Murine gammaherpesvirus 68 open reading frame 31 is required for viral replication

Murine gammaherpesvirus 68 (MHV-68) is genetically related to the human gammaherpesviruses, Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8) and Epstein-Barr virus (EBV). It has been proposed as a model for gammaherpesvirus infection and pathogenesis. Open reading frame 31 (ORF31) is conserved among the Beta- and Gammaherpesvirinae subfamily, and there is no known mammalian homologue of this protein. The function of MHV-68 ORF31 and its viral homologues has not yet been determined. We described here a primary characterization of this protein and its requirement for lytic replication. The native MHV-68 ORF31 was detected at peak levels by 24 h postinfection, and the FLAG-tagged and green fluorescent protein fusion ORF31 were localized in the cytoplasm and nucleus in a diffuse pattern. Two independent experimental approaches were then utilized to demonstrate that ORF31 was required for lytic replication. First, small interfering RNA generated against ORF31 expression blocked protein expression and virus production in transfected cells. Then, two-independent bacterial artificial chromosome-derived ORF31-null MHV-68 mutants (31STOP) were generated and found to be defective in virus production in fibroblast cells. This defect can be rescued in trans by MHV-68 ORF31 and importantly by its KSHV homologue. A repair virus of 31STOP was also generated by homologous recombination in fibroblast cells. Finally, we showed that the defect in ORF31 blocked late lytic protein expression. Our results demonstrate that MHV-68 ORF31 is required for viral lytic replication, and its function is conserved in its KSHV homologue.     

Murine gammaherpesvirus 68 open reading frame 11 encodes a nonessential virion component

Open reading frame 11 (ORF11) is a conserved gammaherpesvirus gene that remains undefined. We identified the product of murine gammaherpesvirus 68 (MHV-68) ORF11, p43, as a virion component with a predominantly perinuclear distribution in infected cells. MHV-68 lacking p43 grew normally in vitro but showed reduced lytic replication in vivo and a delay in seeding to the spleen. Subsequent latency amplification was normal. Thus, MHV-68 ORF11 encoded a virion component that was important for in vivo lytic replication but dispensable for the establishment of latency.     

Murine gammaherpesvirus 68 open reading frame 75c tegument protein induces the degradation of PML and is essential for production of infectious virus

Promyelocytic Leukemia nuclear body (PML NB) proteins mediate an intrinsic cellular host defense response against virus infections. Herpesviruses express proteins that modulate PML or PML-associated proteins by a variety of strategies, including degradation of PML or relocalization of PML NB proteins. The consequences of PML-herpesvirus interactions during infection in vivo have yet to be investigated in detail, largely because of the species-specific tropism of many human herpesviruses. Murine gammaherpesvirus 68 (γHV68) is emerging as a suitable model to study basic biological questions of virus-host interactions because it naturally infects mice. Therefore, we sought to determine whether γHV68 targets PML NBs as part of its natural life cycle. We found that γHV68 induces PML degradation through a proteasome-dependent mechanism and that loss of PML results in more robust virus replication in mouse fibroblasts. Surprisingly, γHV68-mediated PML degradation was mediated by the virion tegument protein ORF75c, which shares homology with the cellular formylglycinamide ribotide amidotransferase enzyme. In addition, we show that ORF75c is essential for production of infectious virus. ORF75 homologs are conserved in all rhadinoviruses but so far have no assigned functions. Our studies shed light on a potential role for this unusual protein in rhadinovirus biology and suggest that γHV68 will be a useful model for investigation of PML-herpesvirus interactions in vivo.     

