Products of US22 Genes M140 and M141 Confer Efficient Replication of Murine Cytomegalovirus in Macrophages and Spleen

A selection strategy, the activator trap, was used in order to identify genes of human cytomegalovirus (HCMV) that encode strong transcriptional activation domains in mammalian cells. This approach is based on the isolation of activation domains from a GAL4 fusion library by means of selective plasmid replication, which is mediated in transfected cells by a GAL4-inducible T antigen gene. With this screening strategy, we were able to isolate two types of plasmids encoding transactivating fusion proteins from a library of random HCMV DNA inserts. One plasmid contained the exon 3 of the HCMV IE-1/2 gene region, which has previously been identified as a strong transcriptional activation domain. In the second type of plasmid, the open reading frame (ORF) UL26 of HCMV was fused to the GAL4 DNA-binding domain. By quantitative RNA mapping using S1 nuclease analysis, we were able to classify UL26 as a strong enhancer-type activation domain with no apparent homology to characterized transcriptional activators. Western blot analysis with a specific polyclonal antibody raised against a prokaryotic UL26 fusion protein revealed that two protein isoforms of 21 and 27 kDa are derived from the UL26 ORF in both infected and transfected cells. Both protein isoforms, which arise via alternative usage of two in-frame translational start codons, showed a nuclear localization and could be detected as early as 6 h after infection of primary human fibroblasts. By performing Western blot analysis with purified virions combined with fractionation experiments, we provide evidence that pUL26 is a novel tegument protein of HCMV that is imported during viral infection. Furthermore, we observed transactivation of the HCMV major immediate-early enhancer-promoter by pUL26, whereas several early and late promoters were not affected. Our data suggest that pUL26 is a novel tegument protein of HCMV with a strong transcriptional activation domain that could play an important role during initiation of the viral replicative cycle.Human cytomegalovirus (HCMV) is an extensively studied human herpesvirus that can cause a broad spectrum of diseases ranging from subclinical infection in normal healthy individuals to congenital malformation in newborns and life-threatening pneumonitis in immunocompromised adults (6). During lytic infection, more than 200 HCMV genes are expressed in a temporally regulated cascade consisting of three major waves of viral gene expression (8). A few of the encoded proteins have been identified as regulatory molecules that are involved in the coordinate expression of genes during the three distinct phases of the viral replicative cycle, termed the immediateearly (IE), early, and late phases (11,36,53,54). The bestcharacterized regulatory proteins are the 72-kDa IE1 (UL123) and the 86-kDa IE2 (UL122) polypeptides that originate from the major IE gene region of HCMV during initial stages of the replicative cycle (23,38,49). Both IE1-p72 and IE2-p86 are able to positively regulate gene expression independently (35,50). This has been...

Section: Discussionmentioning

confidence: 99%

Open Reading Frame UL26 of Human Cytomegalovirus Encodes a Novel Tegument Protein That Contains a Strong Transcriptional Activation Domain

Stamminger

Gstaiger

Weinzierl

et al. 2002

“…Because macrophages and endothelial cells play key roles in CMV dissemination (4,13,36,55,59,64), viral gene products which regulate growth in these cell types should significantly affect MCMV pathogenesis in vivo. This has, for instance, been shown for MCMV mutants with deletions in the m139-to-m141 region, which are growth deficient in macrophages and attenuated in mice (30,31). To directly assess the role of M45 in viral pathogenesis, we investigated the virulence of two M45 mutant viruses (a transposon insertion mutant and a frameshift mutant) in SCID mice.…”

Section: Vol 78 2004mentioning

confidence: 99%

The Ribonucleotide Reductase R1 Homolog of Murine Cytomegalovirus Is Not a Functional Enzyme Subunit but Is Required for Pathogenesis

