Nucleic acid-dependent cross-linking of the nucleocapsid protein of Sindbis virus.
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The assembly of the alphavirus nucleocapsid core is a multistep event requiring the association of the nucleocapsid protein with nucleic acid and the subsequent oligomerization of capsid proteins into an assembled core particle. Although the mechanism of assembly has been investigated extensively both in vivo and in vitro, no intermediates in the core assembly pathway have been identified. Through the use of both truncated and mutant Sindbis virus nucleocapsid proteins and a variety of cross-linking reagents, a possible nucleic acid-protein assembly intermediate has been detected. The cross-linked species, a covalent dimer, has been detected only in the presence of nucleic acid and with capsid proteins capable of binding nucleic acid. Optimum nucleic acid-dependent cross-linking was seen at a protein-to-nucleic-acid ratio identical to that required for maximum binding of the capsid protein to nucleic acid. Identical results were observed when cross-linking in vitro assembled core particles of both Sindbis and Ross River viruses. Purified cross-linked dimers of truncated proteins and of mutant proteins that failed to assemble were found to incorporate into assembled core particles when present as minor components in assembly reactions, suggesting that the cross-linking traps an authentic intermediate in nucleocapsid core assembly. Endoproteinase Lys-C mapping of the position of the cross-link indicated that lysine 250 of one capsid protein was cross-linked to lysine 250 of an adjacent capsid protein. Examination of the position of the cross-link in relation to the existing model of the nucleocapsid core suggests that the cross-linked species is a cross-capsomere contact between a pentamer and hexamer at the quasi-threefold axis or is a cross-capsomere contact between hexamers at the threefold axis of the icosahedral core particle and suggests several possible assembly models involving a nucleic acid-bound dimer of capsid protein as an early step in the assembly pathway. (+info)
Evaluation of recombinant alphaviruses as vectors in gene therapy.
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Alphavirus vectors based on Sindbis virus and Semliki Forest virus (SFV) were characterized as potential gene transfer vectors. Initial studies were performed using vectors engineered to transfer either lacZ or green fluorescent protein (GFP). High levels of gene transfer were achieved in human primary fibroblasts, BHK and 293T cells, with low levels of transduction observed in more than 20 other target cells. Alphavirus-based expression was generally very high, but transient in every cell type. Replication-competent alphavirus was never detected in SFV preparations but could be produced by Sindbis-based vectors at a frequency of up to 3 x 10(-3) infectious units per ml. We constructed a human clotting factor IX (hFIX) cDNA-containing Sindbis virus and compared it with hFIX cDNA-harboring adenoviral and retroviral vectors. In most cases, hFIX levels obtained with Sindbis vector were initially at least an order of magnitude higher than those obtained with other viral vectors. These data demonstrate that alphavirus vectors compare favorably with adenovirus vectors as systems to promote high-level transient gene expression and should be considered as an alternative vector for gene transfer and potential gene therapy studies. (+info)
Isolation and complete nucleotide sequence of a Chinese Sindbis-like virus.
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Infection with alphaviruses is common in the Chinese population. Here we report the isolation of a Sindbis-like virus from a pool of Anopheles mosquitoes collected in Xinjiang, China during an arbovirus survey. This virus, designated XJ-160, rapidly produced cytopathic effects on mosquito and hamster cells. In addition, it was lethal to neonatal mice if inoculated intracerebrally. Serologically, XJ-160 reacted with and was neutralized by an anti-Sindbis antibody. Anti-XJ-160 antibodies were found in several cohorts of Chinese subjects. The complete 11626-base nucleotide sequence of XJ-160 was determined. XJ-160 has diverged significantly from the prototype Sindbis virus, with an 18% difference in nucleotide sequence and an 8.6% difference in amino acids; there are 11 deletions and 2 insertions, involving 99 nucleotides in total. XJ-160 is most closely linked to Kyzylagach virus isolated in Azerbaijan. Both belong to the African/European genetic lineage of Sindbis virus, albeit more distantly related to other members. (+info)
Maintenance of CD8(+) T-cell memory following infection with recombinant sindbis and vaccinia viruses.
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CD8(+) T-cell memory is critical for protection against pathogens poorly controlled by humoral immunity. To characterize two distinct vaccine vectors, the acute and memory CD8(+) T-cell responses to an HIV-1 epitope (p18) expressed by recombinant vaccinia (vp18) and Sindbis (SINp18) viruses were compared. Whereas 9 to 13% of CD8(+) splenocytes were p18 specific during the acute response to vp18, 4% were induced by SINp18 as revealed by class I tetramer staining. Increased T-cell activation by vp18 was confirmed by higher numbers of both p18-specific IFN-gamma-secreting splenocytes and activated CD8(+) and CD4(+) T cells. Although higher frequencies of p18-specific CD8(+) T cells during primary responses correlated with higher frequencies during memory, the overall decline was only two- to threefold during the transition to memory, demonstrating equally efficient maintenance of memory in SINp18- as in vp18-immune mice. Despite modest in vivo activation, SINp18-induced CD4(+) T cells secreted substantial amounts of IFN-gamma and IL-2, potentially contributing to sustained CD8(+) memory. Collectively the data indicate that Sindbis virus recombinants provide effective vaccines for inducing protective memory CD8(+) T cells in the absence of the extensive inflammation and replication associated with vaccinia virus. (+info)
Activation of divergent neuronal cell death pathways in different target cell populations during neuroadapted sindbis virus infection of mice.
