Vpr-induced cell cycle arrest is conserved among primate lentiviruses.

We previously reported that expression of human immunodeficiency virus type 1 strain NL4-3 (HIV-1(NL4-3))vpr causes cells to arrest in the G2 phase of the cell cycle. We examined the induction of cell cycle arrest by other HIV-1 isolates and by primary lentiviruses other than HIV-1. We demonstrate that the vpr genes from tissue culture-adapted or primary isolates of HIV-1 are capable of inducing G2 arrest. In addition, we demonstrate that induction of cell cycle arrest is a conserved function of members of two other groups of primate lentiviruses, HIV-2/simian immunodeficiency virus strain sm (SIVsm)/SIVmac and SIVagm. vpr from HIV-1, HIV-2, and SIVmac induced cell cycle arrest when transfected in human (HeLa) and monkey (CV-1) cells. vpx from HIV-2 and SIVmac did not induce detectable cell cycle arrest in either cell type, and SIVagm vpx was capable of inducing arrest in CV-1 but not HeLa cells. These results indicate that induction of cell cycle perturbation is a general property of lentiviruses that infect primates. The conservation of this viral function throughout evolution suggests that it plays a key role in virus-host relationships, and elucidation of its mechanism may reveal important clues about pathology induced by primary lentiviruses.     

Demonstration of binding of dengue virus envelope protein to target cells.

The nature of the initial interaction of dengue virus with target cells and the extent to which this interaction defines tropism are unknown. Infection of some cells may involve antidengue antibody-mediated immune adherence to cells bearing immunoglobulin Fc receptors; however, this mechanism does not explain primary infection or the infection of cells without Fc receptors. We hypothesized that dengue virus envelope protein mediates initial binding to target cells. To test this hypothesis, a recombinant chimeric form of dengue type 2 virus envelope protein was used as a probe to investigate binding to the surfaces of potential target cells. Envelope protein was expressed amino terminal to the heavy-chain constant region of human immunoglobulin G containing the Fc receptor binding motif; the binding mediated by envelope determinants was distinguishable from the binding mediated by immunoglobulin Fc determinants. We found that the recombinant chimera bound to Vero, CHO, endothelial, and glial cells through envelope protein determinants and to monocytes and U937 cells by Fc-Fc receptor interactions. The highest level of binding was to Vero cells; binding was dose and time dependent and saturable. Examination of partial-length recombinant envelope proteins indicated that the binding motif was expressed between amino acids 281 and 423. Recombinant envelope protein inhibited infection of Vero cells by dengue virus, indicating the functional significance of the interaction of envelope protein and target cells in infectivity. These results suggest that envelope protein binding to a non-Fc receptor could explain the cell and tissue tropism of primary dengue virus infection.     

Neutrophil-mediated suppression of virus replication after herpes simplex virus type 1 infection of the murine cornea.

Herpes simplex virus type 1 (HSV-1) infection of the murine cornea induces the rapid infiltration of neutrophils. We investigated whether these cells could influence virus replication. BALB/c mice treated with monoclonal antibody (MAb) RB6-8C5 experienced a profound depletion of neutrophils in the bloodstream, spleen, and cornea. In these animals, virus titers in the eye were significantly higher than those in the immunoglobulin G-treated controls at 3 days postinfection. By day 9, virus was no longer detectable in the controls, whereas titers of 10(3) to 10(6) PFU were still present in the neutrophil-depleted hosts. Furthermore, virus spread more readily to the skin and brains of MAb RB6-8C5-treated animals, rendering them significantly more susceptible to HSV-1-induced blepharitis and encephalitis. Only 25% of the treated animals survived, whereas all of the controls lived. Although MAb RB6-8C5 treatment did not alter the CD4+ T-cell, B-cell, natural killer cell, or macrophage populations, the CD8+ T-cell population was partially reduced. Therefore, the experiments were repeated in severe combined immunodeficiency mice, which lack CD8+ T cells. Again virus growth was found to be significantly elevated in the eyes, trigeminal ganglia, and brains of the MAb RB6-8C5-treated hosts. These results strongly indicate that in both immunocompetent and immunodeficient mice, neutrophils play a significant role in helping to control the replication and spread of HSV-1 after corneal infection.     

Kaposi's sarcoma-associated herpesvirus contains G protein-coupled receptor and cyclin D homologs which are expressed in Kaposi's sarcoma and malignant lymphoma.

A new human herpesvirus was recently identified in all forms of Kaposi's sarcoma (Kaposi's sarcoma-associated herpesvirus [KSHV] or human herpesvirus 8), as well as in primary effusion (body cavity-based) lymphomas (PELs). A 12.3-kb-long KSHV clone was obtained from a PEL genomic library. Sequencing of this clone revealed extensive homology and colinearity with the right end of the herpesvirus saimiri (HVS) genome and more limited homology to the left end of the Epstein-Barr virus genome. Four open reading frames (ORFs) were sequenced and characterized; these are homologous to the following viral and/or cellular genes: (i) Epstein-Barr virus membrane antigen p140 and HVS p160, (ii) HVS and cellular type D cyclins, (iii) HVS and cellular G protein-coupled receptors, and (iv) HVS. Since there is considerable evidence that cyclin D1 and some G protein-coupled receptors contribute to the development of specific cancers, the presence of KSHV homologs of these genes provides support for a role for KSHV in malignant transformation. All ORFs identified are transcribed in PELs and Kaposi's sarcoma tissues, further suggesting an active role for KSHV in these diseases.     

