Targeted Mutations in the Fusion Peptide Region of La Crosse Virus Attenuate Neuroinvasion and Confer Protection against Encephalitis.

La Crosse virus (LACV) is a major cause of pediatric encephalitis and aseptic meningitis in the Midwestern, Mid-Atlantic, and Southern United States, where it is an emerging pathogen. The LACV Gc glycoprotein plays a critical role in the neuropathogenesis of LACV encephalitis as the putative virus attachment protein. Previously, we identified and experimentally confirmed the location of the LACV fusion peptide within Gc and generated a panel of recombinant LACVs (rLACVs) containing mutations in the fusion peptide as well as the wild-type sequence. These rLACVs retained their ability to cause neuronal death in a primary embryonic rat neuronal culture system, despite decreased replication and fusion phenotypes. To test the role of the fusion peptide in vivo, we tested rLACVs in an age-dependent murine model of LACV encephalitis. When inoculated directly into the CNS of young adult mice (P28), the rLACV fusion peptide mutants were as neurovirulent as the rLACV engineered with a wild-type sequence, confirming the results obtained in tissue culture. In contrast, the fusion peptide mutant rLACVs were less neuroinvasive when suckling (P3) or weanling (P21) mice were inoculated peripherally, demonstrating that the LACV fusion peptide is a determinant of neuroinvasion, but not of neurovirulence. In a challenge experiment, we found that peripheral challenge of weanling (P21) mice with fusion peptide mutant rLACVs protected from a subsequent WT-LACV challenge, suggesting that mutations in the fusion peptide are an attractive target for generating live-attenuated virus vaccines. Importantly, the high degree of conservation of the fusion peptide amongst the Bunyavirales and, structurally, other arboviruses suggests that these findings are broadly applicable to viruses that use a class II fusion mechanism and cause neurologic disease.

Severe fever with thrombocytopenia virus glycoproteins are targeted by neutralizing antibodies and can use DC-SIGN as a receptor for pH-dependent entry into human and animal cell lines.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is a novel bunyavirus that recently emerged in China. Infection with SFTSV is associated with case-fatality rates of up to 30%, and neither antivirals nor vaccines are available at present. Development of antiviral strategies requires the elucidation of virus-host cell interactions. Here, we analyzed host cell entry of SFTSV. Employing lentiviral and rhabdoviral vectors, we found that the Gn/Gc glycoproteins (Gn/Gc) of SFTSV mediate entry into a broad range of human and animal cell lines, as well as human macrophages and dendritic cells. The Gn/Gc proteins of La Crosse virus (LACV) and Rift Valley Fever Virus (RVFV), other members of the bunyavirus family, facilitated entry into an overlapping but not identical range of cell lines, suggesting that SFTSV, LACV, and RVFV might differ in their receptor requirements. Entry driven by SFTSV Gn/Gc was dependent on low pH but did not require the activity of the pH-dependent endosomal/lysosomal cysteine proteases cathepsins B and L. Instead, the activity of a cellular serine protease was required for infection driven by SFTSV and LACV Gn/Gc. Sera from convalescent SFTS patients inhibited SFTSV Gn/Gc-driven host cell entry in a dose-dependent fashion, demonstrating that the vector system employed is suitable to detect neutralizing antibodies. Finally, the C-type lectin DC-SIGN was found to serve as a receptor for SFTSV Gn/Gc-driven entry into cell lines and dendritic cells. Our results provide initial insights into cell tropism, receptor usage, and proteolytic activation of SFTSV and will aid in the understanding of viral spread and pathogenesis.

HIV-1-related central nervous system disease: current issues in pathogenesis, diagnosis, and treatment.

HIV-associated central nervous system (CNS) injury continues to be clinically significant in the modern era of HIV infection and therapy. A substantial proportion of patients with suppressed HIV infection on optimal antiretroviral therapy have impaired performance on neuropsychological testing, suggesting persistence of neurological abnormalities despite treatment and projected long-term survival. In the underresourced setting, limited accessibility to antiretroviral medications means that CNS complications of later-stage HIV infection continue to be a major concern. This article reviews key recent advances in our understanding of the neuropathogenesis of HIV, focusing on basic and clinical studies that reveal viral and host features associated with viral neuroinvasion, persistence, and immunopathogenesis in the CNS, as well as issues related to monitoring and treatment of HIV-associated CNS injury in the current era.

Orthobunyavirus entry into neurons and other mammalian cells occurs via clathrin-mediated endocytosis and requires trafficking into early endosomes.

