Strategies for the Identification and Analysis of Viral Immune-Evasive Genes — Cytomegalovirus as an Example

Gutermann, Anja; Bubeck, Anja; Wagner, Markus; Reusch, Uwe; Ménard, Carine; Koszinowski, Ulrich H.

doi:10.1007/978-3-642-59421-2_1

Cited by 15 publications

(17 citation statements)

References 53 publications

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“…The observation of a large number of insertions within genes M36 and M43 and fewer insertions in other genes demonstrated that, in this new library, the distribution of transposon insertions was not completely random and that it differed from that of the library generated previously (17). All the mutant genomes used in this study were analyzed for genome integrity by restriction enzyme digestion (Fig.…”

Section: Concept Of Us22 Gene Family Mutant Analysismentioning

confidence: 88%

“…Transposon insertion is technically easier but requires a collection of at least 2,000 mutants to obtain a library that covers all CMV genes, since the distribution of insertion sites is not completely random (17). Even with a library of 2,600 clones, it was not possible to isolate at least two independent mutants for each of the genes under study.…”

Section: Discussionmentioning

confidence: 99%

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Role of Murine Cytomegalovirus US22 Gene Family Members in Replication in Macrophages

Ménard

Wagner

Ruzsics

et al. 2003

J Virol

157

233

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The large cytomegalovirus (CMV) US22 gene family, found in all betaherpesviruses, comprises 12 members in both human cytomegalovirus (HCMV) and murine cytomegalovirus (MCMV). Conserved sequence motifs suggested a common ancestry and related functions for these gene products. Two members of this family, m140 and m141, were recently shown to affect MCMV replication on macrophages. To test the role of all US22 members in cell tropism, we analyzed the growth properties in different cell types of MCMV mutants carrying transposon insertions in all 12 US22 gene family members. When necessary, additional targeted mutants with gene deletions, ATG deletions, and ectopic gene revertants were constructed. Mutants with disruption of genes M23, M24, m25.1, m25.2, and m128 (ie2) showed no obvious growth phenotype, whereas growth of M43 mutants was reduced in a number of cell lines. Genes m142 and m143 were shown to be essential for virus replication. Growth of mutants with insertions into genes M36, m139, m140, and m141 in macrophages was severely affected. The common phenotype of the m139, m140, and m141 mutants was explained by an interaction at the protein level. The M36-dependent macrophage growth phenotype could be explained by the antiapoptotic function of the gene that was required for growth on macrophages but not for growth on other cell types. Together, the comprehensive set of mutants of the US22 gene family suggests that individual family members have diverged through evolution to serve a variety of functions for the virus.Herpesviruses are large and complex DNA viruses, widely found in nature. Human cytomegalovirus (HCMV), an important human pathogen, defines the betaherpesvirus family. Mouse CMV (MCMV) and rat CMV serve as biological model systems for HCMV. HCMV, MCMV, and rat CMV display the largest genomes among the herpesviruses (13,34,43). These genomes are essentially colinear over the central 180 kb of the 230-kb genomes. Betaherpesviruses, which include the CMVs as well as human herpesviruses 6 and 7, differ from alpha-and gammaherpesviruses by the presence of additional gene families such as the US22 gene family, which are mainly clustered at the ends of the genome (29, 30).The US22 family was first described in HCMV (13). This gene family comprises 12 members in both HCMV and MCMV and 11 in rat CMV. Members of the US22 gene family are characterized by stretches of hydrophobic and charged residues as well as up to four conserved sequence motifs which are specific for betaherpesviruses. Motif I differs between the HCMV US and UL family members (30). In MCMV, m128 and m139 to m143 share the HCMV US-like motif I, while M23, M24, m25.1, m25.2, M36, and M43 share UL-like motif I. Motifs I and II have consensus sequences, while motifs III and IV are less well defined but have stretches of nonpolar residues (18, 24). The m139 to m141 genes contain all four of these motifs, whereas m142 and m143 (and IRS1/TRS1 of HCMV) lack motif II. In addition, m139, m140, m142, and m143 each have an acidic domain, common to he...

