The HSV-1 protein ICP34.5 interacts with the cellular phosphatase PP1α to mediate the dephosphorylation of eIF2α and thus antagonizes the PKR signaling pathway () (20). Recently, we utilized mutant viruses lacking one or more viral genes to show that the deletion of either the gK or UL20 gene produced significantly greater defects in virion envelopment and overall virus replication than deletion of the carboxyl terminus of either gD, UL11, gM, or gE alone or in various combinations (44). Further analysis of Us11 mutants revealed that Us11 is also required for maximal rates of translation when expressed in its natural context late in infection (M. Pearson from pMAL-c2X [New England Biolabs (NEB)] to contain a PreScission Protease site inserted into the XmnI restriction site, between sequences encoding maltose-binding protein (MBP) and its fusion partner (in this case, US11) (18). The objective of this study was to discover whether US11 had the potential to bind any other transcripts from HSV-infected cells that would be relevant to its role in the virus lytic cycle. Later during infection, the US11 protein accumulates into nucleoli and is also found in RNP fibrils as well as in clusters of interchromatin granules. For the PRV construct, the UL11 gene was amplified from the PRV Becker genome with a forward primer (5′-GCCTGCGAGATCTCCGTGCTGCTGATCGTCACG-3′) complementary to the sequence 100 bp upstream of the start codon and a reverse primer (5′-CAAACGGGTTTATTGGAATTCGTACGCCCGCGAG-3′) complementary to the 3′ end of the UL11 gene.

HSV-1 UL11 was recently shown to form a protein complex with gE, UL16, and UL21 that may be required for efficient virus spread (43). To test this hypothesis, we used CD11c-diphtheria toxin (DT) receptor-green fluorescent protein (GFP)-transgenic (Tg) mice, generated on a C57BL/6 (B6) background, in which DCs can be transiently depleted in vivo by treatment with low doses of DT (15). Cell lines and viruses.The retroviral packaging cell lines PE501, PA317, and PG13 were grown in Dulbecco’s modified Eagle medium with 10% heat-inactivated fetal bovine serum (FBS; HyClone Laboratories, Logan, Utah) (41, 42). This segment of Us11 localizes to the nucleolus, associates with polysomes, and contains a region important for interaction with PKR in an RNA-dependent manner (5, 27, 39, 40). Translation of most transcripts is cap dependent and initiated by the cap-binding complex eIF4F (composed of eIF4E, eIF4G, eIF4A, eIF4B, and eIF4H) binding to the mRNA 5′ m7G-cap via its eIF4E subunit. Once there, CD1d cycles between the cell surface and endosomal compartment through multiple rounds of endocytosis and recycling steps while surveying lipid antigens for NKT cell recognition (3, 9, 15). UL16 is a 373-aa tegument protein that is also conserved among all herpesviruses (24, 34, 48, 55, 58, 73).

Using a murine model of acute hematogenous HSV-1 infection, we previously reported that viral neuroinvasion was reduced in mice lacking ApoE compared to wild-type mice and that the ApoE dose was directly linked to the HSV-1 DNA concentration detected in the nervous system (8). ↵† Supplemental material for this article may be found at Recognized vehicles of transmission are saliva, semen, cervical fluid, or vesicle fluid from active lesions (63). LK Gilbert et al. Phone: (415) 476-4419. The infant showed normal growth and development at 4 years of age. These findings have implications on the role of BER in viral genome maintenance during lytic replication and reactivation from latency.

Weller, J. Thus, both MLH1 and MSH2 are required but appear to participate in distinct events in the virus life cycle. Mailing address: Department of Microbiology, University of Minnesota Medical School, MMC 196, 420 Delaware St. Eight vidarabine-treated patients (22 percent) and three acyclovir-treated patients (9 percent) had moderate debility. In these cells, PKR is activated but eIF-2α is not phosphorylated, and the phosphatase is not redirected to dephosphorylate eIF-2α. The carboxyl-terminal half of the protein consists of a regular tripeptide repeat of the sequence RXP and constitutes a completely novel RNA-binding domain. Laboratory abnormalities at diagnosis associated with mortality were high creatinine, low platelet counts, prolonged partial thromboplastin time, and a high percentage of band forms on the blood smear.

