Long-term in vivo expression of a broad and potent entry inhibitor could circumvent the need for a conventional vaccine for HIV-1. Adeno-associated virus (AAV) vectors can stably express HIV-1 broadly neutralizing antibodies (bNAbs)1,2. However even the best bNAbs neutralize 10–50% of HIV-1 isolates inefficiently (IC80 > 5 μg/ml), suggesting that high concentrations of these antibodies would be necessary to achieve general protection3–6. Here we show that eCD4-Ig, a fusion of CD4-Ig with a small CCR5-mimetic sulfopeptide, binds avidly and cooperatively to the HIV-1 envelope glycoprotein (Env) and is more potent than the best bNAbs (geometric mean IC50 < 0.05 μg/ml). Because eCD4-Ig binds only conserved regions of Env, it is also much broader than any bNAb. For example, eCD4-Ig efficiently neutralized 100% of a diverse panel of neutralization-resistant HIV-1, HIV-2, and SIV isolates, including a comprehensive set of isolates resistant to the CD4-binding site bNAbs VRC01, NIH45-46, and 3BNC117. Rhesus macaques inoculated with an AAV vector stably expressed 17 to 77 μg/ml of fully functional rhesus eCD4-Ig for 40 weeks, and these macaques were protected from multiple infectious challenges with SHIV-AD8. Rhesus eCD4-Ig was also markedly less immunogenic than rhesus forms of four well characterized bNAbs. Our data suggest that AAV-delivered eCD4-Ig can function like an effective HIV-1 vaccine.

We have discovered a ubiquitin (Ub)-specific cysteine protease encoded within the N-terminal approximately 500 residues of the UL36 gene product, the largest (3164 aa) tegument protein of herpes simplex virus 1 (HSV-1). Enzymatic activity of this fragment, UL36USP, is detectable only after cleavage of UL36USP from full-length UL36 and occurs late during viral replication. UL36USP bears no homology to known deubiquitinating enzymes (DUBs) or Ub binding proteins. Sequence alignment of the large tegument proteins across the family Herpesviridae indicates conservation of key catalytic residues amongst these viruses. Recombinant UL36USP exhibits hydrolytic activity toward Ub-AMC and ubiquitinated branched peptides in vitro. In addition, recombinant UL36USP can cleave polyUb chains and appears to be specific for Lys48 linkages. Mutation of the active site cysteine residue (Cys65) to alanine abolishes this enzymatic activity. The lack of homology between UL36USP and eukaryotic DUBs makes this new family of herpesvirus ubiquitin-specific proteases attractive targets for selective inhibition.

The largest tegument protein of herpes simplex virus 1 (HSV-1), UL36, contains a novel deubiquitinating activity embedded in it. All members of the Herpesviridae contain a homologue of HSV-1 UL36, the N-terminal segments of which show perfect conservation of those residues implicated in catalysis. For murine cytomegalovirus and Epstein-Barr virus, chosen as representatives of the beta-and gammaherpesvirus subfamilies, respectively, we here show that the homologous modules indeed display deubiquitinating activity in vitro. The conservation of this activity throughout all subfamilies is indicative of an important, if not essential, function.Modification of proteins by ubiquitin (Ub) plays a pivotal role in a multitude of cellular processes, including proteolysis, cell cycle control, receptor internalization, and sorting within the endo/lysosomal system (7,14,16). Ubiquitination is achieved by an enzymatic cascade comprising a Ub-activating enzyme (E1), several Ub-carrier proteins (E2s), and hundreds of Ub ligases (E3s). Ubiquitination can be reversed by several families of enzymes collectively designated deubiquitinating enzymes (DUBs) (1,15).A number of viruses have evolved strategies to manipulate the ubiquitination status of host cell proteins, both through conjugation and deconjugation (2,4,6,10,13). Recently, we reported the identification of a novel viral ubiquitin-specific protease (USP), UL36 USP , encoded by the herpes simplex virus 1 (HSV-1) genome (9). UL36USP is a polypeptide of approximately 420 amino acids (aa) carried within the N-terminal portion of UL36, the largest tegument protein (3,164 aa) of HSV-1. This activity was detected through the use of mechanism-based, activesite-directed probes and confirmed by expression in Escherichia coli of a corresponding fragment that cleaves ubiquitin-based substrates. UL36USP activity peaks at late stages of viral replication and appears to require proteolytic processing from fulllength UL36 (9). The N-terminal UL36 fragment is well conserved in alphaherpesviruses, and a low homology to corresponding genes of the betaherpesvirus and gammaherpesvirus subfamilies was apparent in sequence alignments, but with strict conservation of the proposed catalytic residues. DUB activity may therefore be well conserved across the herpesvirus family and, if this is proven to be correct, would suggest an important function for this type of activity.We therefore set out to investigate the possible DUB activity of two phylogenetically distant homologues of HSV-1 UL36 USP , each representing a different subfamily of the Herpesviridae. We chose UL36 homologues encoded by mouse cytomegalovirus (MCMV, M48) and Epstein-Barr virus (EBV, BPLF1) as representatives of the beta-and gammaherpesvirus subfamilies, respectively. In order to assess the degree of homology between UL36 from HSV-1 and its MCMV and EBV counterparts, a sequence alignment was generated, covering the first 336 aa (the numbering refers to HSV-1) of UL36 (Fig. 1). Overall, the homology to HSV-1 is rather low, with on...

Elucidation of maternal immune correlates of protection against congenital cytomegalovirus (CMV) is necessary to inform future vaccine design. Here, we present a novel rhesus macaque model of placental rhesus CMV (rhCMV) transmission and use it to dissect determinants of protection against congenital transmission following primary maternal rhCMV infection. In this model, asymptomatic intrauterine infection was observed following i.v. rhCMV inoculation during the early second trimester in two of three rhCMV-seronegative pregnant females. In contrast, fetal loss or infant CMV-associated sequelae occurred in four rhCMV-seronegative pregnant macaques that were CD4 + T-cell depleted at the time of inoculation. Animals that received the CD4 +