Effect of trimerization motifs on quaternary structure, antigenicity, and immunogenicity of a noncleavable HIV-1 gp140 envelope glycoprotein.

The external domains of the HIV-1 envelope glycoprotein (gp120 and the gp41 ectodomain, collectively known as gp140) contain all known viral neutralization epitopes. Various strategies have been used to create soluble trimers of the envelope to mimic the structure of the native viral protein, including mutation of the gp120-gp41 cleavage site, introduction of disulfide bonds, and fusion to heterologous trimerization motifs. We compared the effects on quaternary structure, antigenicity, and immunogenicity of three such motifs: T4 fibritin, a GCN4 variant, and the Escherichia coli aspartate transcarbamoylase catalytic subunit. Fusion of each motif to the C-terminus of a noncleavable JRCSF gp140(-) envelope protein led to enhanced trimerization but had limited effects on the antigenic profile and CD4-binding ability of the trimers. Immunization of rabbits provided no evidence that the trimerized gp140(-) constructs induced significantly improved neutralizing antibodies to several HIV-1 pseudoviruses, compared to gp140 lacking a trimerization motif. However, modest differences in both binding specificity and neutralizing antibody responses were observed among the various immunogens.

Chimeras of duck and heron hepatitis B viruses provide evidence for functional interactions between viral components of pregenomic RNA encapsidation.

Packaging of hepadnavirus pregenomic RNA (pgRNA) into capsids, or encapsidation, requires several viral components. The viral polymerase (P) and the capsid subunit (C) are necessary for pgRNA encapsidation. Previous studies of duck hepatitis B virus (DHBV) indicated that two cis-acting sequences on pgRNA are required for encapsidation: epsilon, which is near the 5' end of pgRNA, and region II, located near the middle of pgRNA. Later studies suggested that the intervening sequence between these two elements may also make a contribution. It has been demonstrated for DHBV that epsilon interacts with P to facilitate encapsidation, but it is not known how other cis-acting sequences contribute to encapsidation. We analyzed chimeras of DHBV and a related virus, heron hepatitis B virus (HHBV), to gain insight into the interactions between the various viral components during pgRNA encapsidation. We learned that having epsilon and P derived from the same virus was not sufficient for high levels of encapsidation, implying that other viral interactions contribute to encapsidation. Chimeric analysis showed that a large sequence containing region II may interact with P and/or C for efficient encapsidation. Further analysis demonstrated that possibly an RNA-RNA interaction between the intervening sequence and region II facilitates pgRNA encapsidation. Together, these results identify functional interactions among various viral components that contribute to pgRNA encapsidation.

Underrepresentation of the 3' region of the capsid pregenomic RNA of duck hepatitis B virus.

The pregenomic RNA (pgRNA) of hepadnaviruses is packaged into capsids where it is reverse transcribed to yield mature DNA genomes. This report describes differences between the 3' region and other regions of the pgRNA isolated from capsids. Analysis of capsid pgRNA isolated by using an established method involving micrococcal nuclease treatment demonstrated reduced levels of the 3' region of the pgRNA compared to the 5' region. This underrepresentation of the 3' region was partly a result of microccocal nuclease digestion of the 3' region because isolation of capsid pgRNA by an alternative method that did not involve nuclease treatment led to a greater, but not complete, recovery of the 3' region. These results indicate that the 3' region of the capsid pgRNA is susceptible to micrococcal nuclease digestion during its isolation and that the 3' region can still be underrepresented when capsid pgRNA is isolated without nuclease digestion. Additional experiments show that the 3' ends of capsid pgRNA isolated by micrococcal nuclease treatment are heterogeneously dispersed from nucleotide 2577 to the poly(A) tail. These data provide evidence that the 3' region of the capsid pgRNA has biochemical properties different from those of its 5' region. Possibly, the 3' region of the pgRNA is not packaged into the interior of the capsid but rather is associated with a part of the capsid where it is susceptible to microccocal nuclease digestion.

Characterization of the cis-acting contributions to avian hepadnavirus RNA encapsidation.

Previous analysis of duck hepatitis B virus (DHBV) indicated the presence of at least two cis-acting sequences required for efficient encapsidation of its pregenomic RNA (pgRNA), epsilon and region II. epsilon, an RNA stem-loop near the 5' end of the pgRNA, has been characterized in detail, while region II, located in the middle of the pgRNA, is not as well defined. Our initial aim was to identify the sequence important for the function of region II in DHBV. We scanned region II and the surrounding sequence by using a quantitative encapsidation assay. We found that the sequence between nucleotides (nt) 438 and 720 contributed to efficient pgRNA encapsidation, while the sequence between nt 538 and 610 made the largest contribution to encapsidation. Additionally, deletions between the two encapsidation sequences, epsilon and region II, had variable effects on encapsidation, while substitutions of heterologous sequence between epsilon and region II disrupted the ability of the pgRNA to be encapsidated efficiently. Overall, these data indicate that the intervening sequences between epsilon and region II play a role in encapsidation. We also analyzed heron hepatitis B virus (HHBV) for the presence of region II and found features similar to DHBV: a broad region necessary for efficient encapsidation that contained a critical region II sequence. Furthermore, we analyzed variants of DHBV that were substituted with HHBV sequence over region II and found that the chimeras were not fully functional for RNA encapsidation. These results indicate that sequences within region II may need to be compatible with other viral components in order to function in pgRNA encapsidation.

Identification and characterization of a novel replicative intermediate of heron hepatitis B virus.

We have identified and characterized a novel intracellular DNA replicative intermediate that is synthesized by heron hepatitis B virus (HHBV) and not by other avian hepadnaviruses. The new DNA form is synthesized in all host cells tested. The HHBV nucleic acid template, and not HHBV proteins, is responsible for the formation of the new form. The new form is comprised of a full-length minus-strand DNA and an incomplete plus-strand DNA whose 5' ends are mapped to DR2, predominantly. The 3' ends of its plus-strand are located between nucleotides 946 and 1046. Genetic analysis indicates that the sequences responsible for the formation of the new form lie between nucleotides 910 and 1364. The endogenous polymerase activity of capsids isolated from cells converted the new form into RC DNA. Intracellular capsids containing the new form are secreted inefficiently as virions, in comparison to RC- and DL DNA-containing capsids. Our analysis suggests that the new form is an incomplete RC DNA molecule that is due to a specific block or pause in the synthesis of plus-strand DNA. Our analysis also suggests that capsids become competent for efficient secretion sometime after the synthesis of 1500 nucleotides of plus-strand DNA.