B cell activating factor receptor (BAFFR)-/- mice have a profound reduction in mature B cells, but unlike μMT mice, they have normal numbers of newly formed, immature B cells. Using a West Nile virus (WNV) challenge model that requires antibodies (Abs) for protection, we found that unlike wild-type (WT) mice, BAFFR-/- mice were highly susceptible to WNV and succumbed to infection within 8 to 12 days after subcutaneous virus challenge. Although mature B cells were required to protect against lethal infection, infected BAFFR-/- mice had reduced WNV E-specific IgG responses and neutralizing Abs. Passive transfer of immune sera from previously infected WT mice rescued BAFFR-/- and fully B cell-deficient μMT mice, but unlike μMT mice that died around 30 days post-infection, BAFFR-/- mice survived, developed WNV-specific IgG Abs and overcame a second WNV challenge. Remarkably, protective immunity could be induced in mature B cell-deficient mice. Administration of a WNV E-anti-CD180 conjugate vaccine 30 days prior to WNV infection induced Ab responses that protected against lethal infection in BAFFR-/- mice but not in μMT mice. Thus, the immature B cells present in BAFFR-/- and not μMT mice contribute to protective antiviral immunity. A CD180-based vaccine may promote immunity in immunocompromised individuals.
NS1= is a C-terminally extended form of the NS1 protein produced only by encephalitic flaviviruses from the Japanese encephalitis virus serogroup. Here we show that West Nile virus (WNV) NS1= and NS1 localize to the same cellular compartments when expressed from plasmid DNAs and also colocalize to viral RNA replication sites in infected cells. Using complementation analysis with NS1-deleted WNV cDNA, we demonstrated that NS1= is able to substitute for the crucial function of NS1 in virus replication. West Nile virus (WNV) is a mosquito-borne flavivirus within the Japanese encephalitis virus (JEV) serogroup. This serogroup also includes other encephalitic flaviviruses, such as JEV, Murray Valley encephalitis virus, and St. Louis encephalitis virus (1). The natural transmission cycle of WNV is between birds and mosquitoes, primarily the Culex species; however, it can cause incidental infections in humans. Since the outbreak of the more pathogenic WNV NY99 strain in New York in 1999 (2), WNV has emerged as a major cause of arboviral encephalitis in the United States (3). WNV strains can be divided into two distinct lineages, lineage 1 and lineage 2. Lineage 1 includes both WNV NY99 and Kunjin (WNV KUN ) (4), the prevalent strain within Australia (5). Despite high sequence similarity to the WNV NY99 strain (ϳ98% on the amino acid level) (6), most WNV KUN strains are highly attenuated, with only a small number of human infections and no fatalities reported (5, 7). Since its isolation in early 1960s, WNV KUN has been used extensively as a model for WNV infection (8, 9).The WNV KUN genome is a single-stranded positive-sense RNA of 11,022 nucleotides (10-12). After translation as a single polyprotein, it is cleaved by host and viral proteases to produce 3 structural (C, prM, and E) and 7 nonstructural (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) proteins (11,12). NS1 is a multifunctional glycoprotein that is involved in viral replication (13-16) and modulation of the immune response (17-22). The key role NS1 plays in RNA replication has been shown previously, with mutations or deletions in the NS1 gene resulting in a lack of detectable RNA replication. This function can be complemented in trans by the expression of NS1 (14, 15).An additional nonstructural protein, NS1=, is produced exclusively by the members of the JEV serogroup due to the presence of a Ϫ1 programmed ribosomal frameshift at the beginning of the adjacent NS2A gene (23)(24)(25). This frameshift, occurring in 30 to 50% of translation events (23,26), results in the formation of a 52-amino-acid C-terminally extended form of NS1. Although a role in neurovirulence has previously been demonstrated (23), no specific functions for NS1= in viral replication or virus-host interactions have been identified. In the present study, we show that the NS1= protein colocalizes with NS1 in viral RNA replication sites in the endoplasmic reticulum (ER) of infected cells and can substitute for the function of NS1 in viral replication.Plasmid DNA-derived expression of NS1 and NS...
