Visualizing Association of the Retroviral Gag Protein with Unspliced Viral RNA in the Nucleus.

Packaging of genomic RNA (gRNA) by retroviruses is essential for infectivity, yet the subcellular site of the initial interaction between the Gag polyprotein and gRNA remains poorly defined. Because retroviral particles are released from the plasma membrane, it was previously thought that Gag proteins initially bound to gRNA in the cytoplasm or at the plasma membrane. However, the Gag protein of the avian retrovirus Rous sarcoma virus (RSV) undergoes active nuclear trafficking, which is required for efficient gRNA encapsidation (L. Z. Scheifele, R. A. Garbitt, J. D. Rhoads, and L. J. Parent, Proc Natl Acad Sci U S A 99:3944-3949, 2002, https://doi.org/10.1073/pnas.062652199; R. Garbitt-Hirst, S. P. Kenney, and L. J. Parent, J Virol 83:6790-6797, 2009, https://doi.org/10.1128/JVI.00101-09). These results raise the intriguing possibility that the primary contact between Gag and gRNA might occur in the nucleus. To examine this possibility, we created a RSV proviral construct that includes 24 tandem repeats of MS2 RNA stem-loops, making it possible to track RSV viral RNA (vRNA) in live cells in which a fluorophore-conjugated MS2 coat protein is coexpressed. Using confocal microscopy, we observed that both wild-type Gag and a nuclear export mutant (Gag.L219A) colocalized with vRNA in the nucleus. In live-cell time-lapse images, the wild-type Gag protein trafficked together with vRNA as a single ribonucleoprotein (RNP) complex in the nucleoplasm near the nuclear periphery, appearing to traverse the nuclear envelope into the cytoplasm. Furthermore, biophysical imaging methods suggest that Gag and the unspliced vRNA physically interact in the nucleus. Taken together, these data suggest that RSV Gag binds unspliced vRNA to export it from the nucleus, possibly for packaging into virions as the viral genome.IMPORTANCE Retroviruses cause severe diseases in animals and humans, including cancer and acquired immunodeficiency syndromes. To propagate infection, retroviruses assemble new virus particles that contain viral proteins and unspliced vRNA to use as gRNA. Despite the critical requirement for gRNA packaging, the molecular mechanisms governing the identification and selection of gRNA by the Gag protein remain poorly understood. In this report, we demonstrate that the Rous sarcoma virus (RSV) Gag protein colocalizes with unspliced vRNA in the nucleus in the interchromatin space. Using live-cell confocal imaging, RSV Gag and unspliced vRNA were observed to move together from inside the nucleus across the nuclear envelope, suggesting that the Gag-gRNA complex initially forms in the nucleus and undergoes nuclear export into the cytoplasm as a viral ribonucleoprotein (vRNP) complex.

Enhanced binding of antibodies generated during chronic HIV infection to mucus component MUC16.

Transmission of HIV across mucosal barriers accounts for the majority of HIV infections worldwide. Thus, efforts aimed at enhancing protective immunity at these sites are a top priority, including increasing virus-specific antibodies (Abs) and antiviral activity at mucosal sites. Mucin proteins, including the largest cell-associated mucin, mucin 16 (MUC16), help form mucus to provide a physical barrier to incoming pathogens. Here, we describe a natural interaction between Abs and MUC16 that is enhanced in specific disease settings such as chronic HIV infection. Binding to MUC16 was independent of IgG subclass, but strongly associated with shorter Ab glycan profiles, with agalactosylated (G0) Abs demonstrating the highest binding to MUC16. Binding of Abs to epithelial cells was diminished following MUC16 knockdown, and the MUC16 N-linked glycans were critical for binding. Further, agalactosylated VRC01 captured HIV more efficiently in MUC16. These data point to a novel opportunity to enrich Abs at mucosal sites by targeting Abs to MUC16 through changes in Fc glycosylation, potentially blocking viral movement and sequestering the virus far from the epithelial border. Thus, next-generation vaccines or monoclonal therapeutics may enhance protective immunity by tuning Ab glycosylation to promote the enrichment of Abs at mucosal barriers.

