Sandra K. Weller

Human Marginal Zone B Cells

Human marginal zone (MZ) B cells are, in a sense, a new entity. Although they share many properties with their mouse counterpart, they also display striking differences, such as the capacity to recirculate and the presence of somatic mutations in their B cell receptor. These differences are the reason they are often not considered a separate, rodent-like B cell lineage, but rather are considered IgM memory B cells. We review here our present knowledge concerning this subset and the arguments in favor of the proposition that humans have evolved for their MZ B cell compartment a separate B cell population that develops and diversifies its Ig receptor during ontogeny outside T-dependent or T-independent immune responses.

The UL6 Gene Product Forms the Portal for Entry of DNA into the Herpes Simplex Virus Capsid

ABSTRACT During replication of herpes simplex virus type 1 (HSV-1), viral DNA is synthesized in the infected cell nucleus, where DNA-free capsids are also assembled. Genome-length DNA molecules are then cut out of a larger, multigenome concatemer and packaged into capsids. Here we report the results of experiments carried out to test the idea that the HSV-1 UL6 gene product (pUL6) forms the portal through which viral DNA passes as it enters the capsid. Since DNA must enter at a unique site, immunoelectron microscopy experiments were undertaken to determine the location of pUL6. After specific immunogold staining of HSV-1 B capsids, pUL6 was found, by its attached gold label, at one of the 12 capsid vertices. Label was not observed at multiple vertices, at nonvertex sites, or in capsids lacking pUL6. In immunoblot experiments, the pUL6 copy number in purified B capsids was found to be 14.8 ± 2.6. Biochemical experiments to isolate pUL6 were carried out, beginning with insect cells infected with a recombinant baculovirus expressing the UL6 gene. After purification, pUL6 was found in the form of rings, which were observed in electron micrographs to have outside and inside diameters of 16.4 ± 1.1 and 5.0 ± 0.7 nm, respectively, and a height of 19.5 ± 1.9 nm. The particle weights of individual rings as determined by scanning transmission electron microscopy showed a majority population with a mass corresponding to an oligomeric state of 12. The results are interpreted to support the view that pUL6 forms the DNA entry portal, since it exists at a unique site in the capsid and forms a channel through which DNA can pass. The HSV-1 portal is the first identified in a virus infecting a eukaryote. In its dimensions and oligomeric state, the pUL6 portal resembles the connector or portal complexes employed for DNA encapsidation in double-stranded DNA bacteriophages such as φ29, T4, and P22. This similarity supports the proposed evolutionary relationship between herpesviruses and double-stranded DNA phages and suggests the basic mechanism of DNA packaging is conserved.

A herpes simplex virus ribonucleotide reductase deletion mutant is defective for productive acute and reactivatable latent infections of mice and for replication in mouse cells.

Herpes simplex virus encodes a ribonucleotide reductase that is not essential for virus growth in dividing cells at 37 degrees. This enzyme has been proposed as a target for antiviral drugs; its utility in this regard could depend upon its importance in vivo. To test the requirement of viral ribonucleotide reductase in a mammalian host, we tested a mutant virus, lacking most of the gene encoding the ribonucleotide reductase large subunit, in a mouse eye model of pathogenesis and latency where the wild-type virus establishes reactivatable latent infections in trigeminal ganglia following corneal inoculation. The deletion mutant was severely impaired in its ability to replicate acutely in the eye and in the trigeminal ganglion and failed to establish reactivatable latent infections. In contrast, a recombinant virus in which the deleted sequences were restored was competent for both acute and latent infections. The defects of the deletion mutant in the mouse may be related to its severely impaired growth at 38 degrees in mouse cells relative to its growth in Vero cells. These results indicate that ribonucleotide reductase is critical for productive acute and reactivatable latent infections in mice and replication in mouse cells at 38 degrees and suggest that caution be exercised in extrapolating from studies conducted in mice to human infections when judging the utility of this enzyme as a target for antiviral chemotherapy.

