Anthrax vaccines: Pasteur to the present.

Anthrax has been a major cause of death in grazing animals and an occasional cause of death in humans for thousands of years. Since the late 1800s there has been an exceptional international history of anthrax vaccine development. Due to animal vaccinations, the rate of infection has dropped dramatically. Anthrax vaccines have progressed from uncharacterized whole-cell vaccines in 1881, to pXO2-negative spores in the 1930s, to culture filtrates absorbed to aluminum hydroxide in 1970, and likely to recombinant protective antigen in the near future. Each of these refinements has increased safety without significant loss of efficacy. The threat of genetically engineered, antibiotic and vaccine resistant strains of Bacillus anthracis is fueling hypothesis-driven research and global techniques--including genomics, proteomics and transposon site hybridization--to facilitate the discovery of novel vaccine targets. This review highlights historical achievements and new developments in anthrax vaccine research.

Substitutions in a homologous region of extracellular loop 2 of CXCR4 and CCR5 alter coreceptor activities for HIV-1 membrane fusion and virus entry.

CXCR4 and CCR5 are the principal coreceptors for human immunodeficiency virus type-1 (HIV-1) infection. Previously, mutagenesis of CXCR4 identified single amino acid changes that either impaired CXCR4's coreceptor activity for CXCR4-dependent (X4) isolate envelope glycoproteins (Env) or expanded its activity, allowing it to serve as a functional coreceptor for CCR5-dependent (R5) isolates. The most potent of these point mutations was an alanine substitution for the aspartic acid residue at position 187 in extracellular loop 2 (ecl-2), and here we show that this mutation also permits a variety of primary R5 isolate Envs, including those of other subtypes (clades), to employ it as a coreceptor. We also examined the corresponding region of CCR5 and demonstrate that the substitution of the serine residue in the homologous ecl-2 position with aspartic acid impairs CCR5 coreceptor activity for isolates across several clades. These results highlight a homologous and critical element in ecl-2, of both the CXCR4 and CCR5 molecules, for their respective coreceptor activities. Charge elimination expands CXCR4 coreceptor activity, while a similar charge introduction can destroy the coreceptor function of CCR5. These findings provide further evidence that there are conserved elements in both CXCR4 and CCR5 involved in coreceptor function.

N-linked glycosylation of CXCR4 masks coreceptor function for CCR5-dependent human immunodeficiency virus type 1 isolates.

The chemokine receptors CXCR4 and CCR5 are the principal coreceptors for infection of X4 and R5 human immunodeficiency virus type 1 (HIV-1) isolates, respectively. Here we report on the unexpected observation that the removal of the N-linked glycosylation sites in CXCR4 potentially allows the protein to serve as a universal coreceptor for both X4 and R5 laboratory-adapted and primary HIV-1 strains. We hypothesize that this alteration unmasks existing common extracellular structures reflecting a conserved three-dimensional similarity of important elements of CXCR4 and CCR5 that are involved in HIV envelope glycoprotein (Env) interaction. These results may have far-reaching implications for the differential recognition of cell type-dependent glycosylated CXCR4 by HIV-1 isolates and their evolution in vivo. They also suggest a possible explanation for the various observations of restricted virus entry in some cell types and further our understanding of the framework of elements that represent the Env-coreceptor contact sites.

Mutagenesis of CXCR4 identifies important domains for human immunodeficiency virus type 1 X4 isolate envelope-mediated membrane fusion and virus entry and reveals cryptic coreceptor activity for R5 isolates.

CXCR4 is a chemokine receptor and a coreceptor for T-cell-line-tropic (X4) and dual-tropic (R5X4) human immunodeficiency virus type 1 (HIV-1) isolates. Cells coexpressing CXCR4 and CD4 will fuse with appropriate HIV-1 envelope glycoprotein (Env)-expressing cells. The delineation of the critical regions involved in the interactions within the Env-CD4-coreceptor complex are presently under intensive investigation, and the use of chimeras of coreceptor molecules has provided valuable information. To define these regions in greater detail, we have employed a strategy involving alanine-scanning mutagenesis of the extracellular domains of CXCR4 coupled with a highly sensitive reporter gene assay for HIV-1 Env-mediated membrane fusion. Using a panel of 41 different CXCR4 mutants, we have identified several charged residues that appear important for coreceptor activity for X4 Envs; the mutations E15A (in which the glutamic acid residue at position 15 is replaced by alanine) and E32A in the N terminus, D97A in extracellular loop 1 (ecl-1), and R188A in ecl-2 impaired coreceptor activity for X4 and R5X4 Envs. In addition, substitution of alanine for any of the four extracellular cysteines alone resulted in conformational changes of various degrees, while mutants with paired cysteine deletions partially retained their structure. Our data support the notion that all four cysteines are involved in disulfide bond formation. We have also identified substitutions which greatly enhance or convert CXCR4's coreceptor activity to support R5 Env-mediated fusion (N11A, R30A, D187A, and D193A), and together our data suggest the presence of conserved extracellular elements, common to both CXCR4 and CCR5, involved in their coreceptor activities. These data will help us to better detail the CXCR4 structural requirements exhibited by different HIV-1 strains and will direct further mutagenesis efforts aimed at better defining the domains in CXCR4 involved in the HIV-1 Env-mediated fusion process.