La Crosse virus (LACV) is a leading cause of pediatric encephalitis and aseptic meningitis in the midwestern and southern United States, where it is considered an emerging human pathogen. No specific therapies or vaccines are available for LACV or any other orthobunyaviruses. Inhibition of LACV entry into cells is a potential target for therapeutic intervention, but this approach is limited by our current knowledge of the entry process. Here, we determined that clathrin-mediated endocytosis is the primary mechanism of orthobunyavirus entry and identified key cellular factors in this process. First, we demonstrated that LACV colocalized with clathrin shortly after infection in HeLa cells; we then confirmed the functional requirement of dynamin- and clathrin-mediated endocytosis for orthobunyavirus entry using several independent assays and, importantly, extended these findings to primary neuronal cultures. We also determined that macropinocytosis and caveolar endocytosis, both established routes of virus entry, are not critical for cellular entry of LACV. Moreover, we demonstrated that LACV infection is dependent on Rab5, which plays an important regulatory role in early endosomes, but not on Rab7, which is associated with late endosomes. These findings provide the first description of bunyavirus entry into cells of the central nervous system, where infection can cause severe neurological disease, and will aid in the design and development of antivirals and therapeutics that may be useful in the treatment of LACV and, more broadly, arboviral infections of the central nervous system.

Depletion of CD4⁺ T cells abrogates post-peak decline of viremia in SIV-infected rhesus macaques.

CD4+ T cells play a central role in the immunopathogenesis of HIV/AIDS, and their depletion during chronic HIV infection is a hallmark of disease progression. However, the relative contribution of CD4+ T cells as mediators of antiviral immune responses and targets for virus replication is still unclear. Here, we have generated data in SIV-infected rhesus macaques (RMs) that suggest that CD4+ T cells are essential in establishing control of virus replication during acute infection. To directly assess the role of CD4+ T cells during primary SIV infection, we in vivo depleted these cells from RMs prior to infecting the primates with a pathogenic strain of SIV. Compared with undepleted animals, CD4+ lymphocyte-depleted RMs showed a similar peak of viremia, but did not manifest any post-peak decline of virus replication despite CD8+ T cell- and B cell-mediated SIV-specific immune responses comparable to those observed in control animals. Interestingly, depleted animals displayed rapid disease progression, which was associated with increased virus replication in non-T cells as well as the emergence of CD4-independent SIV-envelopes. Our results suggest that the antiviral CD4+ T cell response may play an important role in limiting SIV replication, which has implications for the design of HIV vaccines.

Arboviral encephalitides: transmission, emergence, and pathogenesis.

Arthropod-borne viruses (arboviruses) are of paramount concern as a group of pathogens at the forefront of emerging and re-emerging diseases. Although some arboviral infections are asymptomatic or present with a mild influenza-like illness, many are important human and veterinary pathogens causing serious illness ranging from rash and arthritis to encephalitis and hemorrhagic fever. Here, we discuss arboviruses from diverse families (Flaviviruses, Alphaviruses, and the Bunyaviridae) that are causative agents of encephalitis in humans. An understanding of the natural history of these infections as well as shared mechanisms of neuroinvasion and neurovirulence is critical to control the spread of these viruses and for the development of effective vaccines and treatment modalities.

La Crosse virus (LACV) Gc fusion peptide mutants have impaired growth and fusion phenotypes, but remain neurotoxic.

La Crosse virus is a leading cause of pediatric encephalitis in the Midwestern United States and an emerging pathogen in the American South. The LACV glycoprotein Gc plays a critical role in entry as the virus attachment protein. A 22 amino acid hydrophobic region within Gc (1066-1087) was recently identified as the LACV fusion peptide. To further define the role of Gc (1066-1087) in virus entry, fusion, and neuropathogenesis, a panel of recombinant LACV (rLACV) fusion peptide mutant viruses was generated. Replication of mutant rLACVs was significantly reduced. In addition, the fusion peptide mutants demonstrated decreased fusion phenotypes relative to LACV-WT. Interestingly, these viruses maintained their ability to cause neuronal loss in culture, suggesting that the fusion peptide of LACV Gc is a determinant of properties associated with neuroinvasion (growth to high titer in muscle cells and a robust fusion phenotype), but not necessarily of neurovirulence.

Intrathecal humoral responses are inversely associated with the frequency of simian immunodeficiency virus macrophage-tropic variants in the central nervous system.

