Measles virus superinfection immunity and receptor redistribution in persistently infected NT2 cells

Abstract

A recombinant measles virus (MV) expressing red fluorescent protein (MVDsRed1) was used to produce a persistently infected cell line (piNT2-MVDsRed1) from human neural precursor (NT2) cells. A similar cell line (piNT2-MVeGFP) was generated using a virus that expresses enhanced green fluorescent protein. Intracytoplasmic inclusions containing the viral nucleocapsid protein were evident in all cells and viral glycoproteins were present at the cell surface. Nevertheless, the cells did not release infectious virus nor did they fuse to generate syncytia. Uninfected NT2 cells express the MV receptor CD46 uniformly over their surface, whereas CD46 was present in cell surface aggregates in the piNT2 cells. There was no decrease in the overall amount of CD46 in piNT2 compared to NT2 cells. Cell-to-cell fusion was observed when piNT2 cells were overlaid onto confluent monolayers of MV receptor-positive cells, indicating that the viral glycoproteins were correctly folded and processed. Infectious virus was released from the underlying cells, indicating that persistence was not due to gross mutations in the virus genome. Persistently infected cells were superinfected with MV or canine distemper virus and cytopathic effects were not observed. However, mumps virus could readily infect the cells, indicating that superinfection immunity is not caused by general soluble antiviral factors. As MVeGFP and MVDsRed1 are antigenically indistinguishable but phenotypically distinct it was possible to use them to measure the degree of superinfection immunity in the absence of any cytopathic effect. Only small numbers of non-fusing green fluorescent piNT2-MVDsRed1 cells (1 : 300 000) were identified in which superinfecting MVeGFP entered, replicated and expressed its genes. INTRODUCTIONThe presence of measles virus (MV) in patients with subacute sclerosing panencephalitis (SSPE) provides a prime example of the long-term persistence of a human RNA virus. Individuals suffer a lethal condition in which the brain is heavily infected with MV containing a variety of mutations (Baczko et al., 1993). These variants are derived from the MV involved in the acute infection (Rima et al., 1997). The virus persists in an unknown site in the body for a period of two to 30 years (Modlin et al., 1977). In SSPE, the virus primarily infects neurones and oligodendrocytes in the central nervous system and months to years can pass between the onset of symptoms and death. Persistent MV infection has also been implicated in a number of other human diseases including autoimmune chronic hepatitis, Paget's disease, otosclerosis and Crohn's disease. In almost all cases the evidence for such claimed association has not been confirmed by other investigators (reviewed by Rima, 1999). Whilst in SSPE MV infection occurs primarily, though not exclusively, in non-dividing cells, infection of bone marrow precursor cells, for example osteoblasts and osteoclasts, has been implicated, though not confirmed, in diseases such as Paget's disease of bone and otosclerosis (Basle et ...

Limited Interference at the Early Stage of Infection between Two Recombinant Novirhabdoviruses: Viral Hemorrhagic Septicemia Virus and Infectious Hematopoietic Necrosis Virus

“…Superinfection exclusion has been observed in a broad range of viruses, including vaccinia virus (14,18), human immunodeficiency virus (HIV) (36,37), vesicular stomatitis virus (VSV) (32,43,53), Borna disease virus (BDV) (24), measles virus (34), Sindbis virus (28), Semliki Forest virus (44), rubella virus (15), hepatitis C virus (HCV) (40,51), and bovine viral diarrhea virus (BVDV) (31). Mechanisms of exclusion are diverse and have not been determined in all cases, but mechanisms described so far are caused by competition among different viruses for critical replicative pathways (for example, the use of the same receptors for the entry) or depend on the direct interaction of products of the primary infection with the secondary infecting virus.…”

Exclusion of West Nile Virus Superinfection through RNA Replication

“…Superinfection exclusion has been observed among a broad range of viruses, including vaccinia virus (4), human immunodeficiency virus (25,29), vesicular stomatitis virus (VSV) (40,44), Borna disease virus (8), measles virus (24,36), Sindbis virus (12), Semliki Forest virus (SFV) (41), rubella virus (5), hepatitis C virus (HCV) (35,43), and bovine viral diarrhea virus (BVDV) (17). The exclusion could result from impairments of distinct steps of the viral life cycle during superinfection, including entry, translation, RNA replication, or virus budding (7).…”

Clearance of Measles Virus from Persistently Infected Cells by Short Hairpin RNA

“…In order to achieve this goal, we generated persistently infected NT2 cells using rMVHcRed-CAMH. NT2 cells were chosen, as they are a well-characterized pluripotent human testicular embryonic cell line which can be differentiated with retinoic acid in cells with neuronal appearance and which had been persistently infected with MV earlier (25,30). The cells were infected at an MOI of 0.1 and cultured until most of the cells died.…”

“…5A). Since persistently infected NT2 cells do not fuse to each other but maintain their capacity to fuse with uninfected cells expressing appropriate receptors (25), transduced cells were cultivated either in the absence or presence of a fusion-inhibiting peptide (FIP; Z-Phe-Phe-Gly) (33) whereas in cells expressing shN1, shP6, and shL4, the doublepositive cells disappeared and EGFP single-positive cells (bottom right quadrant) appeared. These cells in the bottom right quadrant, which do not express HcRed anymore, are potentially cured cells.…”

“…When these cells were overlaid (in a 1:1,000 dilution) on a monolayer of uninfected Vero cells, cell-to-cell fusion was readily observed (25). We therefore assessed the presence of infection at 15 days in the shN1-, shP6-, and shL4-transduced piNT2-HcRed cultures containing infected (red) and potentially cured (green) cells.…”