Rhesus cytomegalovirus contains functional homologues of US2, US3, US6, and US11

Human cytomegalovirus (HCMV) is a paradigm for mechanisms subverting antigen presentation by major histocompatibility complex (MHC) molecules. Due to its limited host range, HCMV cannot be studied in animals. Thus, the in vivo importance of inhibiting antigen presentation for the establishment and maintenance of infection with HCMV is unknown. Rhesus cytomegalovirus (RhCMV) is an emerging animal model that shares many of the features of HCMV infection. The recent completion of the genomic sequence of RhCMV revealed a significant degree of homology to HCMV. Strikingly, RhCMV contains several genes with low homology to the HCMV US6 gene family of inhibitors of the MHC I antigen presentation pathway. Here, we examine whether the RhCMV US6 homologues (open reading frames Rh182, -184, -185, -186, -187, and -189) interfere with the MHC I antigen-processing pathway. We demonstrate that Rh182 and Rh189 function similarly to HCMV US2 and US11, respectively, mediating the proteasomal degradation of newly synthesized MHC I. The US3 homologue, Rh184, delayed MHC I maturation. Unlike US3, MHC I molecules eventually escaped retention by Rh184, so that steady-state surface levels of MHC I remained unchanged. Rh185 acted similarly to US6 and inhibited peptide transport by TAP and, consequently, peptide loading of MHC I molecules. Thus, despite relatively low sequence conservation, US6 family-related genes in RhCMV are functionally closely related to the conserved structural features of HCMV immunomodulators. The conservation of these mechanisms implies their importance for immune evasion in vivo, a question that can now be addressed experimentally.     

Characterization of Marek's disease virus insertion and deletion mutants that lack US1 (ICP22 homolog), US10, and/or US2 and neighboring short-component open reading frames.

We report the characterization of Marek's disease virus (MDV) strains having mutations in various genes that map to the unique short (US) region of the viral genome. A deletion mutant (GA delta 4.8lac) lacks 4.8 kbp of US region DNA, the deleted segment having been replaced by the lacZ gene of Escherichia coli. This deletion results in the loss of the MDV-encoded US1, US10, and US2 homologs of herpes simplex virus type 1, as well as three putative MDV-specific genes, Sorf1, Sorf2, and Sorf3. Two mutants containing lacZ insertions in the US1 and US10 genes have been constructed, and we have previously reported a US2lac insertion mutant (J. L. Cantello, A. S. Anderson, A. Francesconi, and R. W. Morgan, J. Virol. 65:1584-1588, 1991). The isolation of these mutants indicates that the relevant genes are not required for growth of MDV in chicken embryo fibroblasts. The mutants had early growth kinetics indistinguishable from those of their parent viruses; however, 5 to 7 days after being plated, the US1 insertion mutant (US1lac) and the GA delta 4.8lac deletion mutant showed a 5- to 10-fold decrease in virus growth. This decrease in virus accumulation correlated with a 30 to 50% decrease in plaquing efficiency when these viruses were plated onto established versus fresh chicken embryo fibroblast monolayers compared with a 10 to 15% decrease seen for the parent viruses and for the US10lac or US2lac insertion mutants. Finally, GA delta 4.8lac could be reisolated from chickens, indicating that the deleted genes are not required for the infection of chickens following intra-abdominal inoculation of an attenuated serotype 1 MDV.     

Analysis of a 1-Mb BAC contig overlapping the mouse Nkrp1 cluster of genes: cloning of three new Nkrp1 members, Nkrp1d, Nkrp1e, and Nkrp1f

The murine Nkrp1 gene family encodes three previously identified activation and inhibitory receptors expressed on natural killer (NK) cells. This family includes the gene for NKR-P1C (NK1.1), the most specific serologic marker on C57BL/6-derived NK cells and is localized in a gene cluster in the NK gene complex (NKC). To further analyze the Nkrp1 family, we constructed and analyzed a bacterial artificial chromosome contig. A genomic organization of the Nkrp1 family was obtained and three new Nkrp1 genes were isolated from interleukin-2-activated NK cells. Thus, the Nkrp1 family adds to the repertoire of receptors expressed by NK cells.     