Twin studies demonstrate a host cell genetic effect on productive human immunodeficiency virus infection of human monocytes and macrophages in vitro.

Biological and genetic variability is a prominent feature of human immunodeficiency virus (HIV) strains, especially in tropism, syncytium formation, and replicative capacity. To determine whether there were variable host cell effects on HIV replication in monocytes, three different strains of low-passage-number monocytotropic blood isolates of HIV and the laboratory-adapted strain Ba-L were inoculated into panels of adherent monocytes drawn from 44 different donors, and peak extracellular HIV p24 antigen titers were compared. The clinical HIV strains showed patterns of either moderate or low-level replication in most donor monocytes (20 to 4,000 pg/ml). However, within this range there was marked variation in peak titers in most donors. HIV type 1 Ba-L replicated in all donor monocytes to much higher levels with less variability (30 to 40 ng/ml). Furthermore, replication of 21 clinical blood-derived strains of HIV in blood monocytes and monocyte-derived macrophages (MDM) from pairs of identical twins and age-matched unrelated donors (URD) of the same sex were compared. In all of the seven pairs of identical twins, the kinetics of replication (measured by extracellular HIV p24 antigen) of panels of four clinical HIV type 1 isolates in monocytes were similar within pairs. However, marked and significant differences in kinetics of HIV production occurred within 10 of the 12 unrelated donor pairs (P = 0.0007). The remaining two URD pairs showed similar kinetic patterns, but only one pair had the same HLA-DR genotype. Similar results were observed with monocytes/MDMs obtained from a second bleed of the same donor. Hence, discordant patterns of HIV replication kinetics between URD monocyte pairs contrasted with concordant patterns in identical twin monocytes. These data strongly suggest a host cell genetic effect on productive viral replication in monocytes and MDMs. So far, no consistent genetic linkage of HIV replication pattern with HLA-DR genotype has been observed.     

The fate of immunoglobulin G fed to larvae of Ostrinia nubilalis

Since plants can be transformed genetically to produce functional antibodies, an immunological approach may be developed for controlling their arthropod pests. Specific antibodies would protect plants from arthropods if they could gain access to the pest antigen in sufficient amounts such that the normal function of the antigen is disrupted. In order to study the fate of ingested antibodies in the body of the European corn borer (ECB), Ostrinia nubilalis (Hübner), (Lepidoptera: Pyralidae), we fed the larvae on serum-containing diet. When larvae were fed on the serum-containing diet for various lengths of time between 12 and 96 h, no significant differences were noted in the immunoglobulin G (IgG) concentration in their body. Immediately after the larvae stopped feeding, the concentrations of the IgG in their midgut was about one half that of the diet itself, but it decreased significantly after 6 h and again after 18 h (about 3 and 10 fold, respectively). Immediately after the larvae stopped feeding, the concentration of the IgG in their hemolymph was about 1/500 that in the diet. The concentration of IgG in the hemolymph of ECB larvae was influenced directly by the titer of antibodies in their diet. During the first 6 h after the larvae stop feeding the concentration of IgG in their hemolymph did not decrease significantly; however, it did so after 18 h (about 6 fold). The possibility that specific antibodies will gain access to antigens in the ECB body is discussed.     

Opposite Orientations of an Inverted Duplication and Allelic Variation at the Mouse Agouti Locus

The mouse agouti protein is a paracrine signaling molecule that causes yellow pigment synthesis. A pale ventral coloration distinguishes the light-bellied agouti (A(w)) from the agouti (A) allele, and is caused by expression of ventral-specific mRNA isoforms with a unique 5' untranslated exon. Molecular cloning demonstrates this ventral-specific exon lies within a 3.1-kb element that is duplicated in the opposite orientation 15-kb upstream to produce an interrupted palindrome and that similarity between the duplicated elements has been maintained by gene conversion. Orientation of the palindrome is reversed in A compared to A(w), which suggests that mutation from one allele to the other is caused by intrachromosomal homologous recombination mediated by sequences within the duplicated elements. Analysis of 15 inbred strains of laboratory and wild-derived mice with Southern hybridization probes and closely linked microsatellite markers suggests six haplotype groups: one typical for most strains that carry A(w) (129/SvJ, LP/J, CE/J, CAST/Ei), one typical for most strains that carry A (Balb/cJ, CBA/J, FVB/N, PERA/Rk, RBB/Dn); and four that are atypical (MOLC/Rk, MOLG/Dn, PERA/Ei, PERC/Ei, SPRET/Ei, RBA/Dn). Our results suggest a model for molecular evolution of the agouti locus in which homologous recombination can produce a reversible switch in allelic identity.