A critical phase of the influenza virus life cycle is the regulated translocation of genomic ribonucleoproteins (vRNPs) from the nuclear interior, across the nuclear envelope, and into the cytoplasm. Two viral proteins, M1 and NS2, have previously been implicated as mediators of vRNP export. We show here that vRNP nuclear export is prevented by leptomycin B (LMB), an inhibitor of the cellular factor CRM1. In LMB-treated cells, vRNPs were found in a peripheral nuclear location that localized with the nuclear lamina. vRNPs were not colocalized with either M1 or NS2. In situ extraction of cells late in infection also revealed a peripheral localization of nuclear vRNPs, whereas early in infection vRNPs were dispersed throughout the nuclear interior. We believe that vRNPs at the nuclear periphery represent a novel intermediate in the influenza virus nuclear export pathway.

Viruses generally have one of two mechanisms for entry and uncoating. They can enter the cell either by endocytosis or by direct fusion at the plasma membrane. We have established a novel mink lung (Mv-1) cell line that expresses a dominant-interfering form of dynamin-1 (K44A) under the control of a tetracycline-responsive element and studied the early events in influenza infection using these cells. We found that influenza virus binds equally to both induced and uninduced cells, but in K44A-expressing cells, electron microscopy showed viruses trapped in deep coated pits and irregular-shaped tubular structures that contain discrete coated regions. We also show by immunofluorescence and confocal microscopy that entry of incoming virus into the nucleus is blocked in K44A-expressing cells. Virus replication was assayed by immunofluorescence microscopy and was strongly inhibited at both early and late times postinfection in K44A-expressing cells. Virus infectivity was inhibited by approximately 2 log units in cells expressing K44A dynamin when analyzed by influenza plaque assay. Overall these data show that dynamin is required for efficient influenza virus entry, presumably due to its function in release of vesicles from coated pits.

In this report, we present evidence that R5 human immunodeficiency virus type 1 (HIV-1) replicates more efficiently in primary CD4؉ T cells than X4 HIV-1. By comparing CD3/CD28-costimulated CD4 ؉ T-cell cultures infected by several X4 and R5 HIV-1 strains, we determined that R5-infected CD4 ؉ T cells produce more virus over time than X4-infected CD4 ؉ T cells. In the first comparison, we found that more cells were infected by the X4-tropic strain LAI than by the R5-tropic strain JR-CSF and yet that higher levels of viral production were detected in the R5-infected cultures. The differential viral production was partially due to the severe cytopathic effects of the X4 virus. We also compared cultures infected with the isogenic HIV-1 strains NL4-3 (X4) and 49.5 (R5). We found that fewer cells were infected by the R5 strain, and yet similar levels of viral production were detected in both infected cultures. Cell death played less of a role in the differential viral production of these strains, as the cell viability remained comparable in both X4-and R5-infected cultures over time. The final comparison involved the primary R5-tropic isolate KP1 and the primary dual-tropic isolate KP2. Although both strains infected similar numbers of cells and induced comparable levels of cytopathicity, viral production was considerably higher in the R5-infected culture. In summary, these data demonstrate that R5 HIV-1 has an increased capacity to replicate in costimulated CD4؉ T cells compared to X4 HIV-1.Human immunodeficiency virus type 1 (HIV-1) infects cells by binding to the CD4 receptor and to one of several coreceptors expressed on the surface of target cells (2,13,15,16,18,26). The chemokine receptors CCR5 and CXCR4 serve as the major coreceptors for HIV-1, although several other chemokine receptors have been linked to minor HIV-1 coreceptor usage (2,8,15,18,32,52). Characteristically, non-syncytiuminducing (NSI) isolates utilize CCR5 as a coreceptor and are referred to as R5 strains (2, 14-16). R5 strains often represent the dominant viral population detected during the early stages of clinical HIV-1 infection (9,14,41,43,46,53). In contrast, syncytium-inducing (SI) isolates utilize CXCR4 as a coreceptor and are referred to as X4 strains (18,20,24,25,28,30,40,45,48). X4 strains are typically detected in the later stages of infection and are associated with rapid CD4 ϩ T-cell loss (11,14,24,25,46,48,49). Despite the link between X4 emergence and disease progression, approximately half of all individuals with AIDS continue to harbor predominantly R5 viruses, suggesting that CXCR4 coreceptor usage alone is not responsible for disease progression (9,43,46,53).Several mechanisms have been proposed to explain the R5 dominance of early HIV-1 infection. There is evidence that R5 strains may be transmitted at an increased frequency compared to X4 strains. For example, individuals that carry a 32-bp deletion mutation (⌬32) in the CCR5 gene are highly resistant to HIV-1 infection (10,33,38,44). Although these individuals are suscepti...

To enter human cells, HIV-1 usually uses CD4 and 1 of 2 coreceptors: CCR5 and CXCR4. Interestingly, even though CCR5 is expressed on far fewer T cells than is CXCR4, many patients in early- and late-stage HIV disease maintain high levels of CCR5-tropic (R5) viruses. We hypothesized that such high R5 viral loads may be sustained because, relative to CXCR4-tropic (X4) HIV-1 infection, R5 HIV-1 infection of permissive CD4(+)CCR5(+)CXCR4(+) T cells results in the production of significantly more infectious virus particles per target cell. To investigate this possibility, we compared the levels of virus production per target cell after isogenic R5 and X4 HIV-1 infection of 2 in vitro primary human lymphocyte culture systems: T-cell receptor-stimulated blood-derived CD4(+) T cells and tonsil histoculture (which requires no exogenous stimulation for ex vivo infection). We provide evidence that R5 HIV-1 does indeed compensate for a small target cell population by producing, on average, 5 to 10 times more infectious virus per CCR5(+) target cell than X4 HIV-1. This replicative advantage may contribute to the predominance of R5 HIV-1 in vivo.