Factor(s) present in herpes simplex virus type 1-infected cells can compensate for the loss of the large subunit of the viral ribonucleotide reductase: characterization of an ICP6 deletion mutant

Herpes simplex virus type 1 encodes a ribonucleotide reductase (RR) consisting of two subunits (140 and 38 kDa) whose genes map to coordinates 0.56 to 0.60 on the viral genome. We previously reported the isolation and characterization of a mutant with a lacZ insertion into the large subunit (ICP6) gene (Goldstein and Weller, 1988). Studies with this blue-plaque mutant, hrR3, showed that the viral RR activity is not essential in dividing cells in culture. This mutant, however, synthesizes the N-terminal one-third (434 amino acids) of ICP6 which may have an additional, required function. To test this possibility, a deletion of the ICP6 gene was created by introducing a deleted ICP6 gene into infectious hrR3 DNA and screening for white plaques from a background of blue plaques. Studies with this mutant, ICP6 delta, demonstrated that ICP6 is not required for virus growth and DNA synthesis in dividing cells in culture. However, we show that the ability of ICP6 delta to grow and induce viral DNA synthesis is dependent on the state of the infected cells; ICP6 delta is severely compromised in nondividing cells or in cells at 39.5 degrees. We propose that an alternate pathway(s) for obtaining deoxyribonucleotides is operating in infected cells and can compensate for defects in viral RR. In addition, our experiments suggest that these alternate sources are not available either in nondividing cells or in cells at 39.5 degrees.

Recruitment of Cellular Recombination and Repair Proteins to Sites of Herpes Simplex Virus Type 1 DNA Replication Is Dependent on the Composition of Viral Proteins within Prereplicative Sites and Correlates with the Induction of the DNA Damage Response

ABSTRACT Herpes simplex virus type 1 (HSV-1) DNA replication is associated with nuclear domains called ND10, which contain host recombination proteins such as RPA, RAD51, and NBS1 and participate in the cells response to DNA damage. The stages of HSV-1 infection have been described previously. Infected cells at stage IIIa are observed after the initial disruption of ND10 and display nuclear foci, or prereplicative sites, containing the viral single-stranded-DNA-binding protein (UL29), the origin-binding protein (UL9), and the heterotrimeric helicase-primase. At stage IIIb, the viral polymerase, its processivity factor, and the ND10, protein PML, are also recruited to these sites. In this work, RPA, RAD51, and NBS1 were observed predominantly in stage IIIb but not stage IIIa prereplicative sites, suggesting that the efficient recruitment of these recombination proteins is dependent on the presence of the viral polymerase and other replication proteins within these sites. On the other hand, Ku86 was not found in any of the precursors to replication compartments, suggesting that it is excluded from the early stages of HSV-1 replication. Western blot analysis showed that RPA and NBS1 were (hyper)phosphorylated during infection, indicating that infection induces the host response to DNA damage. Finally, RPA, RAD51, and NBS1 were found to be associated with UL29 foci observed in transfected cells expressing UL29 and the helicase-primase heterotrimer and containing intact ND10. The ability to recruit recombination and repair proteins to various subassemblies of viral replication proteins thus appears to depend on several factors, including the presence of the viral polymerase and/or UL9 within prereplicative sites and the integrity of ND10.

Cloning, sequencing, and functional analysis of oriL, a herpes simplex virus type 1 origin of DNA synthesis.

The herpes simplex virus type 1 genome (160 kilobases) contains three origins of DNA synthesis: two copies of oriS located within the repeated sequences flanking the short unique arm (US), and one copy of oriL located within the long unique arm (UL). Precise localization and characterization of oriL have been severely hampered by the inability to clone sequences which contain it (coordinates 0.398 to 0.413) in an undeleted form in bacteria. We report herein the successful cloning of sequences between 0.398 to 0.413 in an undeleted form, using a yeast cloning vector. Sequence analysis of a 425-base pair fragment spanning the deletion-prone region has revealed a perfect 144-base pair palindrome with striking homology to oriS. In a functional assay, the undeleted clone was amplified when functions from herpes simplex virus type 1 were supplied in trans, whereas clones with deletions of 55 base pairs or more were not amplified.