Human cytomegalovirus US7, US8, US9, and US10 are cytoplasmic glycoproteins, not found at cell surfaces, and US9 does not mediate cell-to-cell spread

Human cytomegalovirus (HCMV) expresses a large number of membrane proteins with unknown functions. One class of these membrane proteins apparently acts to allow HCMV to escape detection by the immune system. The best characterized of these are the glycoproteins encoded within the US2 to US11 region of the HCMV genome that mediate resistance to CD8(+) and CD4(+) T cells. US2, US3, US6, and US11 block various aspects of the major histocompatibility complex (MHC) class I and class II antigen presentation pathways, functioning in cytoplasmic membranes to cause retention, degradation, or mislocalization of MHC proteins. Distantly homologous genes in this region, US7, US8, US9, and US10, are not well characterized. Here, we report expression of the glycoproteins encoded by US7 to US10 by using replication-defective adenovirus (Ad) vectors. US7, US9, and US10 remained sensitive to endoglycosidase H and were exclusively or largely present in the endoplasmic reticulum (ER) as determined by confocal microscopy. US8 reached the Golgi apparatus and trans-Golgi network and was more quickly degraded. Previous studies suggested that US9 could localize to cell junctions and mediate cell-to-cell spread in ARPE-19 retinal epithelial cells. We found no evidence of US9 at cell junctions of HEC-1A epithelial cells. HCMV recombinants lacking US9 produced smaller plaques on ARPE-19 cell monolayers but also exhibited defects in virus replication compared with wild-type HCMV in these cells. Other HCMV recombinants constructed in a similar fashion that were able to express US9 also produced small plaques and some of these exhibited defects in production of infectious progeny in ARPE-19 cells. Thus, there was no correlation between defects in cell-to-cell spread (plaque size) and loss of expression of US9, and it is possible that US9(-) mutants produce smaller plaques because they produce fewer progeny. Together, our results do not support the hypothesis that US9 plays a direct role in HCMV cell-to-cell spread.     

A 15-kilobase-pair region of the human cytomegalovirus genome which includes US1 through US13 is dispensable for growth in cell culture.

The genome of a temperature-sensitive, DNA-negative mutant of human cytomegalovirus was cloned in cosmids and analyzed by restriction endonuclease mapping and Southern blotting. The data presented show that in the mutant genome, nearly half of the short segment was deleted (14.3 to 15.1 kb; map position, 0.83 to 0.9), including the genes for a potential immediate early protein (US3) and a structural glycoprotein of 47 to 52 kDa (US6 through US11). The deleted DNA region was replaced by a 20.8- to 21.6-kb fragment that represented an inverted repetition of the retained portion of the short segment (map position, 0.92 to 1.0), suggesting that US20 through US36 were duplicated in the mutant. Northern (RNA) blots with appropriate probes of total cell RNA extracted from mutant-infected cells confirmed the absence of mRNAs originating from US3 or from US8 through US11. It is concluded that the deleted genes are dispensable for human cytomegalovirus replication in cell culture.     

Horizontal transmission of Marek's disease virus requires US2, the UL13 protein kinase, and gC

Marek's disease virus (MDV) causes a general malaise in chickens that is mostly characterized by the development of lymphoblastoid tumors in multiple organs. The use of bacterial artificial chromosomes (BACs) for cloning and manipulation of the MDV genome has facilitated characterization of specific genes and genomic regions. The development of most MDV BACs, including pRB-1B-5, derived from a very virulent MDV strain, involved replacement of the US2 gene with mini-F vector sequences. However, when reconstituted viruses based on pRB-1B were used in pathogenicity studies, it was discovered that contact chickens housed together with experimentally infected chickens did not contract Marek's disease (MD), indicating a lack of horizontal transmission. Staining of feather follicle epithelial cells in the skins of infected chickens showed that virus was present but was unable to be released and/or infect susceptible chickens. Restoration of US2 and removal of mini-F sequences within viral RB-1B did not alter this characteristic, although in vivo viremia levels were increased significantly. Sequence analyses of pRB-1B revealed that the UL13, UL44, and US6 genes encoding the UL13 serine/threonine protein kinase, glycoprotein C (gC), and gD, respectively, harbored frameshift mutations. These mutations were repaired individually, or in combination, using two-step Red mutagenesis. Reconstituted viruses were tested for replication, MD incidence, and their abilities to horizontally spread to contact chickens. The experiments clearly showed that US2, UL13, and gC in combination are essential for horizontal transmission of MDV and that none of the genes alone is able to restore this phenotype.     

