Mario P. S. Chin

Patterns of Human Immunodeficiency Virus Type 1 Recombination Ex Vivo Provide Evidence for Coadaptation of Distant Sites, Resulting in Purifying Selection for Intersubtype Recombinants during Replication

ABSTRACT High-frequency recombination is a hallmark of HIV-1 replication. Recombination can occur between two members of the same subtype or between viruses from two different subtypes, generating intra- or intersubtype recombinants, respectively. Many intersubtype recombinants have been shown to circulate in human populations. We hypothesize that sequence diversity affects the emergence of viable recombinants by decreasing recombination events and reducing the ability of the recombinants to replicate. To test our hypothesis, we compared recombination between two viruses containing subtype B pol genes (B/B) and between viruses with pol genes from subtype B or F (B/F). Recombination events generated during a single cycle of infection without selection pressure on pol gene function were analyzed by single-genome sequencing. We found that recombination occurred slightly (∼30%) less frequently in B/F than in B/B viruses, and the overall distribution of crossover junctions in pol was similar for the two classes of recombinants. We then examined the emergence of recombinants in a multiple cycle assay, so that functional pol gene products were selected. We found that the emerging B/B recombinants had complex patterns, and the crossover junctions were distributed throughout the pol gene. In contrast, selected B/F recombinants had limited recombination patterns and restricted crossover junction distribution. These results provide evidence for the evolved coadapted sites in variants from different subtypes; these sites may be segregated by recombination events, causing the newly generated intersubtype recombinants to undergo purifying selection. Therefore, the ability of the recombinants to replicate is the major barrier for many of these viruses.

Super-resolution imaging of nuclear import of adeno-associated virus in live cells

Adeno-associated virus (AAV) has been developed as a promising human gene therapy vector. Particularly, recombinant AAV vector (rAAV) achieves its transduction of host cells by crossing at least three physiological barriers including plasma membrane, endosomal membrane, and nuclear envelope (NE). So far, the AAV transduction mechanism has not been explored thoroughly at the single viral particle level. In this study, we employed high-speed super-resolution single-point edge-excitation sub-diffraction (SPEED) microscopy to map the events of single rAAV2 particles infecting live human cells with an unprecedented spatiotemporal resolution of 9–12 nm and 2–20 ms. Data reveal that rAAV2 particles are imported through nuclear pore complexes (NPCs) rather than nuclear membrane budding into the nucleus. Moreover, approximately 17% of the rAAV2 molecules starting from the cytoplasm successfully transverse the NPCs to reach the nucleoplasm, revealing that the NPCs act as a strict selective step for AAV delivery. This study lastly suggests a new pathway to improve AAV vectors for human gene therapy.

Analysis of the Origin and Evolutionary History of HIV-1 CRF28_BF and CRF29_BF Reveals a Decreasing Prevalence in the AIDS Epidemic of Brazil

Background HIV-1 subtype B and subtype F are prevalent in the AIDS epidemic of Brazil. Recombinations between these subtypes have generated at least four BF circulating recombinant forms (CRFs). CRF28_BF and CRF29_BF are among the first two BF recombinants being identified in Brazil and they contributed significantly to the epidemic. However, the evolution and demographic histories of the CRFs are unclear. Methodology/Principal Findings A collection of gag and pol sequences sampled within Brazil was screened for CRF28_BF-like and CRF29_BF-like recombination patterns. A Bayesian coalescent framework was employed to delineate the phylogenetic, divergence time and population dynamics of the virus having CRF28_BF-like and CRF29_BF-like genotype. These recombinants were phylogenetically related to each other and formed a well-supported monophyletic clade dated to 1988–1989. The effective number of infections by these recombinants grew exponentially over a five-year period after their emergence, but then decreased toward the present following a logistic model of population growth. The demographic pattern of both recombinants closely resembles those previously reported for CRF31_BC. Conclusions We revealed that HIV-1 recombinants of the CRF28_BF/CRF29_BF clade are still circulating in the Brazilian population. These recombinants did not exhibit a strong founder effect and showed a decreasing prevalence in the AIDS epidemic of Brazil. Our data suggested that multiple URFs may also play a role in shaping the epidemic of recombinant BF HIV-1 in the region.

HIV-1 Recombination: An Experimental Assay and a Phylogenetic Approach

The generation of genetic diversity is a fundamental characteristic of HIV-1 replication, allowing the virus to successfully evade the immune response and antiviral therapies. Although mutations are the first step towards diversity, mixing of the mutations through the process of recombination increases the variation and allows for the faster establishment of advantageous strains within the viral population. Therefore, studying recombination of HIV-1 provides insights into not only the mechanisms of HIV-1 replication but also into the potential for spread of antiviral drug resistance mutations within and across viral subtypes. This chapter describes, in detail, a highly sensitive recombination assay designed to measure the frequency of recombination between two viruses. This assay allows us to investigate the requirements, mechanisms, and final products of recombination. Additionally, software-based phylogenetic tools are described in this chapter, which allow for the identification of specific recombination events within patient samples or viral progeny from the recombination assay.

Cis–Acting RNA Elements of Human Immunodeficiency Virus

The World Health Organization has indicated that effective control of the HIV/AIDS pandemic is the world’s most urgent public health challenge. The 2009 UNAIDS Global Facts and Figures report estimated that almost 60 million people have been infected with the virus and that 25 million AIDS-related deaths have occurred since the pandemic began in the early 1980s. In 2008, there were approximately 33.4 million people living with HIV, 2.7 million new infections and 2 million deaths from AIDS-related causes. The pathogen that causes this pandemic is the Major (M) group of HIV type 1 (HIV-1). Group M HIV-1 dominates the global pandemic with at least nine subtypes and multiple intersubtype recombinants have been identified to date (Leitner et al., 2005). Many of these recombinants are circulating in multiple geographical regions and are integral parts in the HIV-1 pandemic (McCutchan, 2006; Takebe et al., 2004).