Murine cytomegalovirus open reading frame M27 plays an important role in growth and virulence in mice

Using a Tn3-based transposon mutagenesis approach, we have generated a pool of murine cytomegalovirus (MCMV) mutants. In this study, one of the mutants, RvM27, which contained the transposon sequence at open reading frame M27, was characterized both in tissue culture and in immunocompetent BALB/c mice and immunodeficient SCID mice. Our results suggest that the M27 carboxyl-terminal sequence is dispensable for viral replication in vitro. Compared to the wild-type strain and a rescued virus that restored the M27 region, RvM27 was attenuated in growth in both BALB/c and SCID mice that were intraperitoneally infected with the viruses. Specifically, the titers of RvM27 in the salivary glands, lungs, spleens, livers, and kidneys of the infected SCID mice at 21 days postinfection were 50- to 500-fold lower than those of the wild-type virus and the rescued virus. Moreover, the virulence of the mutant virus appeared to be attenuated, because no deaths occurred among SCID mice infected with RvM27 for up to 37 days postinfection, while all the animals infected with the wild-type and rescued viruses died within 27 days postinfection. Our observations provide the first direct evidence to suggest that a disruption of M27 expression results in reduced viral growth and attenuated viral virulence in vivo in infected animals. Moreover, these results suggest that M27 is a viral determinant required for optimal MCMV growth and virulence in vivo and provide insight into the functions of the M27 homologues found in other animal and human CMVs as well as in other betaherpesviruses.     

Murine gammaherpesvirus 68 contains two functional lytic origins of replication

A 1.25-kbp DNA fragment from the right side of the genome containing the lytic origin of replication (oriLyt) of murine gammaherpesvirus 68 (MHV-68) has been identified by a plasmid replication assay. Here we show that a mutant MHV-68 with a deletion of an essential part of this oriLyt, generated by using an MHV-68 bacterial artificial chromosome, was only slightly attenuated and still able to replicate but that a mutant containing an additional deletion on the left side of the genome was replication deficient. The newly identified region was sufficient to support plasmid replication, thus providing evidence for a second oriLyt.     

Glycoprotein M is an essential lytic replication protein of the murine gammaherpesvirus 68

All herpesviruses encode a homolog of glycoprotein M (gM), which appears to function in virion morphogenesis. Despite its conservation, gM is inessential for the lytic replication of alphaherpesviruses. In order to address the importance of gM in gammaherpesviruses, we disrupted it in the murine gammaherpesvirus 68 (MHV-68). The mutant virus completely failed to propagate in normally permissive fibroblasts. The defective genome was rescued by either homologous recombination to restore the wild-type gM in situ or the insertion of an ectopic, intergenic expression cassette encoding gM into the viral genome. Thus, gM was essential for the lytic replication of MHV-68.     

Disruption of the murine gammaherpesvirus 68 M1 open reading frame leads to enhanced reactivation from latency

Murine gammaherpesvirus 68 (gammaHV68, or MHV-68) is a genetically tractable, small animal model for the analysis of gammaherpesvirus pathogenesis. The gammaHV68 genome is colinear with the genomes of other sequence gammaherpesviruses, containing large blocks of conserved genes interspersed by a number of putative genes without clear homologs in the other gammaherpesviruses. One of these putative unique genes, the M1 open reading frame (ORF), exhibits sequence homology to a poxvirus serine protease inhibitor, SPI-1, as well as to another gammaHV68 gene, M3, which we have recently shown encodes an abundantly secreted chemokine binding protein. To assess the contribution of the M1 ORF to gammaHV68 pathogenesis, we have generated a recombinant gammaHV68 in which the M1 ORF has been disrupted through targeted insertion of a lacZ expression cassette (M1.LacZ). Although M1.LacZ replicated normally in tissue culture, it exhibited decreased splenic titers at days 4 and 9 postinfection in both immunocompetent and immunodeficient mice. Despite decreased levels of acute virus replication, M1.LacZ established a latent infection comparable to wild-type (wt) gammaHV68, but exhibited an approximately fivefold increase in efficiency of reactivation from latency. M1.LacZ also caused severe vasculitis of the great elastic arteries in gamma interferon receptor (IFN-gammaR)-deficient mice with a frequency comparable to wt gammaHV68, but did not cause the mortality or splenic pathology observed with wt gammaHV68 infection of IFN-gammaR-deficient mice. Restoration of M1 ORF sequences into M1.LacZ (M1 marker rescue, or M1.MR) demonstrated that M1.LacZ phenotypic alterations in growth in vivo and latency were not due to the presence of additional mutations located elsewhere in the M1. LacZ genome. Generation of a second M1 mutant virus containing a deletion at the 5' end of the M1 ORF (M1Delta511), but lacking the LacZ expression cassette, revealed the same latency phenotype observed with the M1.LacZ mutant. However, M1Delta511 was not attenuated for acute virus replication in the spleen. We conclude that (i) the induction of arteritis in gammaHV68-infected IFN-gammaR-deficient mice can occur in the absence of splenic pathology and mortality, (ii) replication during acute infection is not the primary determinant for the establishment of latent infection, and (iii) the M1 ORF, or a closely linked gene, encodes a gene product that functions to suppress virus reactivation.     