Strict conservation of the retroviral nucleocapsid protein zinc finger is strongly influenced by its role in viral infection processes: characterization of HIV-1 particles containing mutant nucleocapsid zinc-coordinating sequences.

The retroviral nucleocapsid (NC) protein contains highly conserved amino acid sequences (-Cys-X2-Cys-X4-His-X4-Cys-) designated retroviral (CCHC) Zn2+ fingers. The NC protein of murine leukemia viruses contains one NC Zn2+ finger and mutants that were competent in metal binding (CCCC and CCHH) packaged wild-type levels of full-length viral RNA but were not infectious. These studies were extended to human immunodeficiency virus type 1 (HIV-1), a virus with two NC Zn2+ fingers. Viruses with combinations of CCHC, CCCC, and CCHH Zn2+ fingers in each position of HIV-1 NC were characterized. Mutant particles contained the normal complement of processed viral proteins. Four mutants packaged roughly wild-type levels of genomic RNA, whereas the remaining mutants packaged reduced levels. Virions with mutated C-terminal position NC fingers were replication competent. One interesting mutant, containing a CCCC Zn2+ finger in the N-terminal position of NC, packaged wild-type levels of viral RNA and showed approximately 5% wild-type levels of infectivity when examined in CD4-expressing HeLa cells containing an HIV-1 LTR/beta-galactosidase construct. However, this particular mutant was replication defective in H9 cells; all other mutants were replication defective over the 8-week course of the assay. Two long terminal repeat viral DNA species could be detected in the CCCC mutant but not in any of the other replication-defective mutants. These studies show that the N-terminal Zn2+ finger position is more sensitive to alterations than the C-terminal position with respect to replication. Additionally, the retroviral (CCHC) NC Zn2+ finger is required for early infection processes. The evolutionary pressure to maintain CCHC NC Zn2+ fingers depends mainly on its function in infection processes, in addition to its function in genome packaging.

Genetic analysis of the zinc finger in the Moloney murine leukemia virus nucleocapsid domain: replacement of zinc-coordinating residues with other zinc-coordinating residues yields noninfectious particles containing genomic RNA.

The effect of changing zinc (Zn2+)-coordinating residues in the nucleocapsid protein of Moloney murine leukemia virus was investigated by introducing a His-34-to-Cys or Cys-39-to-His mutation into the putative Zn2+ finger. Mutant virions contained normal levels of properly processed Gag and Env proteins and wild-type levels of full-length viral RNA. However, the specific infectivity of the mutants was approximately 4 x 10(-4) that of wild-type particles. They were probably noninfectious because of the inability of the particles to synthesize cDNA transcripts, since full-length viral DNA could not be detected in Hirt supernatants of NIH 3T3 cells infected with the CCCC or CCHH virus. These mutants will provide an extremely valuable tool for analysis of the role of retroviral Zn2+ fingers in infection processes, independent of viral RNA recognition and packaging.

The two zinc fingers in the human immunodeficiency virus type 1 nucleocapsid protein are not functionally equivalent.

The highly conserved zinc fingers in retroviral nucleocapsid (NC) proteins have the general structure Cys-(X)2-Cys-(X)4-His-(X)4-Cys. Human immunodeficiency virus type 1 (HIV-1) contains two Zn2+ fingers, and mutants were constructed in which the native sequence of each Zn2+ finger was maintained but their positions in the NC protein were changed. Mutants had either two first-finger sequences (pNC1/1), two second-finger sequences (pNC2/2), or reversed first- and second-finger sequences (pNC2/1). Cells transfected with mutant or wild-type clones produced similar levels of Tat, Gag, Pol, and Env proteins, formed syncytia, and shed viruslike particles that were indistinguishable by electron microscopy. However, the pNC2/1 and pNC2/2 mutants were inefficient in packaging genomic RNA (less than 15% of wild-type levels), whereas the pNC1/1 mutant packaged approximately 70% of wild-type levels of RNA. No infectious virus could be detected with either the pNC2/1 or pNC2/2 mutants, whereas the pNC1/1 mutant appeared to sustain a low level of replication and reverted to a competent wild-type-like viral species after a 2- to 4-week lag period. The data strongly suggest that the two Zn2+ fingers of HIV-1 are not functionally equivalent and that the first Zn2+ finger in the Gag precursor plays a more prominent role in RNA selection and packaging. The data also indicate that both Zn2+ fingers in the mature NC protein play as yet unknown roles in viral assembly or the early stages of the viral infection process.

Large-scale purification of gp70 from Moloney murine leukemia virus.

The external envelope glycoprotein, gp70, of the Moloney murine leukemia virus was extracted from NIH 3T3 cells utilizing the detergent n-octyl-beta-D-glycopyranoside. The extracted gp70 was sequentially purified utilizing lectin-affinity, anion-exchange, and molecular-exclusion chromatography techniques. Approximately 10 mg of gp70 was purified by this method and shown to be 95% homogeneous, as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The presence of purified gp70 from Moloney murine leukemia virus was confirmed by amino acid analysis, amino-terminal sequencing, and immunoreactivity with a monoclonal antibody raised against gp70. The procedure is rapid, utilizes commercially available media, and can be used to purify large amounts of retroviral envelope glycoprotein from virus.