SUMMARY Binding of the HIV envelope to the chemokine coreceptors triggers membrane fusion and signal transduction. The fusion process has been well characterized, yet the role of coreceptor signaling remains elusive. Here we describe a critical function of the chemokine coreceptor signaling in facilitating HIV infection of resting CD4 T cells. We find that static cortical actin in resting T cells represents a restriction, and HIV utilizes the Gαi-dependent signaling from the chemokine coreceptor CXCR4 to activate a cellular actin depolymerizing factor, cofilin, to overcome this restriction. HIV envelope-mediated cofilin activation and actin dynamics are important for a post entry process that leads to viral nuclear localization. Inhibition of HIV-mediated actin rearrangement markedly diminishes viral latent infection of resting T cells. Conversely, induction of active cofilin greatly facilitates it. These findings shed new light on viral exploitation of cellular machinery in resting T cells, where chemokine receptor signaling becomes obligatory.

The quiescent nature of most peripheral T cells poses an effective limitation to human immunodeficiency virus (HIV) replication and, in particular, to viral integration into the host chromatin. Two HIV proteins, Nef and Tat, increase T cell activity, but a requirement of integration for viral gene expression would preclude a role for these proteins in resting cells. Here, we report that HIV infection leads to selective transcription of the nef and tat genes before integration. This preintegration transcription in quiescent cells leads to increased T cell activation and viral replication.

An adenovirus previously isolated from a mesenteric lymph node from a chimpanzee was fully sequenced and found to be similar in overall structure to human adenoviruses. The genome of this virus, called C68, is 36,521 bp in length and is most similar to subgroup E of human adenovirus, with 90% identity in most adenovirus type 4 open reading frames that have been sequenced. Substantial differences in the hexon hypervariable regions were noted between C68 and other known adenoviruses, including adenovirus type 4. Neutralizing antibodies to C68 were highly prevalent in sera from a population of chimpanzees, while sera from humans and rhesus monkeys failed to neutralize C68. Furthermore, infection with C68 was not neutralized from sera of mice immunized with human adenovirus serotypes 2, 4, 5, 7, and 12. A replication-defective version of C68 was created by replacing the E1a and E1b genes with a minigene cassette; this vector was efficiently transcomplemented by the E1 region of human adenovirus type 5. C68 vector transduced a number of human and murine cell lines. This nonhuman adenoviral vector is sufficiently similar to human serotypes to allow growth in 293 cells and transduction of cells expressing the coxsackievirus and adenovirus receptor. As it is dissimilar in regions such as the hexon hypervariable domains, C68 vector avoids significant cross-neutralization by sera directed against human serotypes.Vectors based on human adenovirus subgroup C (i.e., types 2 and 5) have realized widespread application in preclinical and clinical models of gene therapy (34). The viruses are rendered replication defective by deletion of E1 sequences. Multiple essential genes are disabled in more advanced versions of adenovirus vectors (7,10,17,31). An important limitation of the use of adenovirus type 2-and adenovirus type 5-based vectors for human applications is that many individuals are immune to the virus as the result of a previous natural infection (6). A manifestation of existing immunity to the virus is B-cell activation, leading to persistent neutralizing antibodies that block vector uptake in vivo and diminish transduction.One approach to accomplish immunologic distinction is to engineer the capsid of an adenovirus type 5-or adenovirus type 2-based vector. Several studies have attempted to accomplish this by exchanging the gene encoding fiber, since the protein is directly involved in receptor binding. While this has been successful in redirecting uptake of vector via a pathway distinct from that directed by the coxsackievirus and adenovirus (CAR) receptor, such chimeric viruses are still cross-neutralized due to blocking antibodies directed against hexon epitopes in the hypervariable regions (11,14,19,28,31). Recent attempts to engineer hexon proteins in chimeric viruses have been complicated by serotype-specific constraints in the hexon structure, which compromise the formation of stable chimeras. Selective modification of the hypervariable regions of hexon have diminished type-specific cross-neutralization in vitro...

Replication of human immunodeficiency virus (HIV) involves

Limited proteolysis is a widely occurring mechanism in protein biosynthesis. Protein precursors can be classified according to their functions, localization within cell compartments, and enzymic cleavage mechanisms. The presecretory proteins represent an important class of very rapidly turning over precursors which play an early role in the sequestration of secretory products and whose cleavage appears to be intimately associated with structures formed at the ribosome-membrane junction during protein synthesis. A model is proposed which predicts that the prepeptide forms a beta-pleated sheet structure with other components of the membrane which results in the transfer of a loop of peptide across the microsomal membrane. Proinsulin is representative of the general class of proproteins that are processed post-translationally within their secretory cells either during the formation and maturation of secretory granules (peptides hormones and neurotransmitters, serum albumins) or during the assembly of macromolecular structures (virus capsules, membrane-associated enzyme complexes). The former group are cleaved by Golgi-associated proteases having tryptic and carboxypeptidase B-like specificity. Some precursors are secreted as such and processed extracellularly either in the circulation or at special sites (procollagens, zymogens, provenoms, vitellogenins).

Retroviruses require integration of their RNA genomes for both stability and productive viral replication. In HIV infection of non-dividing, resting CD4 T cells, where integration is greatly impeded, the reverse transcribed HIV DNA has limited biological activity and a short half-life. In metabolically active and proliferating T cells, unintegrated DNA rapidly diminishes with cell division. HIV also infects the non-dividing but metabolically active macrophage population. In an in vitro examination of HIV infection of macrophages, we find that unintegrated viral DNA not only has an unusual stability, but also maintains biological activity. The unintegrated linear DNA, 1-LTR, and 2-LTR circles are stable for at least 30 days. Additionally, there is persistent viral gene transcription, which is selective and skewed towards viral early genes such as nef and tat with highly diminished rev and vif. One viral early gene product Nef was measurably synthesized. We also find that independent of integration, the HIV infection process in macrophages leads to generation of numerous chemokines.

Lentiviral Nef increases viral replication in vivo, plays a direct role in pathogenesis, and increases viral particle infectivity. We now find that HIV Nef also increases the activation of T cells, a cellular state required for optimal viral replication. This enhancement is stimulant-dependent. As defined by IL-2 generation, activation of T cells stimulated with classical mitogens [phorbol 12-myristate 13-acetate (PMA) ؉ anti-CD3, PMA ؉ phytohemagglutinin, and PMA ؉ ionomycin] is unaffected by the expression of Nef. However, Nef increases IL-2 secretion when cells are stimulated through the T cell receptor and the costimulus receptor (CD28). This increase in activation, which depends on Nef myristylation, is caused by an increase in the number of cells reaching full activation and not by an increase in the amount of IL-2 secreted per cell. These findings demonstrate that Nef lowers the threshold of the dual-receptor T cell activation pathway.

The study indicates that services that enhance access to treatment and respond to the range of social service needs of women are important for effective substance abuse treatment for women with children.