Evidence for translational repression of the SOCS-1 major open reading frame by an upstream open reading frame

The suppressor of cytokine signalling 1 protein (SOCS-1) belongs to a novel family of cytokine inducible factors which function as inhibitors of the JAK/STAT pathway. While SOCS-1 previously has been described as a single-exon gene, here we present evidence for an additional 5' exon, separated by a 509 bp intron from exon 2. Exon 1 and part of exon 2 contain an open reading frame of 115 nt, ending one nucleotide upstream of the major reading frame. Using SOCS-1-promoter/luciferase constructs, we investigated which sequences are involved in the regulation of SOCS-1 expression. Serial promoter deletion clones indicate the localization and functionality of SP1, interferon-stimulated responsive elements (ISRE), and interferon-gamma-activated sites (GAS) promoter elements in the SOCS-1 5' flanking region. We present evidence that the upstream open reading frame (uORF) represses the translation of the downstream major open reading frame (mORF). Mutating the start codon of the uORF relieves this repression. Our data indicate that expression of the SOCS-1 protein is repressed on translational level by a mechanism, which bears similarities to that postulated for genes like retinoic acid receptor beta2 (RARbeta2), S-adenosylmethionine-decarboxylase (AdoMetDC), Bcl-2, and others.     

The internal open reading frame within the nucleocapsid gene of mouse hepatitis virus encodes a structural protein that is not essential for viral replication.

The coronavirus mouse hepatitis virus (MHV) contains a large open reading frame embedded entirely within the 5' half of its nucleocapsid (N) gene. This internal gene (designated I) is in the +1 reading frame with respect to the N gene, and it encodes a mostly hydrophobic 23-kDa polypeptide. We have found that this protein is expressed in MHV-infected cells and that it is a previously unrecognized structural protein of the virion. To analyze the potential biological importance of the I gene, we disrupted its expression by site-directed mutagenesis using targeted RNA recombination. The start codon for I was replaced by a threonine codon, and a stop codon was introduced at a short interval downstream. Both alterations created silent changes in the N reading frame. In vitro translation studies showed that these mutations completely abolished synthesis of I protein, and immunological analysis of infected cell lysates confirmed this conclusion. The MHV I mutant was viable and grew to high titer. However, the I mutant had a reduced plaque size in comparison with its isogenic wild-type counterpart, suggesting that expression of I confers some minor growth advantage to the virus. The engineered mutations were stable during the course of experimental infection in mice, and the I mutant showed no significant differences from wild type in its ability to replicate in the brains or livers of infected animals. These results demonstrate that I protein is not essential for the replication of MHV either in tissue culture or in its natural host.     

Efficient homologous RNA recombination and requirement for an open reading frame during replication of equine arteritis virus defective interfering RNAs

Equine arteritis virus (EAV), the prototype arterivirus, is an enveloped plus-strand RNA virus with a genome of approximately 13 kb. Based on similarities in genome organization and protein expression, the arteriviruses have recently been grouped together with the coronaviruses and toroviruses in the newly established order Nidovirales. Previously, we reported the construction of pEDI, a full-length cDNA copy of EAV DI-b, a natural defective interfering (DI) RNA of 5.6 kb (R. Molenkamp et al., J. Virol. 74:3156-3165, 2000). EDI RNA consists of three noncontiguous parts of the EAV genome fused in frame with respect to the replicase gene. As a result, EDI RNA contains a truncated replicase open reading frame (EDI-ORF) and encodes a truncated replicase polyprotein. Since some coronavirus DI RNAs require the presence of an ORF for their efficient propagation, we have analyzed the importance of the EDI-ORF in EDI RNA replication. The EDI-ORF was disrupted at different positions by the introduction of frameshift mutations. These were found either to block DI RNA replication completely or to be removed within one virus passage, probably due to homologous recombination with the helper virus genome. Using recombination assays based on EDI RNA and full-length EAV genomes containing specific mutations, the rates of homologous RNA recombination in the 3'- and 5'-proximal regions of the EAV genome were studied. Remarkably, the recombination frequency in the 5'-proximal region was found to be approximately 100-fold lower than that in the 3'-proximal part of the genome.     