Cellular targets of the SV40 small-t antigen in human cell transformation.

SV40 LT and ST antigens cooperate to induce the proliferation and eventual transformation of several human cell types. In natural virus infections, ST often enhances the function of LT when both proteins are present, and it can be difficult to completely separate the roles of the individual proteins. By studying ST in the absence of LT or by replacing ST function with combinations of cellular proteins, several themes have emerged which help define the requirement for ST in human cell transformation. These include the activation of transcription of two cyclins, D and A, along with downregulation of the cyclin kinase inhibitor p27. Modification of these key cell cycle regulators may be influenced by the activation of key downstream targets in the PI3K pathway.

Differential binding of IgG and IgA to mucus of the female reproductive tract.

Cells of the endocervix are responsible for the secretion of mucins, which provide an additional layer of protection to the female reproductive tract (FRT). This barrier is likely fortified with IgA as has previously been shown in the gastrointestinal tract and lungs of mice. Mucus associated IgA can facilitate clearance of bacteria. While a similar function for IgG has been proposed, an association with mucus has not yet been demonstrated. Here we find that IgA and IgG are differentially associated with the different types of mucus of the FRT. We observed that while both IgA and IgG are stably associated with cervical mucus, only IgG is associated with cervicovaginal mucus. These findings reveal that antibodies can bind tightly to mucus, where they can play a significant role in the fortification of the mucus barriers of the FRT. It may be possible to harness this interaction in the development of vaccines designed to protect the FRT mucosal barriers from sexually transmitted diseases such as HIV.

The role of the foreskin in male circumcision: an evidence-based review.

HIV sexual transmission via the male genital tract remains poorly defined. Male circumcision was shown to reduce female-to-male transmission in Africa, providing a clue that the foreskin plays a role in the route of transmission. Scientific data in four categories relating to how the foreskin might affect HIV transmission is summarized: (i) surface area, (ii) microbiologic environment, (iii) HIV-1-susceptible cells, and (iv) tissue structure. The relative contribution of each of these areas is yet unknown, and further studies will be crucial in understanding how male circumcision affects HIV transmission in men.

Role of SV40 ST antigen in the persistent infection of mesothelial cells.

Viral DNA is maintained episomally in SV40 infected mesothelial cells and virus is produced at low but steady rates. High copy numbers of the viral DNA are maintained in a WT infection where both early antigens are expressed. In the absence of ST, cells are immortal but non-transformed and the infected cells maintain only a few copies of episomal viral DNA. We show that ST expression is necessary for the maintenance of high copy numbers of viral DNA and that the PP2A binding ability of ST plays a role in genome maintenance. Interestingly, an siRNA to the virus late region downregulates virus copy number and virus production but does not prevent the anchorage-independent growth of these cells. Furthermore, addition of virus neutralizing antibody to culture media also decreases copy numbers of viral DNA in WT-infected cells, suggesting that virus production and re-infection of cells may play a role in maintaining the persistent infection.

Activated CD34-derived Langerhans cells mediate transinfection with human immunodeficiency virus.

Langerhans cells (LCs) are a subset of dendritic cells (DCs) that reside within epidermal and mucosal tissue. Because of their location, LCs are potentially the first cells to encounter human immunodeficiency virus (HIV) during sexual transmission. We report that LCs purified from CD34(+)-derived DCs can facilitate the transinfection of target cells but only after activation. Virions were observed in an intracellular compartment that contains several tetraspanins, in addition to the unique LC markers langerin and CD1a. This reveals that the trafficking of HIV within LCs is reminiscent of that which occurs in mature monocyte-derived DCs and that it varies with the activation state of the cell. The observation that activated LCs can mediate transinfection suggests a potential role for these cells in the known increase in HIV transmission associated with sexually transmitted infections that would cause inflammation of the genital lining.