A long-standing question in retrovirus biology is how RNA genomes are distributed among virions. In the studies presented in this report, we addressed this issue by directly examining HIV-1 RNAs in virions using a modified HIV-1 genome that contained recognition sites for BglG, an antitermination protein in the Escherichia coli bgl operon, which was coexpressed with a fragment of BglG RNA binding protein fused to a fluorescent protein. Our results demonstrate that the majority of virions (>90%) contain viral RNAs. We also coexpressed HIV-1 genomes containing binding sites for BglG or the bacteriophage MS2 coat protein along with 2 fluorescent protein-tagged RNA binding proteins. This method allows simultaneously labeling and discrimination of 2 different RNAs at single-RNA-detection sensitivity. Using this strategy, we obtained physical evidence that virions contain RNAs derived from different parental viruses (heterozygous virion) at ratios expected from a random distribution, and we found that this ratio can be altered by changing the dimerization sequences. Our studies of heterozygous virions also support a generally accepted but unproven assumption that most particles contain 1 dimer. This study provides answers to long-standing questions in HIV-1 biology and illustrates the power and sensitivity of the 2-RNA labeling method, which can also be adapted to analyze various issues of RNA biogenesis including the detection of different RNAs in live cell imaging.Bgl ͉ MS2 ͉ dimerization ͉ DIS ͉ fluorescent protein

Neither the number of HIV-1 proviruses within individual infected cells in HIV-1–infected patients nor their genetic relatedness within individual infected cells and between cells and plasma virus are well defined. To address these issues we developed a technique to quantify and genetically characterize HIV-1 DNA from single infected cells in vivo. Analysis of peripheral blood CD4 + T cells from nine patients revealed that the majority of infected cells contain only one copy of HIV-1 DNA, implying a limited potential for recombination in virus produced by these cells. The genetic similarity between HIV populations in CD4 + T cells and plasma implies ongoing exchange between these compartments both early and late after infection.

Recombination is a major mechanism that generates variation in populations of human immunodeficiency virus type 1 (HIV-1). Mutations that confer replication advantages, such as drug resistance, often cluster within regions of the HIV-1 genome. To explore how efficiently HIV-1 can assort markers separated by short distances, we developed a flow cytometry-based system to study recombination. Two HIV-1-based vectors were generated, one encoding the mouse heat-stable antigen gene and green fluorescent protein gene (GFP), and the other encoding the mouse Thy-1 gene and GFP. We generated derivatives of both vectors that contained nonfunctional GFP inactivated by different mutations. Recombination in the region between the two inactivating mutations during reverse transcription could yield a functional GFP. With this system, we determined that the recombination rates of markers separated by 588, 300, 288, and 103 bp in one round of viral replication are 56, 38, 31, and 12%, respectively, of the theoretical maximum measurable recombination rate. Statistical analyses revealed that at these intervals, recombination rates and marker distances have a near-linear relationship that is part of an overall quadratic fit. Additionally, we examined the segregation of three markers within 600 bp and concluded that HIV-1 crossover events do not exhibit high negative interference. We also examined the effects of target cells and viral accessory proteins on recombination rate. Similar recombination rates were observed when human primary CD4؉ T cells and a human T-cell line were used as target cells. We also found equivalent recombination rates in the presence and absence of accessory genes vif, vpr, vpu, and nef. These results illustrate the power of recombination in generating viral population variation and predict the rapid assortment of mutations in the HIV-1 genome in infected individuals.

All positive-strand RNA [(؉)RNA] viruses replicate their RNA on intracellular membranes, often in association with spherular invaginations of the target membrane. For brome mosaic virus, we previously showed that such spherules serve as compartments or mini-organelles for RNA replication and that their assembly, structure, and function have similarities to the replicative cores of retrovirus and double-stranded RNA virus virions. Some other (؉)RNA viruses conduct RNA replication in association with individual or clustered double-membrane vesicles, appressed double membranes, or other structures whose possible relationships to the spherular invaginations are unclear. Here we show that modulating the relative levels and interactions of brome mosaic virus replication factors 1a and 2a polymerase (2a pol ) shifted the membrane rearrangements associated with RNA replication from small invaginated spherules to large, karmellae-like, multilayer stacks of appressed double membranes that supported RNA replication as efficiently as spherules. Spherules were induced by expressing 1a, which has functional similarities to retrovirus virion protein Gag, or 1a plus low levels of 2a pol . Double-membrane layers were induced by 1a plus higher levels of 2a pol and were suppressed by deleting the major 1a-interacting domain from 2a pol . The stacked, doublemembrane layers alternated with spaces that, like spherule interiors, were 50 -60 nm wide, connected to the cytoplasm, and contained 1a and 2a pol . These and other results suggest that seemingly diverse membrane rearrangements associated with RNA replication by varied (؉)RNA viruses may represent topologically and functionally related structures formed by similar proteinprotein and protein-membrane interactions and interconverted by altering the balances among those interactions. P ositive-strand RNA [(ϩ)RNA] viruses are the largest genetic class of viruses and include many pathogens, such as the severe acute respiratory syndrome (SARS) coronavirus, hepatitis C virus, and potential bioterrorism agents. Such (ϩ)RNA viruses encapsidate messenger-sense genomic RNAs and replicate those genomes through negative-strand RNA intermediates. The RNA replication complexes of (ϩ)RNA viruses invariably form on intracellular membranes, usually in association with vesiculation or other membrane rearrangements. Different (ϩ)RNA viruses use distinct but usually specific membranes, ranging from the outer membranes of the endoplasmic reticulum (ER), later or mixed compartments of the secretory pathway, endosomes, mitochondria, and other organelles (1-7).Many (ϩ)RNA viruses, including alphaviruses, nodaviruses, bromoviruses, and many others form RNA replication complexes at virus-induced, vesicular invaginations of specific intracellular membranes (4-6, 8-11). One such virus is brome mosaic virus (BMV), a member of the alphavirus superfamily of human, animal, and plant viruses. BMV encodes two proteins that direct viral RNA replication in its natural plant hosts or yeast. Viral replication factor ...