The immediate-early protein IE0 of the Autographa californica nucleopolyhedrovirus is not essential for viral replication

The role of the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) immediate-early protein IE0 in the baculoviral infection is not clear. In this study, we constructed the recombinant virus vAcDeltaie0 null for ie0 expression by targeted mutagenesis replacing exon0 with the cat gene. We found that vAcDeltaie0 replicated efficiently in Spodoptera littoralis SL2 cells, which are poorly permissive for AcMNPV. In contrast, in Spodoptera frugiperda SF9 cells, which are fully permissive for AcMNPV, vAcDeltaie0 DNA replication and budded virus production were delayed. These results and recently published data (X. Dai et al., J. Virol. 78:9633-9644, 2004) indicate that ie0 is not essential for AcMNPV replication but enhances it in permissive SF9 cells.     

Murine gammaherpesvirus 68 lacking thymidine kinase shows severe attenuation of lytic cycle replication in vivo but still establishes latency

The lytic cycle functions of gammaherpesviruses have received relatively little attention to date, at least in part due to the lack of a convenient experimental model. The murine gammaherpesvirus 68 (MHV-68) now provides such a model and allows the roles of individual lytic cycle gammaherpesvirus proteins to be evaluated in vivo. We have used MHV-68 to determine the contribution of a gammaherpesvirus thymidine kinase (TK) to viral lytic replication and latency establishment. MHV-68 mutants with a disrupted TK gene grew normally in vitro but showed a severe attenuation of replication in the lungs after intranasal inoculation, with lytic titers at least 1,000-fold lower than those of wild-type and revertant viruses. Nevertheless, the establishment of latency by the TK-deficient mutants, while delayed, was not prevented by their lytic replication deficit. The viral TK clearly plays a crucial role in the capacity of MHV-68 to replicate efficiently in its natural host but does not seem to be essential to establish a persistent infection. The potential of TK-deficient mutants as gammaherpesvirus vaccines is discussed.     

Telomeric silencing of an open reading frame in Saccharomyces cerevisiae

The endmost chromosome I ORF is silenced by a natural telomere position effect. YAR073W/IMD1 was found to be transcribed at much higher levels in sir3 mutants and when its adjacent telomere was removed from it. These results suggest that telomeres play a role in silencing actual genes.     

COX-2 induction during murine gammaherpesvirus 68 infection leads to enhancement of viral gene expression

The murine gammaherpesvirus 68 (MHV-68 or gammaHV-68) model provides many advantages for studying virus-host interactions involved in gammaherpesvirus replication, including the role of cellular responses to infection. We examined the effects of cellular cyclooxygenase-2 (COX-2) and its by-product prostaglandin E(2) (PGE(2)) on MHV-68 gene expression and protein production following de novo infection of cultured cells. Western blot analyses revealed an induction of COX-2 protein in MHV-68-infected cells but not in cells infected with UV-irradiated MHV-68. Luciferase reporter assays demonstrated activation of the COX-2 promoter during MHV-68 replication. Two nonsteroidal anti-inflammatory drugs, a COX-2-specific inhibitor (NS-398) and a COX-1-COX-2 inhibitor (indomethacin), substantially reduced MHV-68 protein production in infected cells. Inhibition of viral protein expression and virion production by NS-398 was reversed in the presence of exogenous PGE(2). Global gene expression analysis using an MHV-68 DNA array showed that PGE(2) increased production of multiple viral gene products, and NS-398 inhibited production of many of the same genes. These studies suggest that COX-2 activity and PGE(2) production may play significant roles during MHV-68 de novo infection.