Integrase has been implicated in human immunodeficiency virus type 1 (HIV-1) nuclear import. Integrase analyses, however, can be complicated by the pleiotropic nature of mutations: whereas class I mutants are integration defective, class II mutants display additional assembly and/or reverse transcription defects. We previously determined that HIV-1 V165A , originally reported as defective for nuclear import, was a class II mutant. Here we analyzed mutants containing changes in other putative nuclear localization signals, including 186 KRK 188 / 211 KELQKQITK 219 and Cys-130. Previous work established HIV-1 K186Q , HIV-1 Q214L/Q216L , and HIV-1 C130G as replication defective, but phenotypic classification was unclear and nuclear import in nondividing cells was not addressed. Consistent with previous reports, most of the bipartite mutants studied here were replication defective. These mutants as well as HIV-1 V165A synthesized reduced cDNA levels, but a normal fraction of mutant cDNA localized to dividing and nondividing cell nuclei. Somewhat surprisingly, recombinant class II mutant proteins were catalytically active, and class II Vpr-integrase fusion proteins efficiently complemented class I mutant virus. Since a class I Vpr-integrase mutant efficiently complemented class II mutant viruses under conditions in which class II Vpr-integrases failed to function, we conclude that classes I and II define two distinct complementation groups and suggest that class II mutants are primarily defective at a postnuclear entry step of HIV-1 replication. HIV-1 C130G was also defective for reverse transcription, but Vpr-integrase C130G did not efficiently complement class I mutant HIV-1. Since HIV-1 C130A grew like the wild type, we conclude that Cys-130 is not essential for replication and speculate that perturbation of integrase structure contributed to the pleiotropic HIV-1 C130G phenotype.

Retroviruses carry two enzymes, reverse transcriptase (RT) and integrase (IN), which function early in the viral life cycle. Soon after infection, RT converts genomic RNA into linear double-stranded cDNA. This DNA, which contains a copy of the viral long terminal repeat (LTR) at each end, is the substrate for IN-mediated DNA recombination. IN initially processes the 3Ј ends of the cDNA adjacent to phylogenetically conserved CA dinucleotides and then inserts these cleaved ends into a target DNA site in a cell chromosome. The cisacting end regions important for integration define the viral attachment (att) sites, which are comprised of U3 and U5 sequences in the upstream and downstream LTRs, respectively. (For a recent review of retroviral integration, see reference 6.)In addition to the linear DNA product of reverse transcription, various types of circular DNA form in retroviral-infected cells.

Vaccine development has the potential to be accelerated by coupling tools such as systems immunology analyses and controlled human infection models to define the protective efficacy of prospective immunogens without expensive and slow phase 2b/3 vaccine studies. Among human challenge models, controlled human malaria infection trials have long been used to evaluate candidate vaccines, and RTS,S/AS01 is the most advanced malaria vaccine candidate, reproducibly demonstrating 40 to 80% protection in human challenge studies in malaria-naïve individuals. Although antibodies are critical for protection after RTS,S/AS01 vaccination, antibody concentrations are inconsistently associated with protection across studies, and the precise mechanism(s) by which vaccine-induced antibodies provide protection remains enigmatic. Using a comprehensive systems serological profiling platform, the humoral correlates of protection against malaria were identified and validated across multiple challenge studies. Rather than antibody concentration, qualitative functional humoral features robustly predicted protection from infection across vaccine regimens. Despite the functional diversity of vaccine-induced immune responses across additional RTS,S/AS01 vaccine studies, the same antibody features, antibody-mediated phagocytosis and engagement of Fc gamma receptor 3A (FCGR3A), were able to predict protection across two additional human challenge studies. Functional validation using monoclonal antibodies confirmed the protective role of Fc-mediated antibody functions in restricting parasite infection both in vitro and in vivo, suggesting that these correlates may mechanistically contribute to parasite restriction and can be used to guide the rational design of an improved vaccine against malaria.

Results of in vitro assays identified residues in the C-terminal domain (CTD) of human immunodeficiencyRetrovirus replication is dependent on the integration of the reverse-transcribed viral genome into a host chromosome. Subsequent to target cell entry, the double-stranded DNA substrate for integration is generated by the viral enzyme reverse transcriptase (RT) upon conversion of the genomic RNA into DNA. Acting on the attachment (att) sites at the cDNA ends, the viral DNA recombinase or integrase (IN) catalyzes two distinct endonucleolytic reactions. For the first reaction, 3Ј processing, human immunodeficiency virus type 1 (HIV-1) IN removes the dinucleotide GT from each end. This exposes a 3Ј hydroxyl moiety in preparation for the second reaction, DNA strand transfer. Upon recognition and binding to a suitable target site, IN uses the 3Ј-OHs to cut the chromosome in a staggered fashion, which at the same time joins the viral ends to the 5Ј-phosphates of the cut. Cellular enzymes are likely involved in the repair of the resultant gapped product, thus fully recombining the viral cDNA with the host (reviewed in references 16 and 38).