HCMV trimer- and pentamer-specific antibodies synergize for virus neutralization but do not correlate with congenital transmission.

Human cytomegalovirus (HCMV) causes substantial disease in transplant patients and harms the development of the nervous system in babies infected in utero. Thus, there is a major focus on developing safe and effective HCMV vaccines. Evidence has been presented that a major target of neutralizing antibodies (NAbs) is the HCMV pentamer glycoprotein gH/gL/UL128-131.

The Human Cytomegalovirus Trimer and Pentamer Promote Sequential Steps in Entry into Epithelial and Endothelial Cells at Cell Surfaces and Endosomes.

Human cytomegalovirus (HCMV) infects a wide variety of human cell types by different entry pathways that involve distinct envelope glycoprotein complexes that include gH/gL, a trimer complex consisting of gHgL/gO, and a pentamer complex consisting of gH/gL/UL128/UL130/UL131. We characterized the effects of soluble forms of these proteins on HCMV entry. Soluble trimer and pentamer blocked entry of HCMV into epithelial and endothelial cells, whereas soluble gH/gL did not.

CD147 Promotes Entry of Pentamer-Expressing Human Cytomegalovirus into Epithelial and Endothelial Cells.

Human cytomegalovirus (HCMV) replicates in many diverse cell types , and entry into different cells involves distinct entry mechanisms and different envelope glycoproteins. HCMV glycoprotein gB is thought to act as the virus fusogen, apparently after being triggered by different gH/gL proteins that bind distinct cellular receptors or entry mediators. A trimer of gH/gL/gO is required for entry into all cell types, and entry into fibroblasts involves trimer binding to platelet-derived growth factor receptor alpha (PDGFRα).

Fibroblast-adapted human CMV vaccines elicit predominantly conventional CD8 T cell responses in humans.

Cytomegalovirus (CMV)-based vaccines have shown remarkable efficacy in the rhesus macaque model of acquired immune deficiency syndrome, enabling 50% of vaccinated monkeys to clear a subsequent virulent simian immunodeficiency virus challenge. The protective vaccine elicited unconventional CD8 T cell responses that were entirely restricted by MHC II or the nonclassical MHC I molecule, MHC-E. These unconventional responses were only elicited by a fibroblast-adapted rhesus CMV vector with limited tissue tropism; a repaired vector with normal tropism elicited conventional responses.

Differentiated Levels of Ganciclovir Resistance Conferred by Mutations at Codons 591 to 603 of the Cytomegalovirus UL97 Kinase Gene.

Diagnostic mutations in the cytomegalovirus UL97 kinase gene are used to assess the level of associated ganciclovir resistance and therapeutic options. The best-known mutations at codons 460, 520, or 591 to 607 individually confer 5- to 10-fold-decreased ganciclovir susceptibility, except that a 3-fold decrease occurs in the case of the amino acid substitution C592G. Less common point and in-frame deletion mutations at codons 591 to 603 remain incompletely characterized.

Human Cytomegalovirus gH/gL Forms a Stable Complex with the Fusion Protein gB in Virions.

Human cytomegalovirus (HCMV) is a ubiquitous virus that is a major pathogen in newborns and immunocompromised or immunosuppressed patients. HCMV infects a wide variety of cell types using distinct entry pathways that involve different forms of the gH/gL glycoprotein: gH/gL/gO and gH/gL/UL128-131 as well as the viral fusion glycoprotein, gB. However, the minimal or core fusion machinery (sufficient for cell-cell fusion) is just gH/gL and gB.

PDGF receptor-α does not promote HCMV entry into epithelial and endothelial cells but increased quantities stimulate entry by an abnormal pathway.

Epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor-α (PDGFRα) were reported to mediate entry of HCMV, including HCMV lab strain AD169. AD169 cannot assemble gH/gL/UL128-131, a glycoprotein complex that is essential for HCMV entry into biologically important epithelial cells, endothelial cells, and monocyte-macrophages. Given this, it appeared incongruous that EGFR and PDGFRα play widespread roles in HCMV entry.

Human cytomegalovirus glycoprotein gO complexes with gH/gL, promoting interference with viral entry into human fibroblasts but not entry into epithelial cells.

A complex of five human cytomegalovirus virus (HCMV) proteins, gH, gL, UL128, UL130, and UL131 (gH/gL/UL128-131), is essential for virus entry into epithelial cells. We previously showed that gH/gL/UL128-131 expressed in epithelial cells interferes with subsequent HCMV entry into cells. There was no interference with only gH/gL or gB.

Cellular FLIP can substitute for the herpes simplex virus type 1 latency-associated transcript gene to support a wild-type virus reactivation phenotype in mice.

Latency-associated transcript (LAT) deletion mutants of herpes simplex virus type 1 (HSV-1) have reduced reactivation phenotypes. Thus, LAT plays an essential role in the latency-reactivation cycle of HSV-1. We have shown that LAT has antiapoptosis activity and demonstrated that the chimeric virus, dLAT-cpIAP, resulting from replacing LAT with the baculovirus antiapoptosis gene cpIAP, has a wild-type HSV-1 reactivation phenotype in mice and rabbits.

Human cytomegalovirus glycoproteins gB and gH/gL mediate epithelial cell-cell fusion when expressed either in cis or in trans.