Sustained simian immunodeficiency virus (SIV) infection of the central nervous system (CNS) depends on macrophage-tropic (M-tropic) strains that are often easily neutralizable. The CNS is often thought of as an immunologically privileged site that fosters replication of M-tropic quasispecies. Yet, there are limited data addressing the intrathecal antibody response or the role of the humoral response, in general, to control M-tropic strains. We investigated the temporal course of the intrathecal fusion inhibitory activity against an M-tropic viral variant and found an inverse relationship between the magnitude of this neutralization and the prevalence of M-tropic populations. These studies suggest a role for the humoral response in the suppression of M-tropic viral species in the CNS in experimental SIV infection.

Planning Future Strategies for Domestic and International NeuroAIDS Research, July 24-25, 2008.

The National Institute of Mental Health in cooperation with the National Institute on Drug Abuse and the National Institute of Neurological Disorders and Stroke organized a meeting on July 24-25, 2008 to develop novel research directions for neuroAIDS research. The deliberations of this meeting are outlined in this brief report. Several critical research areas in neuroAIDS were identified as areas of emphasis. Opportunities for collaborations between large NIH-funded projects were also discussed.

Anx2 interacts with HIV-1 Gag at phosphatidylinositol (4,5) bisphosphate-containing lipid rafts and increases viral production in 293T cells.

The neuronal damage characteristic of HIV-1-mediated CNS diseases is inflicted by HIV-1 infected brain macrophages. Several steps of viral replication, including assembly and budding, differ between macrophages and T cells; it is likely that cell-specific host factors mediate these differences. We previously defined Annexin 2 (Anx2) as an HIV Gag binding partner in human monocyte-derived macrophages (MDMs) that promotes proper viral assembly. Anx2, a calcium-dependent membrane-binding protein that can aggregate phospholipid-containing lipid rafts, is expressed to high levels in macrophages, but not in T lymphocytes or the 293T cell line. Here, we use bimolecular fluorescence complementation in the 293T cell model to demonstrate that Anx2 and HIV-1 Gag interact at the phosphatidylinositol (4,5) bisphosphate-containing lipid raft membrane domains at which Gag mediates viral assembly. Furthermore, we demonstrate that Anx2 expression in 293T cells increases Gag processing and HIV-1 production. These data provide new evidence that Anx2, by interacting with Gag at the membranes that support viral assembly, functions in the late stages of HIV-1 replication.

Mutagenesis of the La Crosse Virus glycoprotein supports a role for Gc (1066-1087) as the fusion peptide.

The La Crosse Virus (LACV) M segment encodes two glycoproteins (Gn and Gc), and plays a critical role in the neuropathogenesis of LACV infection as the primary determinant of neuroinvasion. A recent study from our group demonstrated that the region comprising the membrane proximal two-thirds of Gc, amino acids 860-1442, is critical in mediating LACV fusion and entry. Furthermore, computational analysis identified structural similarities between a portion of this region, amino acids 970-1350, and the E1 fusion protein of two alphaviruses: Sindbis virus and Semliki Forrest virus (SFV). Within the region 970-1350, a 22-amino-acid hydrophobic segment (1066-1087) is predicted to correlate structurally with the fusion peptides of class II fusion proteins. We performed site-directed mutagenesis of key amino acids in this 22-amino acid segment and determined the functional consequences of these mutations on fusion and entry. Several mutations within this hydrophobic domain affected glycoprotein expression to some extent, but all mutations either shifted the pH threshold of fusion below that of the wild-type protein, reduced fusion efficiency, or abrogated cell-to-cell fusion and pseudotype entry altogether. These results, coupled with the aforementioned computational modeling, suggest that the LACV Gc functions as a class II fusion protein and support a role for the region Gc 1066-1087 as a fusion peptide.

Simian immunodeficiency virus envelope compartmentalizes in brain regions independent of neuropathology.

