Mechanisms of Establishment and Maintenance of RNA Virus Persistence in Primary Lymphocytes: a Dissertation

RNA virus persistence in lymphocytes has been studied extensively in vitro, but the influence of lymphocyte homeostatic mechanisms and antiviral immunity on persistence has not been well studied in an in vivo system. It is demonstrated here that vesicular stomatitis virus (VSV), a negative-strand RNA virus, is maintained in B lymphocytes in vivo despite the existence of homeostatic mechanisms that drive the cells to proliferate under conditions of B cell deficiency and a strong antibody response to the virus. It is also shown that antiviral antibodies inhibit VSV reactivation from persistently infected primary B cells in vitro. A model is proposed for virus persistence in vivo in which B cell homeostatic signals drive virus expression in some infected cells, resulting in an antibody response, which maintains virus persistence in B cells. In the course of conducting experiments to define the homeostatic signals that might act on persistently infected B cells in vivo, it was found that a fraction of small, resting splenic B cells proliferates after adoptive transfer into B cell deficient hosts (sublethally irradiated, xid, or SCID). This process, termed homeostatic proliferation, is driven by B cell deficiency since proliferation is limited in B cell sufficient hosts. This reveals the existence of a mechanism by which B cells sense their own numbers. The proliferation is unique in that the replicating cells do not upregulate cell surface markers, such as CD25 and B7-2, associated with antigen or mitogen induced proliferation. They do, however, show transient increases in other activation markers (CD69, CD71), demonstrating the action of an inductive signal. Homeostatic proliferation is a property of both mature and immature B cells, but in competition experiments, only mature B cells inhibit proliferation. xid B cells express a defective form of Bruton\u27s tyrosine kinase (Btk); as a result, these cells proliferate poorly in response to stimulation through a number of cell surface receptors including the BCR, IL-5R, IL-10R, the toll-like receptor RP-105, and CD38. Homeostatic proliferation is severely reduced in xid B cells; thus, this process is regulated by a Btk-dependent inductive signal, which is counterbalanced by an inhibitory signal provided by mature B cells. B cell homeostatic proliferation does not rely on transcription factors (c-rel and p50) critical for conventional proliferation induced by antigen or mitogen (c-rel), or for peripheral B cell survival (p50), suggesting that multiple signals drive this process and that survival and proliferation signals are not identical. VSV persists in small, resting primary B cells for several weeks in vitro, and virus replication is restricted at multiple levels depending on the activation state of the cells. After adoptive transfer of infected B cells into B cell deficient (xid) recipients, viral RNA, but not infectious particles, can be detected by RT-PCR in recipient spleens for at least 72 days. RT-PCR analysis of FACS sorted donor cells stained with CFSE reveals that viral RNA is maintained in transferred B cells but can also found in recipient cells. Infected B cells can undergo homeostatic proliferation and an antibody response is generated to the virus, suggesting that homeostatic signals induce virus expression in some transferred cells. Virus persistence is maintained despite an active immune response to the virus. In fact, persistence may be maintained by antiviral antibody since in vitro treatment of infected primary B cells with anti-VSV antibody inhibits virus reactivation at multiple levels (transcription, protein synthesis, assembly/release of infectious particles). This inhibition is reversible upon antibody removal, demonstrating that functional virus is maintained in antibody treated cells. Antibody specific for a single viral protein (VSV G) is sufficient since inhibition is mediated by monoclonal antibodies specific for a VSV G; neutralizing activity is not required because inhibition occurs with non-neutralizing monoclonal antibodies to VSV G. It is proposed that antibody binding to VSV G on infected B cells generates inhibitory signal(s) that suppress signaling pathways required for virus replication in B cells. Finally, a model of RNA virus persistence in B cells is proposed in which lymphocyte homeostatic signals promote virus expression, leading to the production of antiviral antibodies, which suppress virus replication inside infected B cells and help to maintain persistence

Expression of a human coxsackie/adenovirus receptor transgene permits adenovirus infection of primary lymphocytes

