Human and simian immunodeficiency viruses (HIV/SIVs) use CD4 as the primary receptor to enter target cells. Here, we show that the chimpanzee CD4 is highly polymorphic, with nine coding variants present in wild populations, and that this diversity interferes with SIV envelope (Env)–CD4 interactions. Testing the replication fitness of SIVcpz strains in CD4+ T cells from captive chimpanzees, we found that certain viruses were unable to infect cells from certain hosts. These differences were recapitulated in CD4 transfection assays, which revealed a strong association between CD4 genotypes and SIVcpz infection phenotypes. The most striking differences were observed for three substitutions (Q25R, Q40R, and P68T), with P68T generating a second N-linked glycosylation site (N66) in addition to an invariant N32 encoded by all chimpanzee CD4 alleles. In silico modeling and site-directed mutagenesis identified charged residues at the CD4–Env interface and clashes between CD4- and Env-encoded glycans as mechanisms of inhibition. CD4 polymorphisms also reduced Env-mediated cell entry of monkey SIVs, which was dependent on at least one D1 domain glycan. CD4 allele frequencies varied among wild chimpanzees, with high diversity in all but the western subspecies, which appeared to have undergone a selective sweep. One allele was associated with lower SIVcpz prevalence rates in the wild. These results indicate that substitutions in the D1 domain of the chimpanzee CD4 can prevent SIV cell entry. Although some SIVcpz strains have adapted to utilize these variants, CD4 diversity is maintained, protecting chimpanzees against infection with SIVcpz and other SIVs to which they are exposed.

Genome engineering of bacteriophages provides opportunities for precise genetic dissection and for numerous phage applications including therapy. However, few methods are available for facile construction of unmarked precise deletions, insertions, gene replacements and point mutations in bacteriophages for most bacterial hosts. Here we describe CRISPY-BRED and CRISPY-BRIP, methods for efficient and precise engineering of phages in Mycobacterium species, with applicability to phages of a variety of other hosts. This recombineering approach uses phage-derived recombination proteins and Streptococcus thermophilus CRISPR-Cas9.

The arms race between bacteria and their bacteriophages profoundly influences microbial evolution. With an estimated 1023phage infections occurring per second, there is strong selection for both bacterial survival and phage coevolution for continued propagation. Many phage resistance systems, including restriction-modification systems, clustered regularly interspaced short palindromic repeat-Cas (CRISPR-Cas) systems, a variety of abortive infection systems, and many others that are not yet mechanistically defined, have been described. Temperate bacteriophages are common and form stable lysogens that are immune to superinfection by the same or closely related phages. However, temperate phages collude with their hosts to confer defense against genomically distinct phages, to the mutual benefit of the bacterial host and the prophage. Prophage-mediated viral systems have been described inMycobacteriumphages andPseudomonasphages but are predicted to be widespread throughout the microbial world. Here we describe a new viral defense system in which the mycobacteriophage Sbash prophage colludes with itsMycobacterium smegmatishost to confer highly specific defense against infection by the unrelated mycobacteriophage Crossroads. Sbash genes30and31are lysogenically expressed and are necessary and sufficient to confer defense against Crossroads but do not defend against any of the closely related phages grouped in subcluster L2. The mapping of Crossroads defense escape mutants shows that genes132and141are involved in recognition by the Sbash defense system and are proposed to activate a loss in membrane potential mediated by Sbash gp30 and gp31.IMPORTANCEViral infection is an ongoing challenge to bacterial survival, and there is strong selection for development or acquisition of defense systems that promote survival when bacteria are attacked by bacteriophages. Temperate phages play central roles in these dynamics through lysogenic expression of genes that defend against phage attack, including those unrelated to the prophage. Few prophage-mediated viral defense systems have been characterized, but they are likely widespread both in phage genomes and in the prophages integrated in bacterial chromosomes.

An effective T-cell–based AIDS vaccine should induce strong HIV-specific CD8+ T cells in mucosal tissues without increasing the availability of target cells for the virus. Here, we evaluated five immunization strategies that include Human adenovirus-5 (AdHu5), Chimpanzee adenovirus-6 (AdC6) or -7 (AdC7), Vaccinia virus (VV), and DNA given by electroporation (DNA/EP), all expressing Simian immunodeficiency virus group specific antigen/transactivator of transcription (SIVmac239Gag/Tat). Five groups of six rhesus macaques (RMs) each were vaccinated with DNA/EP-AdC6-AdC7, VV-AdC6-AdC7, DNA/-EP-VV-AdC6, DNA/EP-VV-AdC7, or AdHu5-AdHu5-AdHu5 and were challenged repeatedly with low-dose intrarectal SIVmac239. Upon challenge, there were no significant differences among study groups in terms of virus acquisition or viral load after infection. When taken together, the immunization regimens did not protect against SIV acquisition compared with controls but did result in an ∼1.6-log decline in set-point viremia. Although all immunized RMs had detectable SIV-specific CD8+ T cells in blood and rectal mucosa, we found no correlation between the number or function of these SIV-specific CD8+ T cells and protection against SIV acquisition. Interestingly, RMs experiencing breakthrough infection showed significantly higher prechallenge levels of CD4+C-C chemokine receptor type 5 (CCR5)+HLA-DR+ T cells in the rectal biopsies (RB) than animals that remained uninfected. In addition, among the infected RMs, the percentage of CD4+CCR5+Ki-67+ T cells in RBs prechallenge correlated with higher early viremia. Overall, these data suggest that the levels of activated CD4+CCR5+ target T cells in the rectal mucosa may predict the risk of SIV acquisition in RMs vaccinated with vectors that express SIVGag/Tat.