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Infection of adult mice with neuroadapted Sindbis virus (NSV) results in a severe encephalomyelitis accompanied by prominent hindlimb paralysis. We find that the onset of paralysis parallels morphologic changes in motor neuron cell bodies in the lumbar spinal cord and in motor neuron axons in ventral nerve roots, many of which are eventually lost over time. However, unlike NSV-induced neuronal cell death found in the brain of infected animals, the loss of motor neurons does not appear to be apoptotic, as judged by morphologic and biochemical criteria. This may be explained in part by the lack of detectable caspase-3 expression in these cells. (+info)
The surface conformation of Sindbis virus glycoproteins E1 and E2 at neutral and low pH, as determined by mass spectrometry-based mapping.
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Sindbis virus contains two membrane glycoproteins, E1 and E2, which are organized into 80 trimers of heterodimers (spikes). These trimers form a precise T=4 icosahedral protein lattice on the surface of the virus. Very little is known about the organization of the E1 and E2 glycoproteins within the spike trimer. To gain a better understanding of how the proteins E1 and E2 are arranged in the virus membrane, we have used the techniques of limited proteolysis and amino acid chemical modification in combination with mass spectrometry. We have determined that at neutral pH the E1 protein regions that are accessible to proteases include domains 1-21 (region encompassing amino acids 1 to 21), 161-176, and 212-220, while the E2 regions that are accessible include domains 31-84, 134-148, 158-186, 231-260, 299-314, and 324-337. When Sindbis virus is exposed to low pH, E2 amino acid domains 99-102 and 262-309 became exposed while other domains became inaccessible. Many new E1 regions became accessible after exposure to low pH, including region 86-91, which is in the putative fusion domain of E1 of Semliki Forest virus (SFV) (M. C. Kielian et al., J. Cell Biol. 134:863-872, 1996). E1 273-287 and region 145-158 were also exposed at low pH. These data support a model for the structure of the alphavirus spike in which the E1 glycoproteins are centrally located as trimers which are surrounded and protected by the E2 glycoprotein. These data improve our understanding of the structure of the virus membrane and have implications for understanding the protein conformational changes which accompany the process of virus-cell membrane fusion. (+info)
The role of CD8(+) T cells and major histocompatibility complex class I expression in the central nervous system of mice infected with neurovirulent Sindbis virus.
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Little is known about the role of CD8(+) T cells infiltrating the neural parenchyma during encephalitis induced by neurovirulent Sindbis virus (NSV). NSV preferentially infects neurons in the mouse brain and spinal cord; however, it is generally accepted that neurons can express few if any major histocompatibility complex (MHC) class I molecules. We evaluated the possible roles and interactions of CD8(+) T cells during NSV encephalitis and demonstrated that MHC class I antigen (H2K/D) was expressed on endothelial cells, inflammatory cells, and ependymal cells after intracerebral inoculation of NSV. No immunoreactivity was observed in neurons. On the other hand, in situ hybridization with probes for MHC class I heavy chain, beta2 microglobulin, and TAP1 and TAP2 mRNAs revealed increased expression in a majority of neurons, as well as in inflammatory cells, endothelial cells, and ependymal cells in the central nervous system of infected mice. NSV-infected neurons may fail to express MHC class I molecules due to a posttranscriptional block or may express only nonclassical MHC class I genes. To better understand the role CD8(+) T cells play during fatal encephalitis induced by NSV, mice lacking functional CD8(+) T cells were studied. The presence or absence of CD8 did not alter outcome, but absence of beta2 microglobulin improved survival. Interestingly, the intracellular levels of viral RNA decreased more rapidly in immunocompetent mice than in mice without functional CD8(+) T cells. These observations suggest that CD8(+) T cells may act indirectly, possibly via cytokines, to contribute to the clearance of viral RNA in neurons. (+info)
Differences between C57BL/6 and BALB/cBy mice in mortality and virus replication after intranasal infection with neuroadapted Sindbis virus.
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Neuroadapted Sindbis virus (NSV), given intranasally, caused fatal encephalitis in 100% of adult C57BL/6 mice and 0% of BALB/cBy mice. Most C57BL/6 mice developed severe kyphoscoliosis followed by hind-limb paralysis, while BALB/cBy mice did not. In situ hybridization for detecting NSV RNA and immunohistochemistry for detecting NSV antigen indicated that virus delivered by this route infected neurons of the olfactory region and spread caudally without infection of ependymal cells. Virus antigen was more abundant and infectious virus increased more rapidly and reached higher levels in C57BL/6 mice than in BALB/cBy mice. Surprisingly, infectious virus was cleared faster in C57BL/6 mice, and this was associated with more rapid production of neutralizing antibody. However, viral RNA was cleared more slowly in C57BL/6 mice. In both mouse strains, more infectious virus was present in the lumbar spinal cord than in the cervical spinal cord. These data suggest that genetic susceptibility to fatal NSV encephalomyelitis is determined at least in part by the efficiency of viral replication and spread in the central nervous system. The differences identified in this study provide possible phenotypes for mapping genetic loci involved in susceptibility. (+info)