Molecular analyses and phylogeny of the herpes simplex virus 2 US9 and glycoproteins gE/gI obtained from infected subjects during the Herpevac Trial for Women

Abstract

Herpes simplex virus 2 (HSV-2) is a large double-stranded DNA virus that causes genital sores when spread by sexual contact and is a principal cause of viral encephalitis in newborns and infants. Viral glycoproteins enable virion entry into and spread between cells, making glycoproteins a prime target for vaccine development. A truncated glycoprotein D2 (gD2) vaccine candidate, recently tested in the phase 3 Herpevac Trial for Women, did not prevent HSV-2 infection in initially seronegative women. Some women who became infected experienced multiple recurrences during the trial. The HSV US7, US8, and US9 genes encode glycoprotein I (gI), glycoprotein E (gE), and the US9 type II membrane protein, respectively. These proteins participate in viral spread across cell junctions and facilitate anterograde transport of virion components in neurons, prompting us to investigate whether sequence variants in these genes could be associated with frequent recurrence. The nucleotide sequences and dN/dS ratios of the US7-US9 region from viral isolates of individuals who experienced multiple recurrences were compared with those who had had a single episode of disease. No consistent polymorphism(s) distinguished the recurrent isolates. In frequently recurring isolates, the dN/dS ratio of US7 was low while greater variation (higher dN/dS ratio) occurred in US8, suggesting conserved function of the former during reactivation. Phylogenetic reconstruction of the US7-US9 region revealed eight strongly supported clusters within the 55 U.S. HSV-2 strains sampled, which were preserved in a second global phylogeny. Thus, although we have demonstrated evolutionary diversity in the US7-US9 complex, we found no molecular evidence of sequence variation in US7-US9 that distinguishes isolates from subjects with frequently recurrent episodes of disease.

Fig 1. gI, gE and US9 amino acid sequences of isolates from the Herpevac Trial.

Substitutions in the A) gI, B) gE, and C) US9 amino acid sequences are shown compared to reference sequence HG52. Sequence of the South African reference strain SD90e is also represented. Colored lines indicate whether the pattern of substitutions occurred in non-recurrent and recurrent samples (black), non-recurrent samples only (blue) or recurrent samples only (red). Vertical lines represent substitutions and triangles represent INDELs. The uppermost example of each variant is labeled; those below share the same variation unless indicated otherwise (e.g. I241K vs. I241R). The number of subjects’ samples that match each pattern is indicated at the right in parentheses. The location of the putative US8A polypeptide is also shown. SIG, signal sequence; tm, transmembrane domain.

Fig 2. Mean ratios of non-synonymous (dN) to synonymous (dS) evolutionary substitutions in Us7, Us8, Us8A, and Us9 genes from subjects with non-recurrent vs. recurrent HSV-2 infections.

Bars represent the mean ± SEM of 12 non-recurrent or 8 recurrent samples. P values are shown above the bars. ns, not statistically significant.

Fig 3. Phylogenetic reconstruction and amino acid variants shared among different U.S. HSV-2 clusters.

Amino acid sequences in the SD90e reference strain correspond to gI (highlighted in green), gE (yellow), US8A (orange) and US9 (grey). Each gene segment is also bracketed at the bottom of the figure. Amino acid sequences were ordered according to the Bayesian tree diagrammed on the left side of the figure. Blue and red accession numbers represent, respectively, non-recurrent and recurrent isolates (Table 2). Selected regions with amino acid variation are shown. The position of each amino acid variant shared by non-related clusters (♦) or at which multiple amino acid variants occur ($♦$) is indicated above the reference strain. Bayesian posterior probabilities (≥80%) and maximum-parsimony bootstrap (≥65%) / maximum-likelihood bootstrap (≥65%) values appear above and below the branches, respectively. * High-passage laboratory strain.