Somatic diversification in the absence of antigen-driven responses is the hallmark of the IgM+IgD+CD27+ B cell repertoire in infants

T cell–dependent immune responses develop soon after birth, whereas it takes 2 yr for humans to develop T cell–independent responses. We used this dissociation to analyze the repertoire diversification of IgM+IgD+CD27+ B cells (also known as “IgM memory” B cells), comparing these cells with switched B cells in children <2 yr of age, with the aim of determining whether these two subsets are developmentally related. We show that the repertoire of IgM+IgD+CD27+ B cells in the spleen and blood displays no sign of antigen-driven activation and expansion on H-CDR3 spectratyping, despite the many antigenic challenges provided by childhood vaccinations. This repertoire differed markedly from those of switched B cells and splenic germinal center B cells, even at the early stage of differentiation associated with μ heavy chain expression. These data provide evidence for the developmental diversification of IgM+IgD+CD27+ B cells, at least in very young children, outside of T cell–dependent and –independent immune responses.

DNA Polymerase η Is Involved in Hypermutation Occurring during Immunoglobulin Class Switch Recombination

Base substitutions, deletions, and duplications are observed at the immunoglobulin locus in DNA sequences involved in class switch recombination (CSR). These mutations are dependent upon activation-induced cytidine deaminase (AID) and present all the characteristics of the ones observed during V gene somatic hypermutation, implying that they could be generated by the same mutational complex. It has been proposed, based on the V gene mutation pattern of patients with the cancer-prone xeroderma pigmentosum variant (XP-V) syndrome who are deficient in DNA polymerase η (pol η), that this enzyme could be responsible for a large part of the mutations occurring on A/T bases. Here we show, by analyzing switched memory B cells from two XP-V patients, that pol η is also an A/T mutator during CSR, in both the switch region of tandem repeats as well as upstream of it, thus suggesting that the same error-prone translesional polymerases are involved, together with AID, in both processes.

Herpes Simplex Virus DNA Cleavage and Packaging Proteins Associate with the Procapsid prior to Its Maturation

ABSTRACT Packaging of DNA into preformed capsids is a fundamental early event in the assembly of herpes simplex virus type 1 (HSV-1) virions. Replicated viral DNA genomes, in the form of complex branched concatemers, and unstable spherical precursor capsids termed procapsids are thought to be the substrates for the DNA-packaging reaction. In addition, seven viral proteins are required for packaging, although their individual functions are undefined. By analogy to well-characterized bacteriophage systems, the association of these proteins with various forms of capsids, including procapsids, might be expected to clarify their roles in the packaging process. While the HSV-1 UL6, UL15, UL25, and UL28 packaging proteins are known to associate with different forms of stable capsids, their association with procapsids has not been tested. Therefore, we isolated HSV-1 procapsids from infected cells and used Western blotting to identify the packaging proteins present. Procapsids contained UL15 and UL28 proteins; the levels of both proteins are diminished in more mature DNA-containing C-capsids. In contrast, UL6 protein levels were approximately the same in procapsids, B-capsids, and C-capsids. The amount of UL25 protein was reduced in procapsids relative to that in more mature B-capsids. Moreover, C-capsids contained the highest level of UL25 protein, 15-fold higher than that in procapsids. Our results support current hypotheses on HSV DNA packaging: (i) transient association of UL15 and UL28 proteins with maturing capsids is consistent with their proposed involvement in site-specific cleavage of the viral DNA (terminase activity); (ii) the UL6 protein may be an integral component of the capsid shell; and (iii) the UL25 protein may associate with capsids after scaffold loss and DNA packaging, sealing the DNA within capsids.

The human spleen is a major reservoir for long-lived vaccinia virus–specific memory B cells

The fact that you can vaccinate a child at 5 years of age and find lymphoid B cells and antibodies specific for this vaccination 70 years later remains an immunologic enigma. It has never been determined how these long-lived memory B cells are maintained and whether they are protected by storage in a special niche. We report that, whereas blood and spleen compartments present similar frequencies of IgG(+) cells, antismallpox memory B cells are specifically enriched in the spleen where they account for 0.24% of all IgG(+) cells (ie, 10-20 million cells) more than 30 years after vaccination. They represent, in contrast, only 0.07% of circulating IgG(+) B cells in blood (ie, 50-100,000 cells). An analysis of patients either splenectomized or rituximab-treated confirmed that the spleen is a major reservoir for long-lived memory B cells. No significant correlation was observed between the abundance of these cells in blood and serum titers of antivaccinia virus antibodies in this study, including in the contrasted cases of B cell-depleting treatments. Altogether, these data provide evidence that in humans, the two arms of B-cell memory--long-lived memory B cells and plasma cells--have specific anatomic distributions--spleen and bone marrow--and homeostatic regulation.