Mitochondrial antiviral signaling protein (MAVS) is a critical innate immune signaling protein that directs the actions of the RIG-I-like receptor (RLR) signaling pathway of RNA virus recognition and initiation of anti-viral immunity against West Nile virus (WNV). In the absence of MAVS, mice die more rapidly after infection with the pathogenic WNV-Texas (TX) strain, but also produce elevated WNV-specific IgG concomitant with increased viral burden. Here we investigated whether there was a B cell intrinsic role for MAVS during the development of protective humoral immunity following WNV infection. MAVS -/- mice survived infection from the non-pathogenic WNV-Madagascar (MAD) strain, with limited signs of disease. Compared to wildtype (WT) controls, WNV-MAD-infected MAVS -/- mice had elevated serum neutralizing antibodies, splenic germinal center B cells, plasma cells and effector T cells. We found that when rechallenged with the normally lethal WNV-TX, MAVS -/- mice previously infected with WNV-MAD were protected from disease. Thus, protective humoral and cellular immune responses can be generated in absence of MAVS. Mice with a conditional deletion of MAVS only in CD11c + dendritic cells phenocopied MAVS whole body knockout mice in their humoral responses to WNV-MAD, displaying elevated virus titers and neutralizing antibodies. Conversely, a B cell-specific deletion of MAVS had no effect on immune responses to WNV-MAD compared to WT controls. Thus, MAVS in dendritic cells is required to control WNV replication and thereby regulate downstream humoral immune responses.
West Nile virus (WNV), a mosquito-borne flavivirus, is the major cause of arboviral encephalitis in the USA. As with other members of the Japanese encephalitis virus serogroup, WNV produces an additional non-structural protein, NS19, a C-terminal extended product of NS1 generated as the result of a "1 programmed ribosomal frameshift (PRF). We have previously shown that mutations abolishing the PRF, and consequently NS19, resulted in reduced neuroinvasiveness. However, whether this was caused by the PRF event itself or by the lack of a PRF product, NS19, or a combination of both, remains undetermined. Here, we showed that WNV NS19 formed a unique subpopulation of heat-and low-pH-stable dimers. C-terminal truncations and mutational analysis employing an NS19-expressing plasmid showed that stability of NS19 dimers was linked to the penultimate 10 aa. To examine the role of NS19 heat-stable dimers in virus replication and pathogenicity, a stop codon mutation was introduced into NS19 to create a WNV producing a truncated version of NS19 lacking the last 20 aa, but not affecting the PRF. NS19 protein produced by this mutant virus was secreted more efficiently than WT NS19, indicating that the sequence of the last 20 aa of NS19 was responsible for its cellular retention. Further analysis of this mutant showed growth kinetics in cells and virulence in weanling mice after peripheral infection similar to the WT WNV KUN , suggesting that full-length NS19 was not essential for virus replication in vitro and for virulence in mice.
BAFFR−/− mice are a model of humoral immunodeficiency since they lack mature B cells. We found that in contrast to WT mice BAFFR−/− mice were highly susceptible to WNV. BAFFR−/− mice infected with 100pfu WNV succumbed to infection between 8 to 12 days post-infection and higher virus titers were found in their brains compared to WT mice. Infected BAFFR−/− mice had substantially reduced WNVE-specific IgG responses and delayed but still detectable WNV neutralizing Ab compared to WT mice. Passive transfer of immune sera from previously infected WT mice rescued BAFFR−/− mice as well as μMT mice lacking all B cells, but in contrast to μMT mice that all died around 30 days post-infection, BAFFR−/− mice survived and were able to overcome a second WNV challenge. Furthermore, rescued BAFFR−/− mice receiving immune sera later produced their own WNVE-specific IgG, although at much lower levels than WT mice. These data suggested that protective immunity could be induced in these mature B cell-deficient mice. Indeed, one injection of αCD180-WNVE 30 days prior to WNV infection induced strong neutralizing Ab responses in BAFFR−/− mice and significant protective immunity against a normally lethal infection. In contrast, μMT mice could not be protected by αCD180-WNVE immunization confirming that the vaccination requires B cells to induce protective immunity to WNV infection. BAFFR−/− mice have a profound reduction in mature B cells but, unlike μMT mice, have normal numbers of T1 B cells, which our laboratory has shown previously can make IgG Abs. Interestingly, αCD180 immunization rapidly expands T1/T2 B cells. Thus a CD180-based vaccine may be useful to induce protective immunity in immune compromised individuals. (Supported by NIH grants AI83109 and AI52203).
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