV-76 strain with MHV-76, which reactivated easily at remote p.i. lacking the M3 immune evasion protein replicates better intervals and grew to high titers following reactivation. in the presence of M4 protein, since the M3 negative On the other hand, the rate of spontaneously occurring

26 J. Mistríková, J. Rajčáni

tumors was the highest in mice infected with MHV-60, as p53 and Rb. Disruption of the regulatory events which regularly entered non-productive virus latency controlling cell division is the consequence of interaction rather than productive persistence. In connection with of “oncogenic” viral proteins with the cellular regulatory this, one may hypothesize the following: (A) M2 and proteins [73]. Such interactions regarding MuHV 4 (and M3 immune evasion polypeptides increase the extent related gammaherpesviruses) have been described of virus replication during the acute stage p.i., help to elsewhere [74]. We hypothesize that the MuHV-4 coded disseminate the virus via blood stream and increase its oncoproteins (such as v-Cyclin/ORF72, LANA/ORF73) likelihood of establishing latency. The M3 polypeptide, in combination with those blocking apoptosis (v-bcl-2/ which interacts with several cytokines, may be useful M11) could cause host cell immortalization and/or for virus replication in the spleen and other lymphatic transformation especially in the absence of productive tissues. Even in the absence of M2/M3 polypeptides, the virus growth. However, it is not known whether the immune evasion functions may not be fully abolished MuHV 4 DNA is present in all tumors developing in the due to expression of the MK3 polypeptide and/or related virus-exposed animals. Therefore, the possibility of a proteins. (B) The MHV-76 virus was quickly eliminated complex long term immune stimulation following low from lungs, but grew better following reactivation of rate persistence or abortive virus replication cannot be latency, since the missing M4 glycoprotein could not excluded as an additional cause of slowly progressing signal the onset of virus production. Thus, at remote lymphoproliferation. intervals persistent production of infectious virus occurred due to the absence of M4 signaling. The 24–27 month follow-up of Balb/c mice, which Acknowledgements had been infected with MHV-76, showed neither tumors nor splenomegaly at late intervals p.i. The continuous The results mentioned in this review were obtained in (persistent) virus replication at latency sites does not experiments supported by the grant No. 1/2272/05 of favor transformation. Alternatively, UV light irradiated VEGA (Joined Agency of Ministry of Education and SAS oncogenic DNA viruses had been frequently used to of Slovak Republic) and the research project APVV-51- demonstrate their potential capacity to transform cells 005-005 (Agency for Science and Research Projects of in vitro. This approach aimed to avoid productive virus the Ministry of Education of Slovak Republic). replication and concomitant apoptosis. It is known that oncogenic DNA viruses encode non-structural polypeptides which bind cellular anti-onc proteins such References

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