We undertook genetic approaches to address these issues by infecting two different dominant negative TGF-β receptor type II transgenic mouse lines. RESULTS: There were 16 girls and 16 boys, aged 9-44 months (median age, 19 months). Recent studies using knockout (KO) mice demonstrated that the gD receptors herpesvirus entry mediator (HVEM) and nectin-1 are the primary entry receptors for HSV-2 in the mouse vagina and brain. Further, in vitro studies demonstrated that a recombinant expressed US11 protein binds PKR, blocks the phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF-2α) by activated PKR, and, if provided prior to PKR activation, precluded PKR autophosphorylation. Our study shows that glycosaminoglycans and more specifically heparan sulfate (HS) expressed on the cell surface and extracellular matrix may act as negative regulator of cell-cell fusion.

that UL47 also encoded two virion tegument phosphoproteins, VP13 and VP14 (VP13/14) (G. Us11 is a late gene product of HSV-1, which is also able to preclude the host shutoff by direct inhibition of PKR. Us11 is a late gene product of HSV-1, which is also able to preclude the host shutoff by direct inhibition of PKR. The results suggest that the U(S)11 interaction with PKR at the ribosome is RNA dependent and that the U(S)11 protein contains a substrate domain with homology to eIF-2alpha in close proximity to an essential binding domain. Recent work has suggested that gB is the sole fusogenic glycoprotein, regulated by interactions with the viral glycoproteins gD, gH/gL, and gK, membrane protein UL20, and cellular receptors. Thus, at various times during the viral life cycle and in different intracellular compartments, Us11 has the potential to execute discrete tasks. Similar analyses of the UL47 deletion mutants confirmed an earlier report by McLean et al.

The analysis of these functions, however, is complicated by the fact that Us11 is not essential for viral replication in cultured cells. US11 bound specifically to selected aptamers with an affinity of 70 nM. In vitro studies have demonstrated that nucleolin interacts with US11 and that the C-terminal domain of US11, which is required for US11 nucleolar accumulation, is sufficient for interaction with nucleolin. Gluzman, EMBO J. The results indicate that US9, US10, US11, and US12 gene products are dispensable for its neurovirulence and latency in mice. No detectable modification of the overall pattern of protein synthesis was observed in cells growing at normal temperatures, including no induction of Hsp expression or accumulation. This RNA sequence, designated 12/14, is present in the coterminal HSV-1 mRNAs UL12, UL13, and UL14.

The relationship between the expression of UL11 and resistance to HSV infection in US11cl19 cells has not been defined, but the block to infection with wild-type HSV-1 was overcome by exposing cells with attached virus on their surface to the fusogen polyethylene glycol, suggesting that the block to infection preceded the fusion of viral and cellular membranes. This RNA sequence, designated 12/14, is present in the coterminal HSV-1 mRNAs UL12, UL13, and UL14. US11 significantly inhibited Sendai virus (SeV)-induced IFN-β production, and its double-stranded RNA (dsRNA) binding domain was indispensable for this inhibition activity. In an attempt to better understand the multiple functions of US11, we identified cellular binding partners of this protein by using the yeast two-hybrid system. The goal of the experiments described here was to identify and characterize binding partners (viral or cellular) of UL11. Consistent with this hypothesis, the enhanced Akt activation that occurs during US3-null infection requires VP11/12 and correlates with an increase in SFK-dependent VP11/12 tyrosine phosphorylation. Recombinant viruses were constructed to abolish either gM or UL11 expression in the presence of strong syncytial mutations in either gB or gK.

Conversely, depletion of DCs was associated with reduced latency. Similar analyses also revealed that the inhibitory effect was mediated by an interaction between the C-terminal half of Us11 and the N-terminal domain of PKR. This 96-amino-acid, myristylated protein accumulates on the cytoplasmic face of internal membranes, where it is thought to play a role in nucleocapsid envelopment and egress. A. We determined which structural features of true late promoters are responsible for the stringent requirement for viral DNA replication by inserting a series of simple model constructs into the HSV-1 genome in place of one of the two promoters of the UL24 gene. Certain mutations in glycoprotein B (gB), gK, UL20, and other viral genes drastically enhance virus-induced cell fusion in vitro and in vivo. In addition, HeLa-derived cell lines have been selected for their ability to express Us11 protein constitutively.

The role of DCs as mediators of resistance to HSV-1 infection was investigated using CD11c-diphtheria toxin (DT) receptor-green fluorescent protein transgenic mice, in which DCs can be transiently depleted in vivo by treatment with low doses of DT. The Us11-Beclin 1 interaction was tested in complete medium or after stimulating autophagy by poly(I · C) (top panel). (A) Representative viral plaques of HSV-1(F)-YE102 wild-type virus and the syncytial mutant viruses gBΔ28 and gKsyn20 on Vero cells. (A and B) HEK293T cells (∼5 × 106) were cotransfected with 10 μg of plasmid pEF-Flag-RIG-I (A) or pEF-Flag-MDA-5 (B) or 10 μg of plasmid pMyc-MAVS and 10 μg of plasmid US11-HA or transfected with empty vector. The precise role of each of the seven individual CD11c+ dendritic cell subsets (DCs) identified to date in the response to viral infections is not known. Herpesviruses evade cytotoxic T-lymphocyte (CTL) recognition by expressing genes which interfere selectively with presentation of viral antigens by class I major histocompatibility complex (MHC) molecules. Since viral genes are transcribed by cellular RNA polymerase II (RNA pol II), ICP4 must interact with components of the pol II machinery to regulate viral gene expression.