Simian immunodeficiency virus (SIV) and human immunodeficiency virus (HIV) gp160s obtained from the brain are often genetically distinct from those isolated from other organs, suggesting the presence of brain-specific selective pressures or founder effects that result in the compartmentalization of viral quasi-species. Whereas HIV has also been found to compartmentalize within different regions of the brain, the extent of brain-regional compartmentalization of SIV in rhesus macaques has not been characterized. Furthermore, much is still unknown about whether phenotypic differences exist in envelopes from different brain regions. To address these questions, env DNA sequences were amplified from four SIVmac239-infected macaques and subjected to phylogenetic and phenetic analysis. The authors demonstrated that sequences from different areas of the brain form distinct clades, and that the long-term progressing macaques demonstrated a greater degree of regional compartmentalization compared to the rapidly progressing macaques. In addition, regional compartmentalization occurred regardless of the presence of giant-cell encephalitis. Nucleotide substitution rates at synonymous and nonsynonymous sites (ds:dn rates) indicated that positive selection varied among envelopes from different brain regions. In one macaque, envelopes from some but not all brain regions acquired changes in a conserved CD4-binding motif GGGDPE at amino acids 382 to 387. Furthermore, gp160s with the mutation G383E were able to mediate cell-to-cell fusion in a CD4-independent manner and were more susceptible to fusion inhibition by pooled plasma from infected macaques. Reversion of this mutation by site-directed mutagenesis resulted in reduction of CD4-independence and resistance to fusion inhibition in cell fusion assays. These studies demonstrate that SIV evolution within the brain results in a heterogeneous viral population with different phenotypes among different regions.

Annexin 2: a novel human immunodeficiency virus type 1 Gag binding protein involved in replication in monocyte-derived macrophages.

Human immunodeficiency virus (HIV) replication in the major natural target cells, CD4+ T lymphocytes and macrophages, is parallel in many aspects of the virus life cycle. However, it differs as to viral assembly and budding, which take place on plasma membranes in T cells and on endosomal membranes in macrophages. It has been postulated that cell type-specific host factors may aid in directing viral assembly to distinct destinations. In this study we defined annexin 2 (Anx2) as a novel HIV Gag binding partner in macrophages. Anx2-Gag binding was confined to productively infected macrophages and was not detected in quiescently infected monocyte-derived macrophages (MDM) in which an HIV replication block was mapped to the late stages of the viral life cycle (A. V. Albright, R. M. Vos, and F. Gonzalez-Scarano, Virology 325:328-339, 2004). We demonstrate that the Anx2-Gag interaction likely occurs at the limiting membranes of late endosomes/multivesicular bodies and that Anx2 depletion is associated with a significant decline in the infectivity of released virions; this coincided with incomplete Gag processing and inefficient incorporation of CD63. Cumulatively, our data suggest that Anx2 is essential for the proper assembly of HIV in MDM.

HIV-1 tropism for the central nervous system: Brain-derived envelope glycoproteins with lower CD4 dependence and reduced sensitivity to a fusion inhibitor.

We previously described envelope glycoproteins of an HIV-1 isolate adapted in vitro for growth in microglia that acquired a highly fusogenic phenotype and lower CD4 dependence, as well as resistance to inhibition by anti-CD4 antibodies. Here, we investigated whether similar phenotypic changes are present in vivo. Envelope clones from the brain and spleen of an HIV-1-infected individual with neurological disease were amplified, cloned, and sequenced. Phylogenetic analysis demonstrated clustering of sequences according to the tissue of origin, as expected. Functional clones were then used in cell-to-cell fusion assays to test for CD4 and co-receptor utilization and for sensitivity to various antibodies and inhibitors. Both brain- and spleen-derived envelope clones mediated fusion in cells expressing both CD4 and CCR5 and brain envelopes also used CCR3 as co-receptor. We found that the brain envelopes had a lower CD4 dependence, since they efficiently mediated fusion in the presence of low levels of CD4 on the target cell membrane, and they were significantly more resistant to blocking by anti-CD4 antibodies than the spleen-derived envelopes. In contrast, we observed no difference in sensitivity to the CCR5 antagonist TAK-779. However, brain-derived envelopes were significantly more resistant than those from spleen to the fusion inhibitor T-1249 and concurrently showed slightly greater fusogenicity. Our results suggest an increased affinity for CD4 of brain-derived envelopes that may have originated from in vivo adaptation to replication in microglial cells. Interestingly, we note the presence of envelopes more resistant to a fusion inhibitor in the brain of an untreated, HIV-1-infected individual.

Emerging infectious diseases: the Bunyaviridae.

The Bunyaviridae are a large group of viruses that infect a diversity of arthropod vectors and animal hosts. They have a worldwide distribution and can be the cause of human illness ranging from mild asymptomatic infection to hemorrhagic fever and fatal encephalitis. The growth of the human population, the expansion of agricultural and economic development, climatic changes, and the speed and frequency of global transportation all favor the emergence of bunyaviruses and other arthropod borne viruses. International monitoring of the Bunyaviridae and a greater understanding of their ecology and biology are needed to prepare for future outbreaks.