Characterization of synonymous codon usage bias in the pseudorabies virus US1 gene

In the present study, we examined the codon usage bias between pseudorabies virus (PRV) US1 gene and the US1-like genes of 20 reference alphaherpesviruses. Comparative analysis showed noticeable disparities of the synonymous codon usage bias in the 21 alphaherpesviruses, indicated by codon adaptation index, effective number of codons (ENc) and GC3s value. The codon usage pattern of PRV US1 gene was phylogenetically conserved and similar to that of the US1-like genes of the genus Varicellovirus of alphaherpesvirus, with a strong bias towards the codons with C and G at the third codon position. Cluster analysis of codon usage pattern of PRV US1 gene with its reference alphaherpesviruses demonstrated that the codon usage bias of US1-like genes of 21 alphaherpesviruses had a very close relation with their gene functions. ENc-plot revealed that the genetic heterogeneity in PRV US1 gene and the 20 reference alphaherpesviruses was constrained by G+C content, as well as the gene length. In addition, comparison of codon preferences in the US1 gene of PRV with those of E. coli, yeast and human revealed that there were 50 codons showing distinct usage differences between PRV and yeast, 49 between PRV and human, but 48 between PRV and E. coli. Although there were slightly fewer differences in codon usages between E.coli and PRV, the difference is unlikely to be statistically significant, and experimental studies are necessary to establish the most suitable expression system for PRV US1. In conclusion, these results may improve our understanding of the evolution, pathogenesis and functional studies of PRV, as well as contributing to the area of herpesvirus research or even studies with other viruses.     

Replacement mutagenesis of the human cytomegalovirus genome: US10 and US11 gene products are nonessential.

The US6 gene family, located within the unique short region (US) of the human cytomegalovirus (HCMV) genome, contains six open reading frames (US6 through US11) which may encode glycoproteins, such as gcII (D. Gretch, B. Kari, R. Gehrz, and M. Stinski, J. Virol. 62:1956-1962, 1988). By homologous recombination, several different recombinant HCMV were created which contain a marker gene, beta-glucuronidase, inserted within this gene family. It was demonstrated that beta-glucuronidase has utility as a marker gene for the identification of recombinants in this herpesvirus system, without the occurrence of deletions in other regions of the viral genome. DNA and RNA blot analyses attested to the fidelity of the recombination. Immunoprecipitation experiments using monospecific polyclonal antisera indicated that the US10 and/or US11 gene products were not expressed in the recombinants, as predicted. These results, along with single-cycle growth analyses, indicated that the US10 and US11 gene products are nonessential for virus replication and growth in tissue culture. HCMV recombinants expressing beta-glucuronidase seemed to be genetically stable.     

Genome sequence of benzo(a)pyrene-degrading bacterium Novosphingobium pentaromativorans US6-1

Novosphingobium pentaromativorans US6-1 showed a good ability to degrade high-molecular-weight polycyclic aromatic hydrocarbons. We report the draft genome sequence of strain US6-1, which contains a main chromosome (5,096,413 bp, G+C content of 63.1%) and two plasmids (188,476 and 60,085 bp). The majority of the aromatic-hydrocarbon-degrading genes are encoded in the larger plasmid.     

The MHC class I homolog of human cytomegalovirus is resistant to down-regulation mediated by the unique short region protein (US)2, US3, US6, and US11 gene products

Human CMV encodes four unique short region proteins (US), US2, US3, US6, and US11, each independently sufficient for causing the down-regulation of MHC class I molecules on the cell surface. This down-regulation allows infected cells to evade recognition by cytotoxic T cells but leaves them susceptible to NK cells, which lyse cells that lack class I molecules. Another human CMV-encoded protein, unique long region protein 18 (UL18), is an MHC class I homolog that might provide a mechanism for inhibiting the NK cell response. The sequence similarities between MHC class I molecules and UL18 along with the ability of UL18 to form trimeric complexes with beta(2)-microglobulin and peptides led to the hypothesis that if the US and UL18 gene products coexist temporally during infection, the US proteins might down-regulate UL18 molecules, similar to their action on MHC class I molecules. We show here that temporal expression of US and UL18 genes partially overlaps during infection. However, unlike MHC class I molecules, the MHC class I homolog, UL18, is fully resistant to the down-regulation associated with the US2, US3, US6, and US11 gene products. The specific effect of US proteins on MHC class I molecules, but not on UL18, represents another example of how viral proteins have evolved to evade immune surveillance, avoiding fratricide by specifically targeting host proteins.     