Lembo

Donalisio

Hofer

et al. 2004

Ribonucleotide reductase (RNR) is the key enzyme in the biosynthesis of deoxyribonucleotides. Alpha-and gammaherpesviruses express a functional enzyme, since they code for both the R1 and the R2 subunits. By contrast, betaherpesviruses contain an open reading frame (ORF) with homology to R1, but an ORF for R2 is absent, suggesting that they do not express a functional RNR. The M45 protein of murine cytomegalovirus (MCMV) exhibits the sequence features of a class Ia RNR R1 subunit but lacks certain amino acid residues believed to be critical for enzymatic function. It starts to be expressed independently upon the onset of viral DNA synthesis at 12 h after infection and accumulates at later times in the cytoplasm of the infected cells. Moreover, it is associated with the virion particle. To investigate direct involvement of the virally encoded R1 subunit in ribonucleotide reduction, recombinant M45 was tested in enzyme activity assays together with cellular R1 and R2. The results indicate that M45 neither is a functional equivalent of an R1 subunit nor affects the activity or the allosteric control of the mouse enzyme. To replicate in quiescent cells, MCMV induces the expression and activity of the cellular RNR. Mutant viruses in which the M45 gene has been inactivated are avirulent in immunodeficient SCID mice and fail to replicate in their target organs. These results suggest that M45 has evolved a new function that is indispensable for virus replication and pathogenesis in vivo.Cytomegalovirus (CMV), a betaherpesvirus, is a widespread pathogen responsible for generally asymptomatic and persistent infections in healthy people. It may, however, cause severe disease in the absence of an effective immune response, as in immunologically immature and immunocompromised individuals (41). Strict species specificity has hampered investigation of human CMV (HCMV) in its natural host, and infection of mice with murine CMV (MCMV) has been extensively used as a model for studying the pathogenesis of HCMV infection (40,52,63). The two viruses are biologically similar in replication and pathogenesis (34,50,51), and their homologous genomes display similar genetic organizations and encode analogous gene products with similar functions (56). However, the functions of many viral gene products remain to be explored in order to determine the interactions of CMV with the host cell and its pathogenic mechanisms.CMV replicates most efficiently in the absence of cellular DNA synthesis (23, 39). Moreover, it has evolved mechanisms to inhibit progression through the cell cycle and arrests cells with a G 1 DNA content (5,18,47,53,60,68). The viral DNA polymerase thus has competition-free access to the DNA precursors, and CMV replicates efficiently in vitro in quiescent cells (18,45) and infects terminally differentiated cells in vivo (41, 51). However, since postmitotic cells do not replicate their genomes, the very low levels of deoxyribonucleotides (dNTP) and cell functions involved in DNA synthesis limit viral replication.It has been d...

“…A genome-wide screening for HCMV genes affecting virus growth in various cell types identified the UL24 gene, a member of the US22 gene family coding for tegument proteins, as necessary for efficient HCMV replication in microvascular EC (8); interestingly, MCMV homologues of the US22 family, M140 and M141, had previously been identified as tropism genes required for MCMV replication in macrophages (18). In a distinct, knowledge-driven approach, three genes of the UL131A-128 locus (1,16), which are frequently inactivated during clinical strain adaptation in fibroblasts (7,16), were recently shown to be required for efficient HCMV infections of human umbilical vein endothelial cells (HUVEC) as well as for virus transfer to neutrophils and monocytes from an infected HUVEC monolayer (16).…”

mentioning

confidence: 99%

Human Cytomegalovirus UL130 Protein Promotes Endothelial Cell Infection through a Producer Cell Modification of the Virion

Patrone

Secchi

Fiorina

et al. 2005

Human cytomegalovirus (HCMV) growth in endothelial cells (EC) requires the expression of the UL131A-128 locus proteins. In this study, the UL130 protein (pUL130), the product of the largest gene of the locus, is shown to be a luminal glycoprotein that is inefficiently secreted from infected cells but is incorporated into the virion envelope as a Golgi-matured form. To investigate the mechanism of the UL130-mediated promotion of viral growth in EC, we performed a complementation analysis of a UL130 mutant strain. To provide UL130 in trans to viral infections, we constructed human embryonic lung fibroblast (HELF) and human umbilical vein endothelial cell (HUVEC) derivative cell lines that express UL130 via a retroviral vector. When the UL130-negative virus was grown in UL130-complementing HELF, the infectivity of progeny virions for HUVEC was restored to the wild-type level. In contrast, the infectivity of the UL130-negative virus for UL130-complementing HUVEC was low and similar to that of the same virus infecting control noncomplementing HUVEC. The UL130-negative virus, regardless of whether or not it had been complemented in the prior cycle, could form plaques only on UL130-complementing HUVEC, not control HUVEC. Because (i) both wild-type and UL130-transcomplemented virions maintained their infectivity for HUVEC after purification, (ii) UL130 failed to complement in trans the UL130-negative virus when it was synthesized in a cell separate from the one that produced the virions, and (iii) pUL130 is a virion protein, models are favored in which pUL130 acquisition in the producer cell renders HCMV virions competent for a subsequent infection of EC.Human cytomegalovirus (HCMV) is a betaherpesvirus that establishes life-long, subclinical infections ubiquitously in human populations (5). HCMV causes serious morbidity in settings of immune system immaturity or depression: it is the leading viral cause of defects at birth and generates a potentially life-threatening disease in immunocompromised patients. In patients with HCMV disease, the virus can be demonstrated in a variety of cells, including hematopoietic cells (monocytes-macrophages, dendritic cells, and neutrophils), endothelial cells (EC), epithelial cells, fibroblasts, neurons, smooth muscle cells, and hepatocytes (5, 38). Much recent work has focused on HCMV infections of EC, for several reasons: (i) arterial endothelia, along with CD34 ϩ myeloid progenitors, have been proposed as sites of HCMV persistence and latency (20); (ii) a bidirectional transmission of HCMV between EC and leukocytes can be demonstrated in vitro and may reflect a mechanism of dissemination in vivo (11,14,34,44); (iii) circulating giant EC may similarly contribute directly to dissemination (32); (iv) HCMV infections of the uterine microvasculature and cytotrophoblasts may underlie motherto-fetus transmission and compromise the placental trophic function in affected pregnancies (25, 47); and (v) HCMV may play a role in atherogenesis, postangioplasty restenosis, posttransplantation endot...