HIV-1 and other lentiviruses have the unique property among retroviruses to replicate in nondividing cells. This property relies on the use of a nuclear import pathway enabling the viral DNA to cross the nuclear membrane of the host cell. In HIV-1 reverse transcription, a central strand displacement event consecutive to central initiation and termination of plus strand synthesis creates a plus strand overlap: the central DNA flap. We show here that the central DNA flap acts as a cis-determinant of HIV-1 DNA nuclear import. Wild-type viral linear DNA is almost entirely imported into the nucleus where it integrates or circularizes. In contrast, mutant viral DNA, which lacks the DNA flap, accumulates in infected cells as unintegrated linear DNA, at the vicinity of the nuclear membrane. Consistently, HIV-1 vectors devoid of DNA flap exhibit a strong defect of nuclear import, which can be corrected to wild-type levels by reinsertion of the DNA flap sequence.

Whereas human immunodeficiency virus (HIV) infects various cell types by fusion at the plasma membrane, we observed a different entry route in human primary macrophages, in which macropinocytosis is active. Shortly after exposure of macrophages to HIV-1 and irrespective of viral envelope-receptor interactions, particles were visible in intracellular vesicles, which were identified as macropinosomes. Most virions appeared subsequently degraded. However, fusion leading to capsid release in the cytosol and productive infection could take place inside vesicles when particles were properly enveloped. These observations provide new insights into HIV-1 interactions with a cell target relevant to pathogenesis. They may have implications for the design of soluble inhibitors aimed at interfering with the fusion or entry processes.

Background Mycobacterium tuberculosis, the etiological agent of tuberculosis (TB), has the ability to persist in its human host for exceptionally long periods of time. However, little is known about the location of the bacilli in latently infected individuals. Long-term mycobacterial persistence in the lungs has been reported, but this may not sufficiently account for strictly extra-pulmonary TB, which represents 10–15% of the reactivation cases.Methodology/Principal FindingsWe applied in situ and conventional PCR to sections of adipose tissue samples of various anatomical origins from 19 individuals from Mexico and 20 from France who had died from causes other than TB. M. tuberculosis DNA could be detected by either or both techniques in fat tissue surrounding the kidneys, the stomach, the lymph nodes, the heart and the skin in 9/57 Mexican samples (6/19 individuals), and in 8/26 French samples (6/20 individuals). In addition, mycobacteria could be immuno-detected in perinodal adipose tissue of 1 out of 3 biopsy samples from individuals with active TB. In vitro, using a combination of adipose cell models, including the widely used murine adipose cell line 3T3-L1, as well as primary human adipocytes, we show that after binding to scavenger receptors, M. tuberculosis can enter within adipocytes, where it accumulates intracytoplasmic lipid inclusions and survives in a non-replicating state that is insensitive to the major anti-mycobacterial drug isoniazid.Conclusions/SignificanceGiven the abundance and the wide distribution of the adipose tissue throughout the body, our results suggest that this tissue, among others, might constitute a vast reservoir where the tubercle bacillus could persist for long periods of time, and avoid both killing by antimicrobials and recognition by the host immune system. In addition, M. tuberculosis-infected adipocytes might provide a new model to investigate dormancy and to evaluate new drugs for the treatment of persistent infection.

HIV-1-derived vector ͉ integrase deficient ͉ stable transgene expression

HIV-1 virions are efficiently captured by monocyte-derived immature dendritic cells (iDCs), as well as by cell lines expressing the lectin DC-SIGN. Viral infectivity can be retained for several days, and even enhanced, before transmission to CD4 ؉ lymphocytes. The role of DC-SIGN in viral retention and enhancement of infection is not fully understood and varies according to the cell line expressing the lectin. We studied here the mechanisms underlying this process. We focused our study on X4-tropic human immunodeficiency virus (HIV) strains, since they were widely believed not to replicate in iDCs. However, we first show that X4 HIV replicates covertly and slowly in iDCs. This is also the case in Raji-DC-SIGN cells, which are classically used to study HIV transmission. We used either single-cycle or replicative HIV and measured viral RT and replication to further demonstrate that transfer of incoming virions from iDCs or DC-SIGN ؉ cells occurs only on the short-term (i.e., a few hours after viral exposure). There is no long-term storage of original HIV particles in these cells. A few days after viral exposure, replicative viruses, and not single-cycle virions, are transmitted to CD4؉ cells. The cell-type-dependent activity of DC-SIGN reflects the ability of HIV to replicate covertly in some cells, and not in others.

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The retroviral integrase (IN) protein catalyzes integration of the provirus into a chromosome of the infected cell, an essential step of the viral replication cycle (see references 1 and 4 for recent reviews on integration). IN is translated as part of the Gag-Pol precursor molecule, which is cleaved by the viral protease to allow viral particle maturation. Several in vitro studies have examined the biochemical properties of IN, and significant progress has been made in the understanding of its structure and of the mechanism of the integration reaction (1,4,15,31). IN catalyzes the two steps of the integration process. The first step consists of the elimination of 2 nucleotides from each 3Ј end of the proviral DNA. In the second step, the resulting 3Ј-OH ends of the viral DNA are covalently joined to newly created 5Ј ends in the target DNA (11,18,39).Retroviral IN proteins are composed of three functionally distinct domains (see Fig. 1A), all of which are required for a complete integration reaction. The N-terminal domain contains a zinc finger-like motif that stabilizes the folded structure of IN and enhances the catalytic activity of the enzyme (57). The core domain of retroviral IN contains the DDE motif to which the catalytic activity is attributed. This central domain is also involved in the recognition of the conserved nucleotide sequence at each end of the retroviral DNA. The carboxyterminal domain is the least conserved among retroviruses, possesses intrinsic DNA binding activity, and is required for 3Ј-end processing and strand transfer (10, 51). The functional form of IN is multimeric, as was suggested by in vitro evidence of multimerization and demonstrated by trans-complementation of different IN mutants (16,23,(34)(35)(36)50). We recently demonstrated that the multimerization of human immunodeficiency virus type 1 (HIV-1) IN takes place in infectious viral particles and is dependent on disulfide bond formation (46).Besides the well characterized role in the integration process, IN participates in different steps of the virus cycle. Alterations of IN sequence were found to affect viral particle morphogenesis, reverse transcription, and nuclear import of the preintegration complex (PIC) (17, 28, 55), a nucleoprotein complex composed of viral and probably cellular proteins that carries the viral genome from the cytoplasm to the nucleus of the newly infected cell (9,20,21,37,38,45).HIV-1 IN has karyophilic properties which were demonstrated by the nuclear accumulation of this protein both after transient expression of a Flag-or green fluorescent proteintagged IN (46,47) and after microinjection of HIV-1 IN fused to glutathione S-transferase (GST) (28). The GST-IN fusion protein was additionally shown to bind in vitro to karyopherin-␣ (28), a cellular mediator of nuclear transport which is specific for nuclear localization signal-bearing proteins. The interaction between IN and karyopherin-␣ was suggested to be functionally relevant in vivo, providing one additional mechanism for the nuclear import of HI...

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