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Section: Concept Of Us22 Gene Family Mutant Analysismentioning

confidence: 88%

Section: Discussionmentioning

confidence: 99%

Role of Murine Cytomegalovirus US22 Gene Family Members in Replication in Macrophages

Ménard

Wagner

Ruzsics

et al. 2003

J Virol

157

233

View full text Add to dashboard Cite

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“…HCMV executes multiple immune-evasive activities in infected cells. The viral proteins US2, US3, US6, and US11 cooperate to inhibit MHC-I presentation of viral peptides to CD8 T cells (4,5). The secreted HCMV protein UL21.5 acts as a soluble decoy receptor for the proinflammatory chemokine RANTES (D. Wang, W. Bresnahan, and T.S., unpublished data), and HCMV expresses a viral homologue of IL-10, an antiinflammatory interleukin (6).…”

Section: H Uman Cytomegalovirus (Hcmv) Is a Ubiquitous ␤-Herpesmentioning

confidence: 99%

Human cytomegalovirus UL83-coded pp65 virion protein inhibits antiviral gene expression in infected cells

Browne

Shenk

2003

Proc. Natl. Acad. Sci. U.S.A.

180

173

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The initial interaction of human cytomegalovirus with fibroblasts triggers, and then partially blocks, an innate immune response pathway that leads to the induction of IFN-responsive genes and proinflammatory chemokines. Infection of fibroblasts with human cytomegalovirus inhibited their ability to respond to exogenous IFN. Consistent with the observation that the block did not depend on de novo viral protein synthesis, ectopic expression of the viral UL83-coded pp65, an abundant virion protein, inhibited IFN signaling. Furthermore, DNA array analysis showed that infection with a pp65-deficient mutant virus caused a much stronger induction of many IFN-response and proinflammatory chemokine RNAs than infection with wild-type virus. The nuclear DNA-binding activities of transcription factors NF-B and IRF1 were induced to a much greater extent after infection with the pp65-deficient mutant than with wild-type virus. IFN-stimulated gene factor 3 DNA-binding was modestly enhanced, whereas IRF3 activity was not affected by mutation of pp65. Together, these results imply that pp65, which is delivered to newly infected cells in the virion, antagonizes a pathway that affects NF-B and IRF1 and prevents the accumulation of mRNAs encoded by numerous cellular antiviral genes.

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“…Understanding the role of diVerent immunomodulatory proteins during infection is essential to design novel approaches of immunointervention and therapy of HCMV infection. The cloning of the large HCMV genome into BACs has opened a gateway to precisely look at individual immunomodulatory genes using site-directed deletion [17,18]. ReWnements in BAC technologies allow seamless modiWcation down to single amino acids within proteins of recombinant viruses [33], thus enabling their detailed analysis on the molecular level.…”

Section: Discussionmentioning

confidence: 99%

“…Comparing wt-viruses with mutants that carry targeted deletions or insertions provides valuable information on the functionality of particular genes in the context of viral infection (reviewed in [18]). With respect to MHC class I immune evasion, viruses deWcient in the complete US2-US11 gene region have been used to show that downregulation of MHC class I on the surface of HCMV infected cells is critically dependent on the presence of these genes [1].…”

Section: Rv-us2-11 Infected Cells Still Suppress Ie1-peptide Presentamentioning

confidence: 99%

Recombinant viruses as tools to study human cytomegalovirus immune modulation

Besold

Plachter

2008

Med Microbiol Immunol

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Infections with cytomegaloviruses are characterized by an intricate balance between the expression of immunomodulatory viral proteins and antiviral immune defence. For human cytomegalovirus (HCMV), several proteins have been described that interfere with the recognition of infected cells by CD8 T lymphocytes. Although the modes of action of these proteins have been elucidated on the molecular level, thus rendering them useful models to understand MHC class I peptide loading and transport, their role during viral infection has remained enigmatic. We exemplify here, how HCMV mutants can help to understand the importance of individual immunomodulatory proteins in the context of viral infection.

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Strategies for the Identification and Analysis of Viral Immune-Evasive Genes — Cytomegalovirus as an Example

Cited by 15 publications

References 53 publications

Role of Murine Cytomegalovirus US22 Gene Family Members in Replication in Macrophages

Role of Murine Cytomegalovirus US22 Gene Family Members in Replication in Macrophages

Human cytomegalovirus UL83-coded pp65 virion protein inhibits antiviral gene expression in infected cells

Recombinant viruses as tools to study human cytomegalovirus immune modulation

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