Like one of the family: race,ethnicity, and the paradox of US national identity

The tensions between individual rights promised to US citizens and group discrimination targeted against African Americans and similar racial/ethnic groups constitute one enduring paradox of US society. This essay examines this paradox by exploring how a gendered family rhetoric contributes to understandings of race and US national identity. Using African American women's experiences as a touchstone for analysis, the article suggests that African American women's treatment as second-class citizens reflects a belief that they are 'like one of the family', that is, legally part of the US nation-state, but simultaneously subordinated within it. To investigate these relationships, the article examines 1) how intersecting social hierarchies of race and ethnicity foster racialized understandings of US national identity; 2) how the gendered rhetoric of the American family ideal naturalizes and normalizes social hierarchies; and 3) how gendered family rhetoric fosters racialized constructions of US national identity as a large national family.     

Role of murine cytomegalovirus US22 gene family members in replication in macrophages

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.     

Expression of HA of HPAI H5N1 virus at US2 gene insertion site of turkey herpesvirus induced better protection than that at US10 gene insertion site

Herpesvirus of turkey (HVT) is being widely used as a vector for development of recombinant vaccines and US2 and US10 genes are often chosen as insertion sites for targeted gene expression. However, the different effects of the two genes for generation of recombinant HVT vaccines were unknown. In order to compare the effects of inserted genes in the two sites on the efficacy of the recombinant vaccines, host-protective haemagglutinin (HA) gene of the highly pathogenic avian influenza virus (HPAIV) H5N1 was inserted into either US2 or US10 gene locus of the HVT. The resulting US2 (rHVT-US2-HA) or US10 (rHVT-US10-HA) recombinant HVT viruses were used to infect chicken embryo fibroblasts. Plaques and the growth kinetics of rHVT-US2-HA-infected chicken embryo fibroblasts were similar to those of parental HVT whereas rHVT-US10-HA infected chicken embryo fibroblasts had different growth kinetics and plaque formation. The viremia levels in rHVT-US10-HA virus-infected chickens were significantly lower than those of rHVT-US2-HA group on 28 days post infection. The vaccine efficacy of the two recombinant viruses against H5N1 HPAIV and virulent Marek's disease virus was also evaluated in 1-day-old vaccinated chickens. rHVT-US2-HA-vaccinated chickens were better protected with reduced mortality than rHVT-US10-HA-vaccinated animals following HPAIV challenge. Furthermore, the overall hemaglutination inhibition antibody titers of rHVT-US2-HA-vaccinated chickens were higher than those of rHVT-US10-HA-vaccinated chickens. Protection levels against Marek's disease virus challenge following vaccination with either rHVT-US2-HA or rHVT-US10-HA, however, were similar to those of the parental HVT virus. These results, for the first time, indicate that US2 gene provides a favorable foreign gene insertion site for generation of recombinant HVT vaccines.     

The human cytomegalovirus gene product US6 inhibits ATP binding by TAP

Human cytomegalovirus (HCMV) encodes several genes that disrupt the major histocompatibility complex (MHC) class I antigen presentation pathway. We recently described the HCMV-encoded US6 gene product, a 23 kDa endoplasmic reticulum (ER)-resident type I integral membrane protein that binds to the transporter associated with antigen processing (TAP), inhibits peptide translocation and prevents MHC class I assembly. The functional consequence of this inhibition is to prevent the cell surface expression of class I bound viral peptides and their recognition by HCMV-specific cytotoxic T cells. Here we describe a novel mechanism of action for US6. We demonstrate that US6 inhibits the binding of ATP by TAP1. This is a conformational effect, as the ER lumenal domain of US6 is sufficient to inhibit ATP binding by the cytosolic nucleotide binding domain of TAP1. US6 also stabilizes TAP at 37 degrees C and prevents conformational rearrangements induced by peptide binding. Our findings suggest that the association of US6 with TAP stabilizes a conformation in TAP1 that prevents ATP binding and subsequent peptide translocation.