Herpesviruses use a cascade of interactions with different cell surface molecules to gain entry into cells. In many cases, this involves binding to abundant glycosaminoglycans or integrins followed by interactions with more limited cell surface proteins, leading to fusion with cellular membranes. Human cytomegalovirus (HCMV) has the ability to infect a wide variety of human cell types in vivo.

Baculovirus DNA replication and processing.

In this report, factors involved in baculovirus DNA replication are reviewed. These include factors that are required for DNA synthesis, other factors that have been implicated in genome processing or packaging, and homologs of proteins that are involved in DNA replication or repair in other systems. Conservation of a number of these factors in all baculovirus genomes suggest that many of the observations for specific viral systems may apply to the most if not all members of the Baculoviridae.

Isolation and characterization of the DNA-binding protein (DBP) of the Autographa californica multiple nucleopolyhedrovirus.

DNA-binding protein (DBP) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) was expressed as an N-terminal His(6)-tag fusion using a recombinant baculovirus and purified to near homogeneity. Purified DBP formed oligomers that were crosslinked by redox reagents resulting in predominantly protein dimers and tetramers. In gel retardation assays, DBP showed a high affinity for single-stranded oligonucleotides and was able to compete with another baculovirus SSB protein, LEF-3, for binding sites.

Characterization of baculovirus constructs lacking either the Ac 101, Ac 142, or the Ac 144 open reading frame.

To investigate the role of the gene products encoded from the open reading frames 101, 142, and 144 of Autographa californica multiple nucleopolyhedrovirus (AcMNPV), a set of bacmid knockout and repair constructs were generated. The repair genes were engineered to contain an HA epitope tag at their C-termini. The results of transfection-infection assays and growth curve analyses showed that the Ac 101, 142, and 144 genes were required for infectious virus production.

Characterization of a baculovirus lacking the DBP (DNA-binding protein) gene.

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) encodes two proteins that possess properties typical of single-stranded DNA-binding proteins (SSBs), late expression factor-3 (LEF-3), and a protein referred to as DNA-binding protein (DBP). Whereas LEF-3 is a multi-functional protein essential for viral DNA replication, transporting helicase into the nucleus, and forms a stable complex with the baculovirus alkaline nuclease, the role for DBP in baculovirus replication remains unclear. Therefore, to better understand the functional role of DBP in viral replication, a DBP knockout virus was generated from an AcMNPV bacmid and analyzed.

A baculovirus alkaline nuclease knockout construct produces fragmented DNA and aberrant capsids.

DNA replication of bacmid-derived constructs of the Autographa californica multiple nucleocapsid nucleopolyhedrovirus (AcMNPV) was analyzed by field inversion gel electrophoresis (FIGE) in combination with digestion at a unique Eco81I restriction enzyme site. Three constructs were characterized: a parental bacmid, a bacmid deleted for the alkaline nuclease gene, and a bacmid from which the gp64 gene had been deleted. The latter was employed as a control for comparison with the alkaline nuclease knockout because neither yields infectious virus and their replication is limited to the initially transfected cells.

Characterization of the role of very late expression factor 1 in baculovirus capsid structure and DNA processing.

Very late expression factor 1 (VLF-1) of Autographa californica multiple nucleopolyhedrovirus is a putative tyrosine recombinase and is required for both very late gene expression and budded virus production. In this report, we show that a vlf-1 knockout bacmid was able to synthesize viral DNA at levels similar to that detected for a gp64 knockout bacmid that served as a noninfectious control virus. Additionally, analysis of replicated bacmid DNA by field-inversion gel electrophoresis indicated that VLF-1 is not required for synthesizing high-molecular-weight intermediates that could be resolved into unit-length genomes when cut at a unique restriction site.

Conserved molecular systems of the Baculoviridae.

Although the Baculoviridae are a large and diverse family of viruses, they are united by a number of shared features that form the basis for their unique life cycle. These include the mechanism of cell entry, genome replication and processing, and late and very late gene transcription. In this review, the molecular systems that are conserved within the Baculoviridae and that are responsible these processes are described.

Characterization of the replication of a baculovirus mutant lacking the DNA polymerase gene.

In a previous study, the DNA polymerase gene (dnapol) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) was identified as one of six genes required for plasmid replication in a transient replication assay (M. Kool, C. Ahrens, R.

Characterization of a baculovirus lacking the alkaline nuclease gene.

The Autographa californica multiple nucleocapsid nucleopolyhedrovirus (AcMNPV) alkaline nuclease (AN) associates with the baculovirus single-stranded DNA binding protein LEF-3 and possesses both a 5'-->3' exonuclease and an endonuclease activity. These activities are thought to be involved in DNA recombination and replication. To investigate the role of AN in AcMNPV replication, the lambda Red system was used to replace the an open reading frame with a chloramphenicol acetyltransferase gene (cat) and a bacmid containing the AcMNPV genome in Escherichia coli.

Characterization of a baculovirus with a deletion of vlf-1.

Very late expression factor (VLF-1) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is essential for high levels of expression of the very late genes p10 and polh, and evidence suggests VLF-1 may also be involved in viral DNA replication. In this study, investigations determined whether VLF-1 is essential for viral DNA replication by generating a vlf-1 knockout bacmid containing the AcMNPV genome through homologous recombination in Escherichia coli. Additionally, a vlf-1 repair bacmid was constructed by transposing the vlf-1 ORF and native promoter region into the polh locus of the vlf-1 knockout bacmid.