Replication-defective adenoviruses are effective vehicles for gene transfer, both for the repair of defective genes and for studies of gene function in primary cells. Many cell types, including lymphocytes, are refractory to adenovirus infection because they lack the Coxsackie/adenovirus receptor (CAR) needed for virus attachment. To extend the advantages of adenovirus-mediated gene transfer to primary lymphoid populations and other cell types lacking endogenous CAR, we produced a mouse that expresses human (h) CAR as a transgene under control of a murine MHC class I promoter. hCAR protein is expressed on T and B lymphocytes from a variety of organs (spleen, lymph node, bone marrow, thymus, and peritoneum). These lymphocytes are susceptible to adenovirus infection, as demonstrated by reporter green fluorescent protein gene expression, with the fraction of expressing cells as high as 70%. Some lymphocyte subpopulations required stimulation subsequent to adenovirus infection for reporter expression. This activation requirement is a restriction imposed by the promoter used in the adenovirus construct. In subpopulations requiring activation, the elongation factor 1 promoter was far superior to a hCMV promoter for directing green fluorescent protein production. We also find that hCAR mRNA is produced in nonlymphoid tissues from all founder lines, including tissues that do not express endogenous murine CAR, suggesting the opportunity for effecting gene delivery to and testing gene function in a wide variety of primary cell types previously resistant to gene transfer

Naive B lymphocytes undergo homeostatic proliferation in response to B cell deficit

Naive peripheral B cells are maintained in sufficient numbers and diversity to mount effective immune responses against infectious agents. However, the size and repertoire of this B cell pool is constantly diminished by normal cell turnover and Ag activation. Homeostatic (Ag-independent) proliferation in response to B cell depletion is one mechanism to compensate for this cell loss. We have used purified CFSE-labeled B cells and an adoptive transfer model system to show that immature and mature B cells divide in a variety of B cell-deficient (scid, xid, IL-7(-/-), and sublethally irradiated) hosts. Homeostatic B cell proliferation is T cell independent, and B cells that have replicated by this mechanism retain the antigenic phenotype of naive B cells. Replication is significantly reduced in B cell-sufficient normal or B cell-reconstituted immunodeficient recipients by the action of competing mature follicular B cells. Using xid mice and transcription factor knockouts, we show that the activation signal(s) that lead to homeostatic B cell proliferation require Bruton\u27s tyrosine kinase; however, c-Rel, a Bruton\u27s tyrosine kinase-induced NF-kappaB/Rel transcription factor critical for Ag and mitogen stimulation, is dispensable, indicating the uniqueness of this activation pathway. Survival and replication signals can also be separated, because the transcription factor p50 (NF-kappaB1), which is required for the survival of peripheral B cells, is not necessary for homeostatic replication. Homeostatic B cell proliferation provides an Ag-independent mechanism for the maintenance and expansion of naive B cells selected into the mature B cell pool

Site-Directed Mutagenesis of Large DNA Palindromes: Construction and In Vitro Characterization of Herpes Simplex Virus Type 1 Mutants Containing Point Mutations That Eliminate the oriL or oriS Initiation Function

Technical challenges associated with mutagenesis of the large oriL palindrome have hindered comparisons of the functional roles of the herpes simplex virus type 1 (HSV-1) origins of DNA replication, oriL and oriS, in viral replication and pathogenesis. To address this problem, we have developed a novel PCR-based strategy to introduce site-specific mutations into oriL and other large palindromes. Using this strategy, we generated three plasmids containing mutant forms of oriL, i.e., pDoriL-I(L), pDoriL-I(R), and pDoriL-I(LR), containing point mutations in the left, right, and both copies, respectively, of the origin binding protein (OBP) binding site (site I) which eliminate OBP binding. In in vitro DNA replication assays, plasmids with mutations in only one arm of the palindrome supported origin-dependent DNA replication, whereas plasmids with symmetrical mutations in both arms of the palindrome were replication incompetent. An analysis of the cloned mutant plasmids used in replication assays revealed that a fraction of each plasmid mutated in only one arm of the palindrome had lost the site I mutation. In contrast, plasmids containing symmetrical mutations in both copies of site I retained both mutations. These observations demonstrate that the single site I mutations in pDoriL-I(L) and pDoriL-I(R) are unstable upon propagation in bacteria and suggest that functional forms of both the left and right copies of site I are required to initiate DNA replication at oriL. To examine the role of oriL and oriS site I in virus replication, we introduced the two site I mutations in pDoriL-I(LR) into HSV-1 DNA to yield the mutant virus DoriL-I(LR) and the same point mutations into the single site I sequence present in both copies of oriS to yield the mutant virus DoriS-I. In Vero cells and primary rat embryonic cortical neurons (PRN) infected with either mutant virus, viral DNA synthesis and viral replication were efficient, confirming that the two origins can substitute functionally for one another in vitro. Measurement of the levels of oriL and oriS flanking gene transcripts revealed a modest alteration in the kinetics of ICP8 transcript accumulation in DoriL-I(LR)-infected PRN, but not in Vero cells, implicating a cell-type-specific role for oriL in regulating ICP8 transcription